Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
1
University Of Sindh
Jamshoro
PH.D. Thesis
Biosynthesis of Pectolytic Enzyme by Plant Pathogenic
Fungi Using Agricultural Waste as a Carbon Source
BY
Ghulam Sughra Mangrio
ENZYME AND FERMENTATION RESEARCH
LABORATORY, INSTITUTE OF BIOTECHNOLOGY
AND GENETIC ENGINEERING, UNIVERSITY OF
SINDH, JAMSHORO, PAKISTAN
2014
2
University Of Sindh
Jamshoro
PH.D. Thesis
Biosynthesis of Pectolytic Enzyme by Plant Pathogenic
Fungi Using Agricultural Waste as a Carbon Source
BY
Ghulam Sughra Mangrio
THESIS SUBMITTED TO THE UNIVERSITY OF SINDH IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF THE DEGREE OF DOCTOR OF
PHILOSOPHY IN BIOTECHNOLOGY
ENZYME AND FERMENTATION RESEARCH LABORATORY, INSTITUTE OF BIOTECHNOLOGY AND GENETIC
ENGINEERING, UNIVERSITY OF SINDH, JAMSHORO, PAKISTAN
2014
3
4
TABLE OF CONTENTS
Certificate і
Dedication іі
Acknowledgement ііі
List of Abbreviations iv
Summary vi
List of Figures ix
List of Tables xxi
CHAPTER 1 INTRODUCTION PAGE NO.
Introduction 1
Aim and objectives 9
CHAPTER 2 REVIEW OF LITERATURE 10
Review of literature 10
INTRODUCTION OF FUNGI 26
Aspergillus niger 26
Aspergillus fumigatus 27
Mucor geophillus 28
Penicillium lilacinum 28
CHAPTER 3 MATERIALS AND METHODS 30
Chemicals 30
Microorganism 30
Inoculum 30
Optimization of inoculum size 30
Mineral medium 31
Fermentation medium 31
Sample harvesting 31
Biomass 31
A- Optimization of culture conditions 32
i- Effect of fermentation time period 32
5
ii- Effect of carbon source 32
iii- Effect of nitrogen source 32
iv- Effect of pH 32
v- Effect of Temperature 32
vi- Characterization of crude pectinase 33
Vii- Effect of Time of incubation 33
Viii- Effect of Substrate concentration 33
ix- Effect of Enzyme concentration 33
x- Effect of different pH 33
Xi- Effect of pH stability 33
Xii- Effect of temperature 34
Xiii- Effect of temperature stability 34
Xiv- Effect of Metal ions/compounds 34
B- Preparation of Enzyme 34
i- Ammonium sulphate fractionation 35
ii- Dialysis 35
iii- Preparation of gel Sephadex G-100 35
iv- Gel filtration chromatography 35
v- Ion exchange chromatography 36
C- Characterization of purified pectinase 36
i- Effect of temperature on pectinase activity and stability 36
ii- Effect of pH on pectinasee activity and stability 36
iii- Kinetic determinations 37
iv- Effect of metal ions/compounds 37
D- Analytical methods 37
i- Assay of pectinase activity 37
ii- Protein estimation 38
iii- Determination of total carbohydrate 40
iv- Determination of reducing sugars 40
v- Molecular mass determination 42
6
CHAPTER 4 RESULTS AND DISCUSSION 44
A- Growth conditions and enzyme production 45
i- Effect of size of inoculums 45
ii- Fermentation mode 48
iii- Effect of incubation period 49
iv- Effect of agro-industrial wastes as carbon sources 55
v- Effect of sugars as carbon sources 91
vi- Effect of nitrogen sources 106
vii- Selection of the organism 126
viii- Effect of pH on pectinase production 127
ix- Effect of temperature on pectinase production 129
B- Characterization of crude pectinase Enzyme 130
i- Effect of time of incubation on crude pectinase 130
ii- Effect of substrate concentration on crude pectinase 131
iii- Effect of enzyme volume on crude pectinase 132
iv- Effect of different buffers on crude pectinase 133
v- Effect of pH on crude pectinase 134
vi- Effect of pH stability on crude pectinase 136
vii- Effect of temperature on crude pectinase 137
viii- Effect of temperature of crude Pectinase 139
ix- Effect of metal ions/compounds on crude pectinase 140
x- Effect of different concentration of CaCl2 as activator 141
xi- Effect of thermostability with and without activator 142
C- Purification of enzyme 144
i- Removal of microbial cells and other solid matter 145
ii- Concentration by precipitation 145
iii- Ammonium sulphate fractionation 145
iv- Dialysis 146
D- Chromatography 146
i- Gel filtration chromatography 146
ii- Ion exchange chromatography 147
7
iii- Homogeneity 149
iv- Molecular weight 149
E- Characterization of purified pectinase 151
i- Effect of substrate specificity 151
ii- Effect of substrate concentration on pectinase activity 152
iii- Effect of pH on pectinase activity produced by Aspergillus niger 153
iv- Effect of pH stability on pectinase activity produced by Aspergillus niger 155
v- Effect of temperature on pectinase activity produced by Aspergillus niger 156
vi- Effect of temperature stability on pectinase activity produced by
Aspergillus niger 158
vii- Effect of activators and inhibitors 159
CONCLUSION 163
Further Suggestions 164
REFERENCES 166
8
LIST OF TABLES
4.1 Composition of working resolving and stacking gels. 43
5.1 Effect of size of inoculums on growth and pectinase production by A. fumigates. 46
5.2 Effect of size of inoculum on growth and pectinase production by A. niger.
46
5.3 Effect of size of inoculum on growth and pectinase production by a mixed culture of A. niger + A. fumigatus. 47
5.4 Effect of size of inoculum on growth and pectinase production by M . geophillus. 47
5.5 Effect of size of inoculum on growth and pectinase production by P. lilacinum.
47
5.6 Effect of fermentation mode for the growth and biosynthesis through different filamentous fungi. 48
5.7 A. fumigatus was grown on mineral medium without glucose at 30 ± 2º C pH was adjusted at 6.5. 56
5.8 A mixed culture of A. fumigatus + A. niger was grown on mineral without glucose at 30 ± 2 ºC pH was adjusted at 6.5. 56
5.9 A. niger was grown on mineral medium without glucose at 30± 2ºC pH was adjusted at 6.5. 57
5.10 M. geophillus was grown on mineral medium without glucose at 30 ± 2 ºC pH was adjusted at 6.5. 57
5. 11 P. lilacinum was grown on mineral medium without glucose at 30 ± 2 ºC pH was adjusted at 6.5. 58
5.12 A. fumigatus was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5. 60
5.13 A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5. 60
5.14 A. niger was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5. 61
5.15 M. geophillus was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5. 61
5.16 P. lilicinum was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5. 62
9
5.17 Effect on growth and pectinase production by different filamentous fungi when grown on mineral medium supplemented with 1% glucose and without glucose at 30± 2 ºC and the initial pH was adjusted at 6.5. 63
5.18 A. fumigatus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 64
5.19: A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5. 65
5.20 A. niger was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 65
5.21 M. geophillus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 66
5.22 P. lilacinum was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 66
5.23 A. fumigatus was grown on mineral medium supplemented with 5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 67
5.24 A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5. 68
5.25 A. niger was grown on mineral medium supplemented with 5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 68
5.26 M. geophillus was grown on mineral medium supplemented with 5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5. 69
5.27 P. lilacinum was grown on mineral medium supplemented with 5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5. 69
5.28 Effect on growth and pectinase production by different fungi when grown on mineral medium supplemented with 2.5 and 5% date Syrup at 30 + 2 ºC and pH was adjusted 6.5. 70
5.29 A. fumigatus was grown on mineral medium supplemented with 2.5 % molasses at 30 + 2 ºC and pH was adjusted at 6.5. 71
5.30 A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 72
5.31 A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5 % molasses when incubated at3 0± 2 ºC and pH was adjusted at 6.5. 72
10
5.32 M. geophillus was grown on mineral medium supplemented with 2.5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 73
5.33 P. lilacinum was grown on mineral medium supplemented with 2.5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 73
5.34 A. fumigatus was grown on mineral medium supplemented with 5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 74
5.35 A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 75
5.36 A. niger was grown on mineral medium supplemented with 5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 75
5.37 M. geophillus was grown on mineral medium supplemented with 5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 76
5.38 P. lilacinum was grown on mineral medium supplemented with 2.5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 76
5.39 Effect on growth and pectinase production by different fungi when grown on mineral medium supplemented with 2.5 and 5% molasses at 30 + 2 ºC and pH was adjusted 6.5. 77
5.40 A. fumigatus was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 78
5.41 A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 78
5.42 A. niger was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 79
5.43 M.geophilus was grown on mineral medium supplemented with 2.5% citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 79
5.44 P. lilacinum was grown on mineral medium supplemented with 2. 5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 80
5.45 A. fumigatus was grown on mineral medium supplemented with 5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 81
5.46 A mixed culture of A. niger +A. fumigatus was grown on mineral medium supplemented with 5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 81
5.47 A. niger was grown on mineral medium supplemented with 5 %citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 82
5.48 M. geophilus was grown on mineral medium supplemented with 5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 82
11
5.49 P. lilacinum was grown on mineral medium supplemented with 5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 83
5.50 Effect on growth and pectinase production by different fungi when grown on mineral medium supplemented with 2.5 and 5% citrus pectin at 30 + 2 ºC and pH was adjusted 6.5. 84
5.51 A. fumigatus was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 85
5.52 A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 85
5.53 A. niger was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 86
5.54 M. geophillus was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 86
5.55 P. lilacinum was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 87
5.56 A. fumigatus was grown on mineral medium supplemented with 5% CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 88
5.57 A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 88
5.58 A. niger was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 89
5.59 M. geophillus was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 89
5.60 P. lilacinum was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 90
5.61 Effect on growth and pectinase production by different fungi when grown on mineral medium supplemented with 2.5 and 5% CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 91
5.62 A. niger was grown on mineral medium supplemented with 2.5% fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 93
5.63 A. niger was grown on mineral medium supplemented with 5% fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 93
5.64 P. lilacinum was grown on mineral medium supplemented with 2.5% fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 94
12
5.65 P. lilacinum was grown on mineral medium supplemented with 5% fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 94
5.66 A. niger was grown on mineral medium supplemented with 2.5% maltose and 5% molasses as carbon source at 30 ±2 ºC and pH was adjusted at 6.5. 95
5.67 A. niger was grown on mineral medium supplemented with 5% maltose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 96
5.68 P. lilacinum was grown on mineral medium supplemented with 2.5% maltose and 5% molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 96
5.69 P. lilacinum was grown on mineral medium supplemented with 5% maltose and 5% molasses as carbon source at 30±2 ºC and pH was adjusted at 6.5. 97
5.70 A .niger was grown on mineral medium supplemented with 2.5% sucrose and 5% molasses as carbon source at 30 ±2 ºC and pH was adjusted at 6.5. 98
5.71 A. niger was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 98
5.72 P. lilacinum was grown on mineral medium supplemented with 2.5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 99
5.73 P. lilacinum grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 99
5.74 A. niger was grown on mineral medium supplemented with 2.5%galactose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 100
5.75 A niger was grown on mineral medium supplemented with 5% galactose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 100
5.76 P. lilacinum was grown on mineral medium supplemented with 2.5% galactose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 101
5.77 P. lilacinum was grown on mineral medium supplemented with 5%galactose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 101
5.78 A. niger was grown on mineral medium supplemented with 2.5% starch and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 103
5.79 A. niger was grown on mineral medium supplemented with 5% starch and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 103
5.80 P. lilacinum was grown on mineral medium supplemented with 2.5% starch and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 104
5.81 P. lilacinum was grown on mineral medium supplemented with 5 % starch, 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 104
13
5.82 Effect on growth and pectinase production by A. niger grown on mineral medium supplemented with 5% molasses and sugars (2.5% and 5%) as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 105
5.83 Effect on growth and pectinase production by P.lilacinum grown on mineral medium supplemented with 5% molasses and sugars (2.5% and 5%) as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 106
5.84 A niger was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source and 0.2% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5. 107
5.85 A. niger was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source and 0.4% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5 108
5.86 P. lilacinum was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source and 0.2% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5. 108
5.87 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5. 109
5.88 A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses as carbon source and 0.2% urea at 30 ± 2 ºC and pH was adjusted at 6.5. 110
5.89 A. niger was grown on mineral medium supplemented with 5%sucrose, 5% molasses as carbon source and 0.4% urea at 30 ± 2 ºC and pH was adjusted at 6.5. 110
5.90 P. lilacinum was grown on mineral medium supplemented with 5%sucrose, 5% molasses as carbon source and 0.2% urea at 30 ± 2 ºC and pH was adjusted at 6.5. 111
5.91 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses as carbon source and 0.4% urea at 30 ± 2 ºC and pH was adjusted at 6.5. 111
5.92 A. nigar was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.2% NaNO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 112
5.93 A. niger was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.4% NaNO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 112
5.94 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.2% NaNO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 113
5.95 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.4% NaNO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 113
5.96 A. niger was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.2% KNO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 114
14
5.97 A. niger was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.4% KNO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 114
5.98 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.2% KNO3 at 30 ±2 ºC and pH was adjusted at 6.5 115
5.99 P. lilacinum was grown on mineral medium supplemented with 5% Sucrose, 5%
molasses and 0.4% KNO3 at 30 ± 2 ºC and pH was adjusted at 6.5 115
5.100 A. niger was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.2% NH4NO3 at 30 ± 2 ºC and pH was adjusted at 6.5 116
5.101 A. nigar was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.4% NH4NO3 at 30 ± 2 ºC and pH was adjusted at 6.5 116
5.102 P. lilacinum was grown on mineral medium supplemented with 5% Sucrose, 5%
Molasses and 0.2% NH4NO3 at 30± 2 ºC and pH was adjusted at 6.5. 117
5.103 P.lilacinam was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.4% NH4NO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 117
5.104 A. niger was grown on mineral medium supplemented with 5%sucrose, 5% molasses and 0.2% peptone at 30 ± 2 ºC and pH was adjusted at 6.5. 118
5.105 A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% Peptone at 30 ± 2 ºC and pH was adjusted at 6.5. 118
5.106 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% peptone at 30 ± 2 ºC and pH was adjusted at 6.5. 119
5.107 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% peptone at 30 ±2 ºC and pH was adjusted at 6.5. 119
5.108 A. niger was grown on mineral medium supplemented with 5%sucrose, 5%
molasses and 0.2% (NH4)2SO4 at 30 ± 2ºC and pH was adjusted at 6.5. 121
5.109 A.niger was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.4% (NH4)2SO4 at 30 ± 2 ºC and pH was adjusted at 6.5. 121
5.110 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.2% (NH4)2SO4 at 30 ± 2 ºC and pH was adjusted at 6.5. 122
5.111 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5%
molasses and 0.4% (NH4)2SO4 at 30 ± 2 ºC and pH was adjusted at 6.5 122
5.112 Effect of nitrogen sources on growth and pectinase production by A. niger 124
5.113 Effect of nitrogen sources on growth and pectinase production by P. lilacinum 125
15
5.114 Effect of pH on Biosynthesis of Pectinase by A. niger grown on mineral medium
supplemented with 5% sucrose, 5% molasses and 0.4% (NH4)2SO4 at 30 ± 2 ºC for 72 hours 128
5.115 Effect of Temperature on Biosynthesis of Pectinase by A. niger grown on mineral
medium containing 5% molasses 5% sucrose and 0.4% (NH4)2SO4 while pH was adjusted 6.00 for 72 hours 130
5.116 Purification steps of Pectinase produced by Aspergillus niger 148
16
LIST OF FIGURES
1.1 Structure (main chain) of low and high methylated pectic substances and site of action of enzymes involved in their degradation 05
4.1 Standard graph for Galacturonic acid 38
4.2 Standard Graph for total protein 39
4.3 Standard Graph for total carbohydrate 40
4.4 Standard curve for reducing sugar 41
5.1 A. fumigatus, A. niger , A. niger+ A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 1% glucose as carbon source at 30 ± 2ºC pH was adjusted at 6.5 51
5.2 A. fumigatus, A. niger , A. niger+ A. fumigatus, M.geophilus, P. lilacinum were grown on mineral medium containing 2.5 % date sugar as carbon source at 30 ± 2 ºC pH was adjusted at 6.5 51
5.3 A. fumigatus, A. niger , A. niger+ A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 5 % date sugar as carbon source at 30 ± 2 ºC pH was adjusted at 6.5 52
5.4 A. fumigatus, A. niger, A. niger+ A. fumigatus, M.geophilus, P. lilacinum were grown on mineral medium containing 2.5 % Molasses as carbon source at 30 ± 2 ºC pH was adjusted at 6.5. 52
5.5 A. fumigatus, A. niger , A. niger+ A. fumigatus ,M.geophilus, P. lilacinum were grown on mineral medium containing 5 % Molasses as carbon source at 30 ± 2 ºC pH was adjusted at 6.5. 53
5.6 A. fumigatus, A. niger, A. niger+ A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 2.5 % crude citrus pectin as carbon source at 30 ± 2ºC pH was adjusted at 6.5. 53
5.7 A. fumigatus, A. niger, A. niger+ A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 5 % Crude citrus pectin as carbon source at 30 ± 2 ºC pH was adjusted at 6.5. 54
5.8 A. fumigatus, A. niger , A. niger + A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 5 % Crude citrus pectin as carbon source at 30 ± 2 ºC pH was adjusted at 6.5 54
5.9 A. fumigatus, A. niger, A. niger+ A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 5 % crude citrus pectin as carbon source at 30 ± 2 ºC pH was adjusted at 6.5. 55
17
5.10 Comparison of pectinase production by different organisms grown on 5 % Molasses as a carbon source 92
5.11 Comparison of Pectinase production produced by Aspergillus niger and Penicillium lilacinum 126
5.12 Effect of time of incubation on crude Pectinase 131
5.13 Effect of substrate concentration on crude Pectinase 132
5.14 Effect of enzyme concentration on crude Pectinase 133
5.15 Effect of different buffers on crude Pectinase 133
5.16 Effect of pH on crude Pectinase 135
5.17 Effect of pH stability on crude Pectinase 137
5.18 Effect of temperature on crude Pectinase 138
5.19 Effect of temperature stability on crude pectinase 139
5.20 Effect of metal ions/ compounds on pectinase activity 141
5.21 Effect of different concentrations of CaCl2 142
5.22 Effect of themostability at 60°C on different time periods with and without activator
CaCl2 (15mM) on pectinase Activity produced by Aspergillus niger 143
5.23 Effect of themostability at 70 °C on different time periods with and without activator
CaCl2 (15mM) on pectinase Activity produced by Aspergillus niger 143
5.24 Gel Chromatography 147
5.25 Purifiction of Pectinase (F-3) on ion exchange chromatography 148
5.26 SS-PAGE (10% Polyacrylamide) of the purified enzymes. Lane 1, low Mw Marker; Lane 2, Fraction 1; Lane 3, Fraction 2; Lane 4,Fraction 3; Lane 5, Fraction 4; Lane 6, Crude enzyme 150
5.27 SDS-PAGE (10% Polyacrylamide) of the purified enzymes. Lane 1, low Mw Marker; Lane 2, Fraction 3a; Lane 3, Fraction 3b; Lane 4, Crude enzyme 150
5.28 Effect of substrate specificity on pectinase produced by Aspergillus niger 152
5.29 Effect of substrate concentration on Pectinase activity produced by Aspergillus niger 153
5.30 Effect of pH on pectinase activity produced by Aspergillus niger 155
5.31 Effect of pH stability on pectinase activity produced by Aspergillus niger 156
5.32 Effect of temperature on pectinase activity produced by Aspergillus niger 157
18
5.33 Effect of thermostability on pectinase activity produced by Aspergillus niger 159
5.34 Effect of activators & inhibitors (F-1) 161
5.35 Effect of activators & inhibitors (F-2) 161
5.36 Effect of activators & inhibitors (F-3a) 162
5.37 Effect of activators & inhibitors (F-3b) 162
5.38 Effect of activators & inhibitors (F-4) 163
19
CERTIFICATE
This is to certify that research work entitled “Biosynthesis of pectolytic enzyme
by plant pathogenic fungi using agricultural waste as a carbon source” has
been carried out by Miss Ghulam Sughra Mangrio under my supervision in the
Enzyme and Fermentation Research Laboratory, Institute of Biotechnology and
Genetic Engineering, University of Sindh, Jamshoro, Pakistan. The work
reported in this thesis is genuine and distinct. This dissertation is worthy of
presentation to the University of Sindh for the award of degree of Doctor of
Philosophy in Biotechnology.
Signature of the supervisor Prof. Dr. Muhammad Umar Dhot Professor and Ex Director and Founder
Institute of Biotechnology and Genetic Engineering,
University of Sindh, Jamshoro
20
DEDICATION
I Dedicate
This Little Effort To
My Father
Late Prof. Haji Khan Mangrio
The First Inspiration Towards Life
ACKNOWLEDGEMENTS
All praise for the, “Allah SWT” Who is the only supreme Authority, my
countless thanks to Him for accrediting me to accomplish this important task in
21
my life. All my inspiration and greatest respect to the Prophet of Islam (peace be
upon him) who is a greatest and matchless teacher for human kind. In view of
his saying: “He who does not thank to people is not thankful to Allah”.
I am highly grateful in paying deepest thanks to my highly respected
teacher and supervisor Prof. Dr. Muhammed Umar Dahot Ex- Director and
Founder of Institute of Biotechnology and Genetic Engineering, University of
Sindh, Jamshoro for his kindness, valuable guidance, encouragement and
cooperation. His enthusiastic inspiration and affection enabled me to attain the
objectives without any difficulty.
I feel pleasure to express my sincere gratitude to my colleague Dr. Altaf
Ahmed Simair for his valuable suggestions during research and in writing the
dissertation and most gratefully I want to express my deep appreciation to all the
staff members of the IBGE, University of Sindh for their nice behavior and co-
operation throughout the work and especially I want to say thanks to Mr.
Gulbahar who remained with us even on holidays.
I wish to express my thanks to Mr. Gain Chand Lab. Assistant Department
of Biotechnology, Sindh Agriculture University, Tandojam for his sincere
cooperation and moral support during compilation of my work.
Also, thanks for the generosity of HEC Islamabad and IBGE, University of
Sindh for providing funds for completing this project.
Ghulam Sughra Mangrio
LIST OF ABBREVIATIONS
E Activation energy (kJmol-1)
ATCC American Type of Culture Collection
22
BSA Bovine Serum Albumin
CCP Commercial citrus pectin
conc. Concentration
CSL Corn Steep Liquor
Da Dalton
DNS Dinitrosalicylic acid
endo-PGLLs EndoPolygalacturonate lyase
exo-PGLs Exo Polygalacturonate lyase
g Gram
h Hour
kDa Kilo-Dalton
k0 Frequency factor (min-1)
Km Michaelis-Menten constant (equilibrium constant)
L Litre
M Molar
M.O Microorganism
M.W Molecular weight
min minutes
Ml Millilitre
mm Mili meter
mM Millimolar
OD Optical Density
PG Polygalacturonase
PGA Polygalacturonic Acid
PE Pectin esterases
PL Pectin Lyase
PG Polygalacturonase
PMG Polymethylgalacturonases
Endo-PMG Endopolymethylgalacturonases
Exo-PMG Exopolymethylgalacturonases
Exo-PG Exopolygalacturonase
PME Pectin Methyl Esterase
PME Pectin Methyl Esterase
PMG Polymethylgalacturonase
23
PMGL Polymethylegalacturonate lyase
Endo-PMGL EndoPolymethylegalacturonate lyase
Exo-PMGL ExoPolymethylegalacturonate lyase
PGL Polygalacturonate lyase
SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel
Electrophoresis
(sp.) Species
nm Nanometer
No. Number
rpm Revolutions per minute
SMF Submerged fermentation
SSF Solid state fermentation
t1/2 Half-life time of enzyme (min)
Vm Maximum Forward Velocity of the
U Unit
μm Micrometer
v/v Volume per volume
w/v Weight per volume
w/w weight per weight
μg micrograms
μl microliters
Endo-PGL poly (1,4-α-D-galacturonide) lyase,
Exo-PGL poly (1,4-α-D-galacturonide) exolyase
24
SUMMARY
Present study was carried out in the Enzyme and Fermentation Research
laboratory, Institute of Biotechnology and Genetic Engineering, University of
Sindh, Jamshoro. In this study Agricultural waste was used as a carbon source.
New enzymes have been focused by researchers due to their commercial
applications with desirable biochemical and physico-chemical characteristics and
a low cost of production. Research on the selection of suitable substrates for
fermentation has mainly been centered on agro-industrial residues due to their
potential advantages for filamentous fungi. The utilization of these agro-
industrial wastes, on the one hand, provides alternative substrates and, on the
other, helps in solving pollution problems, which otherwise may cause big
problem for their disposal.
In this study various concentration (2.5% and 5%) of natural sugars were
used and 5% molasses was investigated best substrate/carbon source, the best
pectinase producers were selected as A. niger and P. lilacinum. Different carbon
sources (2.5 and 5% fructose, maltose, sucrose galactose and starch) were
incorporated with 5% (v/v) molasses. The experiments were conducted in
triplicates and the results presented are the mean values. Synthetic sugars along
with 5% molasses were used as a carbon source for the growth and production of
pectinase by submerged fermentation process. Different synthetic and natural
nitrogen sources were also used and optimization of temperature and pH was
carried out to maintain a maximum production of pectinase enzyme.
After screening best substrate (carbon source) which was 5% molasses,
fermentation medium was supplemented with 5% molasses and various sugars 2.5
and 5% (fructose, maltose, sucrose, galactose and starch) were tested to find out
optimum carbon sources. The addition of 5.0% sucrose as carbon source induced
the pectinase production while low production of pectinase was recorded with
25
carbon sources other than sucrose. In this study, A. niger and P. lilacinum exhibit
high pectinase production when grown on media supplemented with 5%
molasses. Aspergillus niger is most efficient among filamentous fungi and results
reveal that enzyme to be produced is highly depended upon substrate and
microorganism. An overview of results obtained show that 5% sucrose, 5%
molasses and 0.4% (NH4)2SO4 the best carbon and nitrogen sources for the
production of pectinase by A. niger. The maximum production of pectinase (26.87
U/ml) was observed at pH 6.0 after 72 h incubation. The optimum temperature for
the maximum production of pectinase was achieved at 35 ºC when maximum
production of pectinase was obtained as 28.25 U/ml. The ammonium sulphate
was selected as a best nitrogen source and Aspergillus niger was selected best
organism, which has produced higher amount of pectinase when cultivated in
comparison to Penicillium lilacinum on same optimal conditions.
The crude Pectinase enzyme was characterized on the basis of various
parameters such as incubation time, substrate concentration, enzyme volume,
pH, pH stability, temperature, temperature stability, and effect of various metal
ions or compounds. The Pectinase activity was noted maximum at 15 minutes of
incubation time, 1.5% citrus pectin and 1ml enzyme volume. The highest enzyme
activity was found at pH 5, whereas pectinase exhibited stability in the range of
pH 4.0 to 7.0. The optimum Pectinase activity was noted at 40˚C temperature
while crude pectinase was 100% stable up to 40 °C but activity declined and
retains more than 30% activity up to 80 °C. CaCl2 (1.5 mM) stimulated the
Pectinase activity as compare to other metal ions /compounds.
Pectinase enzyme was purified with ammonium sulphate precipitation and
dialyzed sample was finally applied on gel filtration chromatography (Sephadex G-
100) and Ion Exchange DEAE A-50. The enzyme was purified 2.5 fold by gel
chromatography on Sephadex G-100 and 2.19 fold by Ion Exchange DEAE A-50.
Four fractions were obtained, Fraction 1, 2, 4 showed single bands while Fraction -3
26
showed multiple bands on SDS Page electrophoresis. Fraction -3 was pooled,
dialyzed and separated on Sephdex A-50 and two fractions 3a and 3b showed single
band. The molecular weights of the purified fractions were detected in the range of
found to be 33000 ± 2000 and 38000± 2000 Daltons. The purified enzyme was
specifically most active with pure pectin, while crude pectin, Lemon pectin and
orange peel given lower activity as compared to (control) i-e pure pectin. The
optimum pH and temperature for pectinase activity for different fractions were
between pH 5.0 and 6.0 and 40°- 50 °C, respectively. The enzyme was stable over the
pH range 3.0-8.0. More than 30 % activity was retained when purified pectinase was
incubated with pH 8.0. The thermostability of each fraction was determined and it
was observed that the pectinase activity in all fractions is heat stable up to
temperatures ranging from 50 to 60 °C and activity decreased as the incubation
temperature was increased above 60 °C. The temperature profile showed that
purified pectinase retained maximum activity up to 60 °C and retain activity more
than 40% when incubated at 90°C for 10 minutes. The pectinase activity of different
fractions (FI, F2, F3a, F3b and, F4) was increased with different metal ions. The
Pectinase activity was stimulated in the presence of CaCl2 in all fractions in the effect
of 110-130. ZnSO4, MnSO4 and Mg SO4 shown higher activity in fractions ( F3a, F3b
and F4), while in fractions F1 and F2 ZnSO4 and MnSO4 shown slight inhibition
effect on pectinase activity, which indicates that the pectinase belongs to metalo-
enzymes.
It is concluded that Aspergillus niger is capable to produce pH stable and
thermostable pectinase for industrial purposes. Pectinase from Aspergillus niger
could convert orange peel pectin successfully, and thus the enzyme could not
only act as an agent for bioconversion but also could replace the use of highly
expensive commercial pectin in food industry.
27
CHAPTER NO.1
INTRODUCTION
Human civilization has been using a variety of enzymes in food and in
other processes since long time. Historically, the enzyme industry was developed
through the use of plant and animal materials and industrial use of enzyme is
now an integral part of a wide variety of commercial processes. The applications
of enzymes ranging from small to complex or large scale in the manufacturing of
chemicals, processed foods and many supermarket products. Due to the increase
in the cost of energy and contaminated water, enzyme technology play
important role in science and technology and their use will be enhanced in
future. The development of stirring tank fermented and the genetic manipulation
of microbial cultures used for overproduction of the desired product. Nowadays,
microorganisms are the major sources of enzymes both in volume and variety.
Genetic engineering technology has helped in accelerating the development and
production of enzyme in both bacteria and fungi. Bacteria have the advantage of
fast growth and short fermentation cycle while fungi are preferred to produce
large quantities of desirable enzymes (Gupta and Mukerji, 2001).
It is most likely that the greatest variety of traditional biotechnological
processes are found in the area of food and nutrition, particularly in the
manufacture of foodstuffs and beverages. These processes can be improved, their
efficiency and yield through the selection of more productive microbial strain,
the control of culture condition, and through the adaptation of fermentation
products to the evaluation of food habits and to the consumers’ changing tastes
(Vibha and Neelam 2010, Gurung et al., 2013).
28
According to Global Industry Analysts, Inc (2011) and Norus (2006) in the
field of Biotechnology enzymes are a very well recognized products and the
production of food and brewing enzymes in the world market is estimated to
spread about $ 1.3 billion by 2015 with the highest sales occurred in the milk and
dairy market, BBC Research (2011).
Pectinases production enjoys about 10% of total enzyme production.
Many microorganisms like yeast, protozoan, bacteria, fungi, insects, nematodes
and plants produce Pectinolytic enzymes, but microbial pectinases are more
significant due to their involvement in the Phytopathology plant– microbe
association and the decay of deceased plant materials as reported by Pedrolli et
al., (2009). Enzymes of Microbial origin are good biocatalysts for different
industrial applications (Hasan et al., 2006 ).
Among the various types of fermentation, which aim of providing a
higher nutritional and economic value to agricultural products and by-products
of the food industries, fermentations in solid medium comprise a number of
biotechnological processes and these are of great interest to developing countries
to adopt a fermentation process for the production of daily used goods. Today,
pectinase enzymes are one of the forthcoming enzymes of commercial zone and
the pectinases of microbial origin account for 25% of the international food
enzymes sales (Jayani et al., 2005). This enzyme has an excessive impact with
extraordinary prospective to be offered to food industry specially to process
fruits and vegetables (Whitaker, 1990). The pectinases are very powerful and
continuously upcoming for the commercial sector, especially for food and juice
industry (Kashyap et al., 2001) these enzymes play a major role in the pulp and
paper industry (Beg et al., 2001 and Vikari et al., 2001).
Enzymes are capable to catalyze and degrade all synthetic products of
living creatures. Enzymes first time reported in the nineteenth century. Since
then their use has highly increased in various industries and laboratories. In the
recent years their use is rapidly increasing in the field of biotechnology,
29
especially in the fields of protein and genetic engineering. There are many
exciting research studies involving enzymes with the development of new
commercial and industrially important processes. Enzymes are widely used in
various emerging industries because of their high catalytic power, a specific
mode of action, stereo specificity, and eco-friendly and capable to reduce energy
requirements etc (Vikari et al., 2001)
The biodiversity of microbes is very significant for many reasons,
starting from aesthetic concern to its usefulness, especially in biotechnology.
The excessive emergent sectors are enzymes for the production of the food and
fuel. The potential of the white biotechnology has an environmental advantage and this
economically beneficial technology is beyond all the questions. Enzymes are biocatalysts
with high selectivity and are utilized in the food industry for centuries and play a
significant role in various other industries such as a detergents, textile, pharmaceuticals,
paper and pulp (Vibha and Neelam, 2010).
Huge amounts of industrial waste residues are produced worldwide by
processing raw agricultural ingredients for foodstuffs. These, in turn, carry out
an excessive BOD load on the atmosphere when discarded. These industrial
wastes produced from the processing of sugar cane, orange, coffee and
rice, which provide proper feed stocks for bioconversion into chemicals as well
as enzymes under fermentation techniques. The other waste produces from
agriculture arises from citrus fruits belong to a significant group of fruit crops
grown all over the globe (Giese et al., 2008).
Fruit processing industries produce a large amount of waste material in
the form of peel, pulp, seeds, etc. Some fresh orange peel is used in shredded
form in the preparation of orange-marmalade. Dried citrus peel is rich in
carbohydrates, proteins and pectin with small amount of fat (Vibha and Neelam,
2011).
Several micro bial conversions have been suggesting the use of
processing food waste to produce valued products like biogas, citric acid,
30
ethanol, chemicals, different enzymes, volatile flavoring agents, fatty acids and
microbial biomass. Citrus skin comprises a considerable quantity of pectin and
that may be utilized as an inducer for the production of pectinase enzyme by
various microorganisms. Exploitation of microorganisms for the production of
enzymes is beneficial and advantageous as climate and seasonal influences
cannot affect them, may also be subjected to genetic and ecological managements
to improve production. To reduce the production cost at the industrial level,
extremely productive strains of micro-organisms are required. Several microbes
are used for enzyme biosynthesis. Pectinolytic enzymes are synthesized by
a huge number of microbes including bacteria and fungi for instance
Bacillus Spp., Clostridium Spp., Pseudomonas Spp., Aspergillus Spp., Monilla laxa,
Fusarium Spp., Verticillium Spp., Penicillium Spp., Sclerotinia libertiana,
Coniothyrium diplodiella, Thermomyces lanuginosus, Polyporus squamosus,. etc
(Vibha and Neelam, 2010).
Pectinase enzymes mostly exist in various living beings like plants,
bacteria, fungi, yeast, insects, nematodes and protozoa. These are negatively
charged acidic glycosidic macromolecules having bigger molecular mass. Pectic
substances occur in the plants as the main components of the middle lamella in
the shape of calcium pectate and magnesium pectate. Pectic material comprises
pectins, pectinic acids, protopectins, and pectic acids. The foremost chain of
pectin is partially methyl esterified 1, 4- D- galacturonan. Demethylated pectin is
known as pectic acid (pectate) or polygalacturonic acid (Alkorta et al., 1998).
31
Figure:-1 Structure (main chain) of low and high methylated pectic substances
and site of action of enzymes involved in their degradation (Sieiro et al., 2012).
The pectinase enzymes attack in several ways on the pectin and these
are widely used in processing of fruit juices, extraction of vegetable oil, processing
of alcoholic drinks and a range of uses in food industries. The commercial
pectinolytic enzyme usually might usually be stimulated at pH 5.0 and 45 to 55
ºC. Pectinase producing microorganisms are broadly dispersed in soil, rotten
fruits, vegetables, decomposed leaves, wood and can also be found in samples
of water taken from decomposing coconut peelings, especially in coastal
areas. Duodenal flora of humans also comprise pectinolytic microbes,
predominantly bacteria, since pectin the dietary fiber have been the substrate
for them (Vibha and Neelam, 2010). Traditionally, pectin of citrus peel and apple
pomace is the commercial source because these sources contain high pectin
content with excellent color properties, citrus peel has often been and perfect
source for pectin manufacturers. Sugar beet and sunflower are most recent
sources for the pectin, the quantity of pectin from various sources differs
significantly like apple pomace 10- 15%, and citrus peel 25-35 %, sugar beet 10-20
% and sunflower 15- 25% (Vibha and Neelam, 2010).
32
Classification:
The classification of pectinase enzymes is established in their mode of
action on the galacturonan chain of the molecules of pectic substances. Primarily,
there are three types of pectinase enzymes (Sakai 1992; Palomaki and Saarilahti
1997).
1) De-esterifying enzymes (pectin esterase): These enzymes produce pectic acid
and methanol by catalyzing the hydrolysis of methyl
2) Depolymerizing enzymes: Depolymerizing enzymes comprise hydrolases
and lyases. Lyases enzymes are called transeliminases, those torn apart the
glycosidic linkages of each pectate (polygalacturonate) or pectin (polymethyl-
galacturonate).These are also sectioned into endo- if its configuration of action
is haphazard or exo if its configuration of action is at the terminal end (Rexova-
Bencova and Markovic 1976; Fogarty and Kelly 1983; Whitaker, 1990; Sakai
1992 and Jayani et al., 2005 ).
3) Protopectinases: This enzyme accelerates the change of protopectin into
soluble pectin or pectinic acids with the consequential parting of plant cells from
one another. Pectinase enzymes are also divided into two types, acidic pectinase
and alkaline pectinase (Kashyap et al ., 2001 )
Acidic pectinase:
The pectinase enzymes used in fruit juices and wine making are known
as acidic pectinases. These pectinases are commonly isolated from fungal
sources. To produce glittering clear juices with the help of enzymes to increase
the yield of juice during pressing, draining and also to eliminate suspending
particles to make particles to make the juice sparkling and clear (Kashyap et al.,
2001) and it also reduces filtration time up to 50% (Blanco et al., 1999). According
to Kashyap et al., (2001) this principle is applied in the following processes:
Preparation of purees and nectars.
Pear juice processing and preparation of purees and nectars.
Strawberry, blackberry, raspberry, apple, orange juice and wine clarification.
33
In cloudy juices more amount of polygalacturonases is complemented to fruit
juices to resolve the cloud some examples are –
Cloud stabilization of orange juice.
Lemon juice clarification, recovery of citrus peel oils, preparation of citrus
salads and dried animal feed from citrus fruits.
Processing of fruits like mango, apricot, guava, papaya, pineapple, banana, etc.
The integral plant cells are preserved by selectively polysaccharides
hydrolysis of the middle lamella (Kashyap et al., 2001). Unicellular goods (Cells
suspension material) are substances produced by the conversion of arranged
tissues, which are utilized as basic material for nectars and pulpy juices for
infant foods, as components for the dairy foodstuffs such as desserts and yogurt
and as protoplasts for several biotechnological uses. The enzymes exploited in
this manner are mentioned as 'macerases'. This practice is written as
maceration. The best enzyme used for the maceration consists of cellulases and
hemicellulases in accumulation of the pectinase enzymes for the maceration of
plant tissues, saachariffication and liquefaction of biomass and isolation of
protoplasts. Filamentous fungi, especially Aspergillus niger is most often used
for industrial synthesis of pectinase enzymes (Kotzekidov, 1991; Barnby et al.,
1990; Naidu and Panda, 1998). The enzymes of fungal origin are used in food
industry as fungi are very powerful producers of pectinase enzymes and the
plus point is their pH (ranges from 3 - 5.5) which is equivalent to various fruit
juices. A number of researchers have conveyed that banana pulp can efficiently
be clarified by depectinazation using pectinase enzyme (Viquez et al., 1981;
Koffi et al., 1991; Yusof and Ibrahim, 1994; Brasil et al., 1995; Alvarez et al., 1998;
Ceci and Lozano, 1998; Vaillant et al., 1999; Lee et al., 2006)
Alkaline pectinases:
These enzymes are mostly utilized in treatment of fiber crops for
pectolytic pretreatment of waste water in food industry particularly fruit juices,
production of paper and pulp, oil extraction and coffee and tea fermentation.
34
Pectic enzymes are also involved in wood preservation; here enzyme
preparations or specific bacteria that produce these enzymes are used Ward and
Fogarty, (1973).
In view of the diverse applications of these acidic and alkaline
pectinases, they form the backbone of the biotechnology industry. The various
ongoing researches are likely to find the application of these extremely important
enzymes.
35
Aims and Objectives:
Pectinases are industrially important enzymes and their demand is
increasing in line with the emerging markets of processed food especially in
processed fruits vegetables, juices and wines industry. Keeping in view the
commercial significance of pectinase and biomass proteins, present project was
chalked out with following objectives:
The major object of this study was to discover the potent producer for
pectinase enzyme by various filamentous fungi.
To grow filamentous fungi on agro-industrial waste for the biosynthesis of
pectinase to explore the cost effective production protocols.
To compare the production rate of pectinase among fungi on natural and
synthetic media with optimal conditions.
To check the final pH and thermostable conditions for pectinase enzyme.
To purify the pectinase enzyme and characterize the enzyme in terms of
optimal pH, temperature, molecular weight and Michaels constant.
36
CHAPTER NO.2
REVIEW OF LITRARURE
Microorganisms under appropriate cultural condition synthesize many
enzymes (including pectinase) and other useful products. The effect of
various factors or different cultural conditions must be optimized before the
production of the final product. Different research workers have used various
microorganisms for the production of pectinase and some of the work done
is as under.
Aguilar and Huitron (1986, 1987) reported the results of many important
aspects of fungal pectinase production by Aspergillus niger. Aspergillus was
isolated from Mexican soil and experiments were conducted through fed
batch culture fermentation. The effect of galacturonic acid, glucose, and the
influence of pH on the production of pectinase was determined through strain
Aspergillus sp CH-Y- 1043.
Solis-Pereyra et al., (1993, 1996) reported a comparative study in which the
effect of high initial concentration of glucose and the effect of different
carbon sources was analyzed for the production of pectinase enzyme by
Aspergillus niger in the solid state fermentation (SSF) and submerged
fermentation (SmF).
Maldonado and Strasser (1998) have conducted research and reported a
study to compare solid state fermentation (SSF) and submerged fermentation
(SmF) for the production of pectin estrases and poly galaturonase by A.niger .
This study showed that through solid state fermentation more pectinase was
produced.
Kashyap et al., (2000) have used Bacillus sp. DT7 (isolated from soil) isolate
bacteria a potent producer of extracellular pectinase enzyme, characterized as
37
pectin lyase (PL) enzyme. In optimized conditions Bacillus sp. DT7 produced 53
units/ml of pectin lyase, which was higher as compared to reported in the
literature. The enzyme was purified through gel filtration and ion exchange
Chromatography. The molecular mass of the purified enzyme was determined
as about 106 kDa. Highest enzyme activity was recorded at temperature 60 ºC
and pH 8.0. Calcium chloride (CaCl2) 100 mM and mercaptoethanol boosted the
activity of the purified enzyme.
Teixeira et al., (2000) have used Aspergillus japonicus 586 for the production of
pectinase estrases, endo and exo-polygalacturonases and tested the effect of
various carbon sources (different concentrations) in a liquid media (Manachini
solutions). The medium was inoculated with 5× 106 spoers /ml and kept under
(140 rpm) agitation at 30 °C for 122 hours. After every 24 hours the culture
broth was separated by filtration and evaluation of pectinestrase was carried
out from Culture broth (Enzyme extract) of A. japonicus 586, which showed
best activity in presence of 0.5% pectin. The higher endopolygalacturonase was
observed in presence of 0.2% pectin and 0.2%, glycerol while 0.5% pectin was
used along with 0.5% glucose showed highest activity of exopolygalacturonase.
Concentration of carbon sources significantly affected the pectinestrase, endo-
and exo-polygalacturonase activities. A repression effect was exhibited on all
the analyzed enzymes when high concentrations of pectin, saccharose and
glucose were used in the culture medium.
Shubakov and Elkina, (2002) have compared the production of
polygalacturonases (PGs) through fungal species like Aspergillus niger ACM
F-1119 and Penicillium dierckxii ACIM F- 152. Polygalacturonases (PGs)
produced from both of the microorganisms and it was found that for both
microorganisms sugar beet pectin showed a powerful inducing effect and
zosteran pectin was also an active inducer for P. dierckxii. The most effective
nitrogen source was found ammonium sulphate (2.2g/l) for both organisms.
A. niger shown highest PG production in a medium with preliminary pH value
38
3.0-4.0 while P. dierckxii was unsuccessful to depend significantly on
preliminary pH value of the medium. However, Penicillium dierckxii was
observed to be more active producer of PG as compared to Aspergillus niger.
Malvessi and da Silveira (2004) have investigated a liquid media
supplemented with wheat bran, salts and inducer (pectin) and found that it was
suitable to produce exo and endo- polygalacturonases by Aspergillus oryzae CCT
3940.The higher production of polygalacturonase enzyme was observed in
comparison to rinds of citrus fruits used as inducer. The highest enzyme
activities were recorded at initial pH 4.0 (control) when it reduced somewhat
lower than pH 3.0 the enzyme produced 159 Units of endopolygalacturonase
mL-1 at 83 hours and 45 units of exo- polygalacturonase mL-1 at 64 h .The
optimal values of pH and temperature for the production of
exopolygalacturonase (4.5/57 ºC) and endopolygalacturonase (4.3 /40 ºC) were
recorded respectively.
Martin et al., (2004) have produced pectinases from newly isolated strains of
fungal origin under solid state fermentation process; the Penicillium sp EGC5
and Moniliella SB9 synthesized pectin lyase (PL) and polygalacturonase strain
Penicillium (PG) respectively on a medium contained combination of orange
bagasse and wheat bran as a substrate. The strain Moniliella produced
Polygalacturonase and pectin lyase showing optimal activity at pH 4.5 and
10 at 55 °C and 45 °C respectively, when same enzymes were produced by
Penicillium EGC5 shown an optimal activity at pH 4.5 - 5 and 9 at 40 °C,
respectively.
Phutela et al., (2005) have screened 120 different isolates of thermophilic
fungal strain for the production of pectinase and polygalacturonase. The fungal
strain was recognized as Aspergillus fumigatus Fres. MTCC 4163. Various
optimum parameters for pectinase and polygalacturonase (PG) production were
determined under solid state fermentation (SSF) system. Maximum enzyme
activities were obtained in cultures when grown in a medium comprising wheat
39
bran, sucrose, yeast extract and ammonium sulphate after 2-3 days of incubation
when temperature was maintained as 50 ºC. Maximum enzyme activities of 1116
U/g-1 for pectinase and 1270 Ug-1 for polygalacturonase were acquired at pH
4.0 and 5.0 respectively.
Joshi et al., (2006) have compared pectin methyl esterase (PME) production by
Aspergillus niger using apple pomace as a substrate under solid state
fermentation (SSF) and submerged fermentation (SmF). Optimal temperature
25 °C and pH 4.0 were found for maximum enzyme production was recorded
within 96 hours under both solid state and submerged fermentation systems.
0.2% ammonium sulphate under solid state fermentation (SSF) and 0.2%
diammonium hydrogen phosphate in submerged fermentation contributed the
maximum production of Pectin Methyl Esterase (PME). The 0.5% Sodium
chloride in solid state fermentation and 2.0% manganese sulphate in submerged
fermentation as an additive contributed the maximum production of pectin
methyl esterase (PME). Solid state fermentation produced as about 2.3 times
higher pectin methyl estrase (PME) activity as compared to submerged
fermentation, under optimized parameters of fermentation.
Patil and Dayanand (2006 a) have assessed pectin rich agro-wastes, which were
locally available as lemon rind, sorghum stalk and sunflower head for the
production of pectinase by Aspergillus niger DMF 27 and Aspergillus niger DMF
45 under submerged fermentation (SmF) and solid state fermentation (SSF)
systems. Maximum patience was produced when Agro wastes were combined
with carbon and nitrogen sources. By adding ammonium sulphate the synthesis
level of pectinase was increased increased with all the substrates in both
submerged fermentation (SmF) and solid state fermentation (SSF) systems.
Kabli (2007) exploited Kluyveromyces marxianus for the production of
pectinases and enzymes were partially purified through fractional precipitation
with ammonium sulphate and at 65% ammonium sulphate the most active
fractionation was obtained. In the next step the purification was done through
40
gel filtration on Sephadex G- 75 followed by ion exchange chromatography on
CM- Sephdex C- 50. Through separation 4 peaks of protopectinase were
obtained, the second peak containing most of the recovered protein and highest
protopectinase activity. The enzyme allowed reacting with propectin sources
and the enzyme showed different hydrolytic activities. Enzyme concentration,
effect of substrate, pH and temperature was determined to characterize the
enzyme. The enzyme showed stability up to 50 ºC at pH 4 and 7, the effect of
metal ions on enzyme activity was also tested.
Maller et al., (2007) have reported that pectinolytic enzymes are mostly
produced by many Aspergillus sp. but no reports are available on Aspergillus
niveus. The objectives were to optimize culture conditions and physico-chemical
parameters for the enzyme production by A. niveus and the partial purification
of the enzyme through ion exchange chromatography. The assays were carried
out with 1% Polygalacturonic acid in 100 mM sodium acetate buffer pH. DNS
method was used to quantify the reducing sugar. Czapeck medium was
optimized when optimized when supplemented with 1% pectin (Sigma) at 30 ºC
for 9 days under stationary conditions, or 2 days under agitation. The citrus fruit
peels shown to be good inducers for polygalacturonases and also low levels of
pectin and pectate lyases polygalacturonase shown highest activity at 55ºC
when pH was adjusted at 4.0 while enzyme was thermostable for 90 min at 60ºC.
The enzyme was activated by 1mM Mn++ (17%) and EDTA (10%). 80%
ammonium sulphate precipitation was used to purify the polygalacturonase and
elution done in DEAE cellulose followed by Biogel P 100.
Arotupin et al., (2008) have isolated Aspergillus repens from cultivated soils,
which synthesized pectin methyl esterase (PME) in the liquid culture medium.
The enzyme was partially purified by ammonium sulphate precipitation
and dialyzed. The dialysate fraction of the enzyme was isolated by molecular
exclusion and ion exchange chromatography.The molecular mass of pectin
methyl esterase (PME) was found to be 141.3 9 kDa.The optimum pH and
41
temperature of the enzyme activity were 6.5 and 30 ºC respectively.The
enzyme activity was stimulated by Na+, K+, Ca2+, Mg2+ and Zn2+, whereas
EDTA, PbCl2, HgCl2 and IAA showed inhibition effect to the enzyme
activity.With the increase of substrate concentration up to 4 mg/ml enzyme
activity was also increased. The Line weaver-Burk plot of pectin hydrolysis
showed approximately 1.3 mg/ml.
Li et al., (2008) have isolated Bacillus gibsonii, designated as S-2 (CGMCC1215)
to produce alkaline pectinases using sugar beet pulp as a substrate. Three endo
PGs were purified through ultra-filtration, ammonium sulphate precipitation
and ion– exchange chromatography followed by characterization of the enzyme
. The three purified alkaline endo PGs, designated as S-I, S-II, and S-III and
their molecular weights were detetmined as 38 kDa on SDS-PAGE. The Km
value and optimal temperature for optimal enzyme activities of S-I, S-II and S-III
were 1.2 mg/ mL and 60 °C, 0.9 mg /mL and 55 °C, 1.1mg/mL and 60°C
respectively. Maximum enzyme activity was found at pH 10.5, metal ions
such as Mg 2+ and Ca 2+ enhanced the activities of S-I, S- II while S -III was
suppressed by Ca 2+, and Mn 2+ while Zn 2+ ions inhibited the activity of all
three alkaline enzymes.
Rashmi et al., (2008) have isolated 34 strains of Aspergillus niger and screened
for pectinase production, the best producers to be found as Aspergillus niger
isolate JGIm2, Aspergillus niger isolate JGIm3 and Aspergillus niger isolate
JGIm5. Optimal synthesis of the enzyme found in a medium incorporated with
5% pectin at 48 hours. The enzyme was partially purified through ethanol
precipitation at optimum temperature (45 °C) and pH (4.0). The Km and Vmax
values were calculated as about 0.178 g/dl and 11.621 U/mg proteins
respectively.
Reda et al., (2008) have worked with Bacillus firmus-1-4071 and found that this
bacteria is capable to synthesize very high amount of the polygalacturonase
enzyme under solid state fermentation (SSF) system. Fifty one isolates of
42
bacteria from fermented clayed Solanum tuberosum (potatoes) were screened
for their potential to synthesize pectinase using pectin as a substrate under
(SSF) conditions. The results showed that all the isolates were producing
pettiness' and out of which twenty isolates showed good pectinase production
by using agro- industrial wastes viz Solanum tuberosum, Solanum mélange and
Echoria cresips on citrus peels mixtures at 30 ºC and pH was adjusted at 6 by
the technique of pectin clearing zones (PCZ).
Prodanović and Anton (2008) have investigated the opportunity of the
purification and partitioning of pectinase enzymes obtained from Penicillium
cyclopium by partitioning in polymer/polymer and polymer/salt aqueous two-
phase systems. In the system with 10% (w/w) polyethylene glycol, 1500/5%
(w/w) dextran, 500,000/85% (w/w) crude enzyme, the highest values
for partitioning factors were obtained. The partitioning constant, top phase yield
and purification factor were obtained as about 2.11, 85.68% and 11.28
respectively for the endo-pectinase activity. On the other hand partitioning
constant was 1.89 followed by the , top phase yield of 84.28% purification factor
3.82 for the exo- pectinase activity. In the system with 10% (w/w)
polyethylene glycol 6000/15% (w/w) ammonium sulphate 75% (w/w) crude
enzyme, purification factor 37.85 and 19.52 for exo- and endo- pectinase
repectively in the bottom phase were achieved.
Martínez-Trujillo et al., (2009) have evaluated growth and pectinase
synthesis by a fungal strain Aspergillus flavipes FP-500 at various initial pH
values using various carbon sources like pectin, galacturonic acid,
polygalacturonic acid arabinose, rhamnose, xylose, glycerol and glucose.
Aspergillus flavipes FP-500 has shown the production of exo-pectinases, endo-
pectinases and pectin lyases. Exo-pectinases and pectin lyase (PL) were
produced at basal level as constitutive enzymes.Endo-pectinases are inducible
enzymes and only can be produced when pectin is present as an inducer.
43
Results revealed that pectinases produced in an intensive mode by A. flavipes
FP- 500.
Mohsen et al., (2009) have purified polygacturonase (PG) obtained from
Aspergillus niger U-86 through ammonium sulphate fractionation followed by
gel filtrationon sephadex G.75.SDS-PAGE of the purified enzyme revealed
two bands having molecular mass of 35000 and 38000 DA. The purified
enzyme was stable at the pH (3.0-6.0) and at 30 ºC. The Km value was
calculated as about 1.42 mg/ml.
Pedrolli et al., (2009) have reported that pectinase enzymes act upon pectic
polysaccharides producing simple molecules. The enzyme has long been used to
clarify along with the increase in the amount of fruit juices. These enzymes are
particularly distributed into two major groups that attack on pectin “smooth”
sections or on pectin “hairy” sections. Pectinases are one of the most broadly
dispersed enzymes in fungi, bacteria as well as in plants.
Banu et al., (2010) have selected ten molds, which were isolated from
metropolitan waste samples and screened for the production of pectinase
enzyme. These molds were grown on solid media (YPSS) containing pectin. The
Penicillium chrysogenum was selected on the basis of the clear zone and the
production of pectinase was conducted in submerged fermentation conditions.
The higher amount of the enzyme production was obtained by Penicillium
chrysogenum using sucrose and ammonium per sulphate as carbon and
nitrogen source respectively at pH 6.5 and 35 °C. The maximum activity of the
pectinase enzyme by Penicillium chrysogenum was recorded at pH 6.5 and 50 °C.
The enzyme was thermostable up to 40 °C. Magnesium chloride (MgCl2) and
calcium chloride (CaCl2) ions had a slight effect on the activity pectinase
enzyme. Km and Vmax values of the enzyme were 1.0 mg/ml and 85 U/mg
protein, respectively. The molecular mass of the enzyme was found as a bout of
31000 Da on SDS-PAGE.
44
Martin et al., (2010) have isolated 34 thermophilic and thermotolarent strains
of fungi from soil, organic manure and industrial waste heap based on their
aptitude to be grown at 45 ºC in pectin containing liquid medium. About 50%
of these fungi were recognized such as Aspergillus Monascus, Thermomyces,
Thermomucor, Chaetomium, Neosartia and Scopilariopsis. All the strains synthesized
pectinase under the solid state fermentation system. Maximum activity of the
enzyme was achieved in a culture medium inoculated with thermophilic strain
N31, which was recognized as Thermomucor Indicae –seudaticae cultured on a
medium incorporated with a blend of orange bagasse and wheat bran (1:1) with
70% preliminary moisture using solid state fermentation (SSF) system. This
fungus synthesized the highest amount of Exo-polygalacturonase 120 U/ml in
solid state fermentation (SSF) system while in submerged fermentation (SmF) it
was capable to produce only 13.6 U/ml of the enzyme. When crude
polygalacturonase was characterized produced under SSF and and SmF, it was
found that the crude enzyme from SmF was more thermostable than the enzyme
produced under SSF system and revealed maximum stability in acidic pH.
Patil and Chaudhari (2010) isolated pectinase producing organisms from pectin
industry waste using the selective isolation technique. On the morphological
basis culture was identified as Penicillium sp. Which was found a potential
producer of patience and it has produced a significant amount of extracellular
pectinase enzyme under submerged fermentation process. The produced
enzyme was identified as Polygalacturonase (PG). On partial optimization,
highest production of enzymes was achieved at 35 ºC in a medium comprising
pectin at pH 6.0 within 72 hours. The enzyme purification was done by
ammonium sulphate precipitation, size exclusion and ion exchange
chromatography and then its molecular weight were determined as 35000 DA
by SDS-PAGE. Under optimized conditions activity of purified
Polygalacturonase (PG) has shown as 98.66 U/ml which is almost 12 fold
45
higher than crude. Pectinase production was very cost effective and orange
bagasse gave 64.50 units/gm that was higher than the other natural substrate.
Suresh and Viruthagiri (2010) have produced pectinase through solid-state
fermentation (SSF) process by Aspergillus niger using sugar cane bagasse and
wheat bran as a substrate. Media and fermentation parameters were optimized
for the highest yield of pectinase enzyme. Different combinations of substrate
were used for highest production of pectinase. About 90% of wheat bran and
10% of sugarcane bagasse gave highest production of pectinase within 96 hours.
The optimum pH was found as 5 and temperature 40 °C. The kinetics study of
the pectinase showed Km 294.12 and Vmax 2.33 U/ml.
Thakur et al., (2010) have worked with Mucor circineloides for the synthesis of
extracellular pectinase enzymes. The enzyme biosynthesis was boosted when
different synthesis factors were optimized. High pectinase activity was
achieved within 48 hours at 30 ºC when pH was maintained at 4.0. In this
study (1% w/v) methyl ester and (0.1% w/v) casein hydrolysate respectively,
were used as carbon and nitrogen sources. The pectinase enzyme was purified
to homogeneity (13.3 fold) by Sephacryl S-100 gel filtration Chromatography.
The molecular mass of the enzyme was determined as 66 kDa on SDS-PAGE.
Km and Vmax values were determined as 2.2 mM and 4.81 U/ml at
0.1% and 0.5% (w/v) substrate concentration phenolic acids (0.05 mm), metal
ions such as Mn+2, Co+2, Mg+2, Fe+3, Al+3, Hg+2, and Cu+2, and thiols showed
inhibitory influence to the enzyme activity whereas (0.1% w/v)
polygalacturonic acid at pH 5.5 and 42 ºC showed highest enzyme activity.
Damásio et al., (2011) have isolated fungi from decaying plants and soil of
Brazil for pectinase enzyme production. The best producer was Rhizopus
microsporus var. rhizopodiformis, evaluated for the pectinase production under
various environmental and nutritional situations. The production of pectinase
enzyme was examined at optimum temperature of 40 ºC. The medium was
supplemented with 28 different carbon sources. The inducer influence of
46
different agro-industrial wastes like sugar cane bagasse, wheat flour and
corncob on polygalacturonase (PG) enzyme activity was found as 4-, 3- and 2-
fold greater respectively in comparison to control (pectin). In a medium
supplemented with glucose a constitutive Pectin lyase (PL) activity observed.
Rhizopus microsporus showed maximum production 6of PG (57. 7U /mg) and
PL (88. 6U /mg) respectively in a medium containing lemon rind. PG showed
optimal temperature at 65 ºC and total thermostability at 55 ºC for 90 min. Half-
life of enzyme at 70 ºC was 68 min. These results indicated the great usefulness
of the different agro- industrial wastes by R. microsporus for the production of
pectinase enzyme. The enzyme can be helpful for cost effective in production
and could be helpful related to the waste disposal.
Hendges et al., (2011) have produced endo-polygalacturonase by a
strain Aspergillus niger T0005/007-2 in solid medium within 96 h using a
cylindrical double surface bioreactor with 170 mm of height. In the standard
conditions (static ) the cell concentration nearly 292 mg.g-¹dm (mg per g of dry
medium) was acquired, while in experiments under enforced aeration of 21.4
and 2.8 L.min-1. Kg-1mm (L of air per minute per Kg of moist medium) and with
the central shaft the fungal biomass achieved around 100 mg. g-1dm. Maximum
endopolygalacturonase activity was acquired with the central-shaft
system, 78U .g-1dm (units per g of dry medium). Enforced aeration and
pressurepulse exhibited no optimistic effect on the synthesis of endo-PG, 45U .
g-1dm and 28U .g-1dm, respectively. The enzyme showed thermostability up to
40 ºC, 50% activity decreased after 120 minutes at 50 ºC.
Janani et al., (2011) isolated bacteria from agricultural waste dump soils in
Vellore, Tamilnadu and South India to screen them out for pectinase production.
Out of total ten bacterial strains only 3 strains were positive in pectinase
depolymerization assay plates. The enzyme The partial purification of enzyme
was carried out through ammonium sulphate precipitation following by
dialysis. The strains were identified as Bacillus sp. and they were capable to
47
synthesize high amounts of pectinase under submerged and semi-solid
fermentation systems. Maximum enzyme production was acquired in the
medium supplemented with wheat bran as substrate compared to rice bran.
The optimal temperature for enzyme synthesis was observed as 30° C.
Joshi et al., (2011) have used solid state fermentation system for the optimized
parameters to produce pectinase (Pectin methyl esterase) by Aspergillus niger.
It was found that It was found that the partially purified enzyme by ammonium
sulphate fractionation (20-80% concentration) was stable up to 60 days and its
thermostability was up to 50 ºC and became totally deactivated at 90 ºC. The
partially purified PME presented maximum activity at pH 3.5. The enzyme
was tested for extraction and clarification of juice of different fruits like plum,
peach, pear and apricot. It was determined that the pectin esterase enzyme
produced from apple pomace had required activity and it enhanced the quality
of evaluated fruit juices.
Maller et al., (2011) have grown A. niveus on liquid or solid media incorporated
with agro- industrial trashes as carbon source. For this purpose, Czapeck media
was complemented with a number of 28 carbon sources and amongst those
orange rind was the top polygacturonase inducing substrate. While in
submerged fermentation lemon rind was found the best as polygalacturonase
inducer. When the results of submerged fermentation and soild state
fermentation were compared, it was detected that polygalacturonase level were
4.4-fold higher under solid state fermentation (SSF) system, when both SSF
and SmF were supplemented with lemon peel. Highest PG activity was
recorded when temperature was 55 °C and pH 4.0 whereas enzyme stability
was found at 60 °C for 90 min at pH 3.0-5.0.
Murad and Azzaz (2011) have observed that pectinases are distributed in
microbes and higher plants. Those are highly popular and upcoming enzymes
for the commercial sector and microbial pectinases are estimated to be 25% of
sales among overall food enzyme. Microbial pectinase enzymes may be
48
synthesized from bacteria, yeast and fungi. Aspergillus niger among fungal
species is exploited at industrial level for the production of pectinase enzyme.
Praveen et al., (2011) have studied the synthesis of pectinase through
Aspergillus niger NCIM 548 under solid state fermentation process with a
high concentration of nutrients, micronutrients along with a large surface area
and using Ficus religiose leaves as a substrate. Size of inoculum, pH,
temperature, particle size and moisture content were optimized to acquire
the highest production of the enzyme. The highest production of pectinase was
recorded at pH 5.0 and at 30 ºC under solid state fermentation (SSF) system.
Optimized carbon and nitrogen sources were 4% glucose and 0.3% ammonium
sulphate respectively. Highest production of pectinase was 34. 12U /ml in solid
state fermentation
Rajendran et al., (2011) have used Fusarium sp., to produce novel pectinase,
which was isolated from the natural environment. The isolate was subjected to
varying parameters of incubation time, substrate concentration, pH and
temperature for optimal production of the enzyme. The maximum production
of pectinase was observed up to 40 U/ml when the culture was maintained at
27 °C. At pH 6 and at an initial substrate concentration of 0.5% whilst the
productivity was about 46 U/ml and 40 U/ml of pectinase enzyme
respectively. The enzyme was produced at optimized conditions, and enzyme
was purified using acetone precipitation. After characterization it was found
that the crude enzyme was capable to retain its activity at 48 °C when the pH
range was in-between 4 and 8 respectively.
Geetha et al., (2012) have utilized fruit rind waste (one of the contaminating
solid trashes) for the production of pectinase enzymes, and also isolated
microorganisms like bacterial and fungal origin from the fruit peel waste. The
microorganisms were recognized as Bacillus sp. and Pseudomonas sp. and the
fungal species were recognized as Aspergillus niger, Aspergillus flavus and
Penicillium chrysogenum. About all the identified microorganisms were able to
49
produce sufficient amount of pectinolytic enzymes like pectin estrase and
pectate lyase. Various levels of pectin inducer were observed and all the fungal
and bacterial species produced the maximum amount of the enzyme when 1%
pectin was used in the media. Highest amount of pectate lyase and pectin
esterase were produced by Bacillus sp. and A. niger when grown in the medium
supplemeted with 1% pectin. A. flavus was grown in submerged fermentation;
it produced higher pectin esterase and pectate lyase. Maximum enzyme was
produced under solid state fermentation in contrast to submerged fermentation.
Aspergillus performed better as compared to all other organisms.
Kumar and Sharma (2012) have used Bacteria for the production of various
commercial enzymes along with pectinase enzyme. The pectinase producing
bacteria were isolated from two decomposing fruit materials like apple and
oranges and were screened for pectinolytic activities. The best producer (O1,
i.e. Orange 1) was Cocci sp. The optimized temperature was 35 ºC while
optimum pH was found 8.0 with 120 rpm agitation (supporting aerobic
conditions) using orange as substrate and incubation time was 72 h. This process
requires surfactant for achieving maximum enzyme activity 13.96 U/ml in
crude enzyme extracts. The study suggested that orange substrate is strong
bacterial candidate for industrial production of pectinase enzymes.
Ogunlade and Oluwayemisi (2012) have isolated three strains of A. niger
from banana peels metropolis of Ibadan, Nigeria, and used them to
depolymerize citrus pectin. Best pectinolytic produced on the medium
supplemented with pectin as substrate under the solid state fermentation and
submerged fermentation process. Fermentation process were compared higher
amount of pectinase enzyme produced in solid state fermentation than
submerged fermentation. Different treatments were applied to banana peel
used as carbon source, higher amount of pectinase was produced when banana
peel was treated as compared to control and it proved that pretreatement of
agro-waste increases pectinase production.
50
Panda et al., (2012) have isolated twenty five fungal strains from the soil of
Similipal Bioreserve Forest and screened for cellulytic and pectinolytic activity
and potential producers found as Aspergillus sp. In the samples. Aspergillus
niger and Aspergillus flavus showed high pectinase and cellulase activity
which showed Potency index of IFcel and IFpect respectively. The other
parameters like temperature and pH were optimized from the predominant
strains and predicted from the study that both the fungal strains may be utilized
for the industrial purpose of large scale production of the enzyme.
Patil et al., (2012) screened vegetable to obtain pectinase producing
microorganisms from carrot. The best isolate was identified as a unique strain of
Bacillus sp. based on morphological, biochemical tests and using 16S rRNA
gene sequencing studies. This isolate was selected for further bulk production
using the optimized conditions of pH 9 and temperature of 50 ºC to produce
maximum yield of 49.58 % pectinase from carrot waste.
Tariq and Reyaz (2012) used Penicillium chrysogenum strain MTCC *160 for
pectinase production under solid state fermentation system and enzyme activity
was found 15U/ mL . The pectinase activity was increased when carbon sources
were added and it showed 82.5 U/mL in sucrose, 67.5 U/mL in glucose and
79.1U/mL in lactose. The pectinase activity was also increased in presence of
nitrogen sources like ammonium sulphate 65 U/mL, peptone 37.5 U/mL and
yeast extract 31.6 U/mL. The pectinase activity was maximum at substrate
concentration of 7g and activity found 25 U/mL.
Vasanthi and Meenakshisundaram (2012) have reported that citrus fruit
processing industries yield a huge quantity of waste material, which create
difficulties of waste disposal and finally produces contamination. During
process of citrus fruits a huge amount of waste consist peels , pulp and seeds are
produced. Dried citrus rind is rich in pectin and could be utilized pectin inducer
by microorganisms. The orange peel used for the biosynthesis of pectinase
enzyme by A. niger under solid state fermentation system. The parameters were
51
optimized as 4% substrate concentration, temperature 30 ºC, time period 48 h,
pH 5, nitrogen source 0.3% ammonium sulphate whereas moisture holding
capacity was optimized as 50 %. The pectinase synthesized by Aspergillus niger
was purified by using acetone, ammonium sulphate precipitation and dialysis.
The molecular weight of the purified enzyme determined by SDS- PAGE in the
range of 35- 60 KDa .
52
INTRODUCTION OF FUNGI
In nature, microorganisms have been proficient with enormous potential
for the production of a range of daily used goods including enzymes, which have
been commercially exploited over the years. Pectinase enzymes are known to
produce by many organisms and are beneficial for invading host tissues.
Additionally these enzymes are indispensable in the deterioration of deceased
plant materials by microorganisms and accordingly help to reutilize carbon
compounds on the globe (Alaea et al., 1989). Pectinases are important for the
plants as they assist in cell wall extension and tempering of some plant tissues
throughout maturation and storage period. Pectinase enzymes also maintain
environmental equilibrium by reutilizing of waste materials of plants and
degrade pectin via depolymerization and desertification reactions (Wood and
Kellogg, 1988 and Hölker et al., 2004). This is inducible enzyme, which is
produced by microorganisms throughout their growth on cellulosic materials
(Lee and Koo, 2001). Pectinases widely studied and sold in enormous volumes
for their usage in various industrial applications (Ogle et al., 2001).
Aspergillus niger
The black Aspergilli are perhaps more common than any other group
within the genus. They are worldwide in dissemination and occur in and upon
the utmost range of substrates, including grains, forage products, rotten fruits
and vegetables, exposed cotton textiles and fabrics, leather, dairy products and
other protein rich substrate, and decomposing vegetation in the field. These are
numerous in soils from tropical and subtropical zones. Because of their
cosmopolitan and their roles in natural processes of decomposition and their
extensive use in physiological and nutritional studies of the fungi, and their
important applications in industry they are very useful. Colony of A. niger grows
53
on the Czapek's medium at 24-26 °C, slow growing, attaining diameters of 4.0 to
5.0 cm in 3-4 days, deeply velvety plane or nearly somewhat zonate. It consist
fairly compact white basal mycelium, which may extend 2 to 3 m beyond the
central area of abundant sporulation and remains white even in age at contigious
margins of adjacent colonies, conidial heads are in slightly grayish black brown
shades, borne on long conidiophores, which commonly have a metallic sheen at
low magnification reverse white; exudates and odor lacking. Conidial heads
globose to radiate, mostly 200 to 300\1 in diameter, but ranging up to 500\x
globose heads of a single strains, spores of lighter color terminal in the chains,
conidiophores smooth, long and coarse, commonly 2 to 3 mm but up to 5 to 6
mm high with diameters that reach 30u but are mostly 15 to 20u usually lightly
colored in brown shades, comparatively thin walled with wall thickness
generally 1 U and only rarely reaching 2. Vesicles globose rather variable in size,
most-commonly 40 to 60 p in diameter ranging from 20 m to 80u, fertile over
their entire surface,, sterigmata in two rows, primaries mostly 15 to 30 u long but
ranging from 20 u to 80 \1 fertile over their entire surface- sterigmata in two
rows, primaries mostly 15 to 30u, long but ranging up to 50/1-, conidia globose
with hyaline echinulations when first formed, becoming progressively darker
androugher and finally appearing longitudinally striate from conspicuous bars
of coloring material. Sclerotia produced in some strains, occasionally dominating
the colony appearance, globose to subglobose, cream colored at first then pinkish
buff and black in age. A. niger produce enzymes that could degrade rutin. It
produces citric acid, antibiotics, amylolytic, pectolytic and lipolitic enzymes.
Aspergillus fumigatus
It can grow even at higher temperatures (the most predominant mold
present in moist plant materials undergoing rapid decomposition). Colonies of A.
fumigatus spread broadly over Czapek's solution, with superficial character
changing from silky to deeply felted, while at first, becoming green with the
54
expansion of conidial heads, and it is colorless from the reverse, in other strains
showing changing amounts of yellow, green or even dark red brown shades
produced abundantly and distributed in mass like fume giving the characteristic
name of the fungus A. fumigatus. A. fumigatus is helpful in enzyme production,
decomposing resins, antibiotic production like fumigation and spinulosin.
Mucor geophillus
This is one of the largest genera of the order include number of species. It
is thermophilic fungus and its name was proposed by oudemans (Oudemans
and Koning, 1902). The genus belongs to the kingdom thallophyta and it is
included in order mucorales and family mucroacease (Gilman, 1998). It was
found from the soil of Holland, United States and lowa (Oudemans and Koning,
1902, Abbott, 1923, 1926), Mycelium snow white, very tardily gray, finally pales
olive. Sporangiophores simple or branched in cymes, carrying two to three
branches sporangiaglobes, at first yellow, then olivaceous, leaving acollars after
the destruction of membrane, 50-350µ m diameter, wall small warts. It is
columella globose, voluminous and pale gray. Chlamydospores on the branches
of the mycelium 20µ in diameter, at a time in a more or less extended series
zygospore very like chlamydospores about 30µ in diameter (Gilman, 1957). The
phialides had the shape of typical Purpureocillium lilacinum phialides, or were
very long (up to 30 µm) and Acremonium - like. Conidia, which were primarily
cylindrical and contains 1verticillate branches with whorls of two to four
phialides.
Penicillium lilacinum
(Purpureocillium lilacinum) (Thom, 2010) (Luangsa- ard et al., 2011)
Colonies after 7 days at 25 ℃ 24 ~26 mm in diameter, pentagonal sulcation in the
center, with radial sulcation, white to pale vinaceous, and reverse buff to pale
luteous. Conidiophores were erect, arising mainly from sub-merged hyphae,
with occasional formation of synnemata. Formation of typical conidial structures
55
was observed near the agar, with either solitary phialides or 2~4 in verticils,
which varied in length and occasionally slightly curved or ellipsoidal, measuring
3~17 (~20) × 1.2~2.5 µm, formed in 'slimy heads' on these Acremonium-like
structures with variable size. Chlamydospores were absent Samson (1974).
56
CHAPTER NO.3
MATERIALS AND METHODS
Chemicals: The chemicals of analytical grade used in this study were
purchased from Merck Chemicals, ICN chemicals, Sigma chemicals, Fluka
chemicals and Oxoid etc. All solutions were prepared in double distilled water.
Microorganism: Mucor geophillus was obtained from Research laboratory Shah
Abdul Latif University of Khairpur, whereas Aspergillus niger, Aspergillus
fumigatus and Penicillium lilacinum were isolated and identified in Fermentation
and Enzyme Laboratory, Institute of Biotechnology and Genetic Engineering,
University of Sindh, Pakistan. The slants were maintained on medium
comprising (g/l) glucose 20, peptone 10, agar 20 and distilled water. The
components of stock culture were thoroughly mixed and kept in culture vessels
sterilized at 15 pounds /cm² for 20 minutes at 121 ºC. The sterilized slants
were inoculated with Aspergillus niger, A. fumigatus, Mucor geophillus and
Penicillium lilacinum and incubated at 37 ºC to obtain luxuriant growth.
Inoculum: Other than Mucor geophillus, the rest of fungi (Aspergillus niger ,
Aspergillus fumigatus and Penicillium lilacinum) used in this work were isolated
from soil as these are potent producers of pectinase enzymes and were
maintained on agar slants. Sterilized distilled water was added to each slant of
3-4 days old to scrap spores. The spore suspension was adjusted to a final
concentration by adding sterilized water to stock culture to obtain 5 x 106
spores/ml.
Optimization of inoculum size: The Erlenmeyer flasks (250 ml) comprising 50
ml growth medium (pH 6.5) were plugged with cotton and autoclaved. After
sterilization, spore suspension in the series of 0. 5 ml to 2.0 ml (0.5, 1.0, 1.5 and
57
2.0) was added under aseptic conditions to each Erlenmeyer flasks and
incubated in orbital shaker (129 rpm) at 30 ± 2 °C for the optimization of
inoculum proportion for the synthesis of pectinase. After adjusting the inoculum
size in the subsequent study the growth media were inoculated with 1.0ml
inoculum to optimize the different parameters for the production of pectinase.
Mineral medium: The mineral medium as reported by Burrel et al., (1966) was
slightly modified and used for the growth of fungi and production of pectinase,
which contains (g/L) glucose 10.0 g, KH2PO4 1.0 g, MgSO4 0.25 g, FeSO4 .7H2O
6.32 mg, ZnSO4 . 7H2O 1.1 mg, MnCl2. 2H2O 3.5 mg, CaCl2.2H2O 46.7 mg and
NH4NO3 2.4 g or (NH4) 2 SO4 2.12 g. The pH of medium was adjusted at 6.5.
Fermentation medium: Submerged fermentation was carried out in 250 ml
Erlenmeyer flasks comprising 50 ml of mineral medium with and without 1%
glucose, molasses, date syrup, pectin (raw and synthetic), sucrose, fructose,
maltose, glucose, starch, corn steep liquor, urea, sodium nitrate, potassium
nitrate, ammonium nitrate and ammonium sulphate was taken in a 250 ml
flask. The pH of the medium was adjusted 6.5. These flasks were plugged with
cotton wool and sterilized at 121 ºC for 25 minutes at 15 pounds/cm 2 after
cooling at room temperature. The sterilized medium was inoculated with 1.0 ml
inoculum of different filamentous fungi such as A. niger, A. fumigatus, P.
lilacinum, and M. geophillus in separate flasks. The inoculated flasks were
incubated in an orbital shaking incubator (Gallenkamp) at 30 ± 2 ºC.
Sample harvesting: The samples were harvested after 24 hour interval up to
240 hours and filtered through Whatman No.1 filter paper. The filtrate was then
centrifuged at 5000 rpm for 10 minutes to remove undissolved matter and
impurities. The supernatant was separated carefully with the help of
autopippitte. The spore free filtrate thus obtained was assayed for pectinase
activity, the total and reducing sugars.
Biomass: Mycelial mass obtained after filtration was washed with distilled
water, dried in oven at 80 °C till constant weight.
58
A- Optimization of culture conditions: Growth medium was prepared to study
different parameters for the optimization of pectinase production.
i- Effect of fermentation time period
The Fermentation time period was optimized for the production of Pectinase
by A. niger, A. fumigatus, and a mixed culture of A. niger + A. fumigatus, M.
geophillus and P. lilacinum grown on different carbon sources like 1% glucose
and 2.5 and 5.0% date sugar, molasses, citrus pectin and pure sugars.
ii- Effect of carbon source: i) The results of control (1% glucose) was compared
with 2.5 and 5 % of date syrup, molasses, citrus pectin (synthetic pectin) were
used as carbon sources. ii) Different sugars like fructose, maltose, glucose,
galactose, sucrose and starch were used in two different concentrations 2.5 and 5
% along with selected agricultural waste (5 % molasses) incorporated in
fermentation medium to check their effect on extracellular pectinase production
while the inoculated flasks were incubated at 30 ± 2 ºC.
iii- Effect of nitrogen source: After optimizing carbon source five different
nitrogen sources like corn steep liquor, urea, sodium nitrate, potassium nitrate,
ammonium nitrate and ammonium sulphate were used in the range of 0.2 and
0.4% and selected carbon source was supplemented in the fermentation
medium.
iv- Effect of pH: pH was optimized in order to achieve maximum production
of pectinase. Optimized medium adjusted with various pH values ranging from
2.0 to 11.0 before sterilization and were inoculated with desired microorganism
and incubated on orbital shaker at 120 rpm for 96 hours at 30 ± 2 °C. The final
pH was checked after completion of fermentation process by pH meter and
maximum production of pectinase was found at pH 6.0.
v- Effect of Temperature : The production of pectinase was checked at different
temperatures rangimg from 20 to 45 °C under the similar culture conditions as
discussed above and 35 °C was optimum temperature for the maximum
synthesis of pectinase enzyme.
59
vi- Characterization of crude pectinase : The crude enzyme was characterized
on the basis of different parameters such as incubation time, substrate
concentration, enzyme concentration, pH, pH stability, temperature,
temperature, temperature stability and the effect of metal ions on Pectinase
activity.
vii- Effect of time of incubation : The pectinase activity was observed and
recorded at different time periods (10 – 60 minutes) with 5 minutes difference
by using 1.0 ml of pectinase enzyme (from Broth) and 1.0 ml citrus pectin (1.0%)
was used as substrate. The reaction mixture was incubated at 37 ºC for different
time periods.
viii- Effect of substrate concentration: The effect of substrate concentration was
observed for the rate of enzymatic reaction of pectinase by using citrus pectin
as a substrate with different concentration ranging from 0.5 - 2.5%. The reaction
mix consists of 1. 0 ml of culture broth and 1. 0 ml of substrate of different
concentration and the reaction mixture was incubated at 37 °C for 15 minutes.
ix- Effect of enzyme concentration : The effect of enzyme concentration (0.2–
1.4 ml culture broth) on the rate of enzyme reaction was studied by incubating
with 1.0 ml citrus pectin (1.5%) and the reaction mixture was incubated at 37
°C for 15 minutes.
x- Effect pH : The effect of pH on pectinase activity was tested by evaluating the
enzyme activity at various pH in the range of (3 –10) using 1.0 ml sample of
crude enzyme samples and 1.0 ml citrus pectin (1.5% dissolved in different
range of buffers).
xi- Effect of pH stability: The effect of pH on stability of pectinase enzyme
was checked by measuring % of relative activity at 37 °C when 1.0 ml enzyme
was mixed with 0.2 ml citrus pectin (1.5 %) pH ranging between 3–10 using,
sodium citrate buffer 0.1M incubated for 10 minutes. After 10 minutes 0.5 ml
substrate was added and again incubated for 15 minutes.
60
xii- Effect of temperature: The pectinase activity of culture broth was studied at
different temperatures in the range of 20 °C to 100 °C. The enzyme activity
carried out by reported assay method.
xiii- Effect of temperature stability: The thermo stability of Pectinase enzyme
was tested from culture broth samples by measuring the % of rmaining activity
after heating the enzyme 1.0 ml in the presence of 0.2 ml Citrus pectin (1.5%
dissolved in sodium citrate buffer pH 5.0) at different temperatures ranging
between 20° C to 100 °C for 10 minutes. After 10 minutes the assay was carried
out by adding 1.0 ml 1.5 % citrus pectin as substrate and incubated at 40 °C for
15 minutes.
xiv- Effect of Metal ions / Compounds on crude pectinase: Various metal ions
and compounds in 5mM concentrations were reacted with enzyme (1.0 ml) in
sodium citrate buffer pH 5.0 for 10 minutes at optimum temperature prior to
addition of substrate and remaining activities were determined by adding 1.0 ml
citrus pectin (1.5%) as substrate and incubated at 40 °C for 15 minutes. The
thermo stability of enzyme was also studied at different time periods (10-60
minutes) with and without activator (15 mM CaCl2) at 60 and 70°C.
B- Preparation of enzyme: Erlenmeyer flask (500 ml) containing mineral
medium along with 5% molasses, 5% sucrose and 0.4% ammonium sulphate
and media was sterilized, cooled and inoculated with pre-grown culture of
the Aspergillus niger. The flasks were incubated at 35 °C. The culture was
harvested after 96 hours. The broth was filtered through Whatman No.1 filter
paper, vortexed thoroughly and centrifuged at 6 ,000 rpm for 15 min, 4 °C. The
enzyme was precipitated by the method of Saxena et al., (2003) from the culture
supernatant by adding ammonium sulphate to 60% saturation. This was left
overnight and the precipitates were collected by centrifugation at 6,000
rpm for 15 min. The precipitate obtained were dissolved in sodium citrate buffer
(pH 5 .0) and dialyzed against the same buffer for 24 h. Dialysis was carried out
using cellulose tubing (molecular weight cut off 10, 000 Da). Pectinase enzyme
61
from Aspergillus niger was purified according to the method reported by
Guessous et al., (2001). The technique adopted included fractionation by
ammonium sulphate followed by dialysis and gel filtration chromatography.
Purity was checked using polyacrylamide gel electrophoresis.
i- Ammonium sulphate fractionation: The culture broth at optimized culture
condition was obtained and centrifuged at 6 ,000 rpm for 20 minutes in a
refrigerated condition. Solid ammonium sulphate (GR grade MERCK) was
slowly added to the supernatant up to 60 % saturation. Addition of ammonium
sulphate was carried out with continuous stirring in an ice bath, and then it
was kept at 4. 0 °C for overnight. The precipitates were obtained by
centrifugation at 6 ,000 rpm for 20 minutes at 4. 0 °C. The precipitates were
dissolved in 30 ml of sodium citrate buffer (0.1 M, pH 5.0). The protein content
of the fraction was determined by the method of Lowry et al., (1951).
ii- Dialysis: The precipitates obtained after treatment with ammonium sulphate
were dialysed against 0.1M sodium citrate buffer (pH 5.0) for overnight. Dialysis
was carried out using cellulose tubing (molecular weight cut off 10 ,000 Da).
After dialysis, the sample was concentrated by polyethylene glycol. The
dialyzed sample was concentrated to 20.0 ml by PEG.
iii- Preparation of gel Sephadex G-100: 10.0 grams of Sephadex G-100 (Sigma
Chemicals, USA) was suspended in 150 ml distilled water and placed for
overnight. The solution was stirred occasionally. Then 0.1M solution of NaOH
and 0.1 N HCl was used to suspend the gel after soaking in acid and base. The
gel was again soaked in distilled water for 3 hours and then distilled water was
decanted. Finally the gel was equilibrated with 0.1 M sodium citrate buffer,
pH 5 .0
iv- Gel filtration chromatography: The dialyzed enzyme (10 ml) was applied
on Sephadex G-100 (Sigma Chemicals, USA) column (65 x 1.5cm). Elution of the
62
enzyme was carried out with 0. 1 M sodium citrate buffer (pH 5.0) At a flow rate
of 2 ml /min and the fraction of 5.5 ml was collected by a fraction collector
(Tokyo, Rikakikai Co. Ltd) using EYELA 9900, UV-Vis. Detector. The absorbanc
of protein was monitored at 280 NM. The fractions were collected and the four
active pools were obtained. The pool fractions were stored at 4°C for further
analysis.
v- Ion exchange chromatography: Concentrated enzyme (5 ml) of fraction -3
was loaded into an anion exchange DEAE Sephadex A-50 (Sigma
Chemicals, USA) column (30 x 1.5 cm) at a flow rate of 0.5 ml /min.
Equilibration and elution were performed first with 0. 1 M sodium citrate buffer
to remove unbound proteins and then with a linear salt gradient from 0. 0 to 1.0
N NaCl was used to elute pectinase enzyme. Fractions of 5.5 ml were collected
and analyzed for Pectinase activity and protein content. The active fractions
were pooled and used for the studies described below.
C- Characterization of purified Pectinase
i- Effect of Temperature on Pectinase activity and stability: The optimal
temperature for the purified pectinase was obtained by assaying the enzyme
activity at different temperatures increasing from 20 ºC to 100 ºC. In order to
assess the stability, the enzyme solution (1.0mL) was heated at 20 °C, 30 °C, 40
°C, 50 °C, 60 °C, 70 °C, 80 ºC, 90 ºC and 100 ºC for 10 minutes intervals over the
period of incubation. The residual enzyme activity was measured following the
procedure described above.
ii- Effect of pH on Pectinase activity and stability: The relative pectinase
activity using 1. 5 % (w/v) citrus pectin was determined at various pH. The
range of pH varied from 3 to 11. To test the pH stability, the purified enzyme
using respective buffer having pH ranging from 3 to 10 as described above and
were incubated for 80 minutes at room temperature. The residual enzyme
activity was estimated at 10 minutes interval during the 80 minute period of
incubation.
63
iii- Kinetic determinations: Initial reaction rates Pectin hydrolysis were
determined at different substrate concentrations ranging from 0. 4 g to 2.4 g/100
ml in 0.1 M citrate buffer (pH 5.0) at 40 °C for 15 minutes.
iv- Effect of Metal ions / Compounds: Various metal ions and compounds with
5mM concentrations were reacted with enzyme (1.0 ml) and 1.5 ml sodium
citrate buffer pH 5.0 for 10 minutes at optimum temperature prior to addition of
substrate and remaining activities were determined using 1.0 ml (1.5 %) Citrus
pectin as substrate and incubated at 60 °C for 15 minutes.
D- Analytical methods
i- Assay of Pectinase Activity: Pectinase activity was determined by the
spectrophotometer method as reported by Bailey et al., (1992). A brief
description of the method is given below.
One unit of Pectinase activity was described as the amount of Pectinase
producing 1 μmole of reducing sugar per 1 ml under standard test conditions.
Reagents:
Sodium citrate Buffer (0.1 M. pH 5.0): Dissolve citric acid monohydrate,
C6 H8 O7 . H2O (M. wt. 210.14) 0.1M-solution contains 21.01 g/l. Trisodium
citrate dihydrate, C6 H5 O7 Na3.2H2O, (M. wt. 294.12) 0.1M-solution contains
29.41 g/l, and then mixed to the desired pH.
Substrate-1% pectin: Dissolve 1.0 % citrus pectin (Fluka) in about 100 ml of
citrate buffer pH 5.0, stirrer to dissolve the substrate and continued stirring to
room temperature. Dilute to volume in a 100 ml volumetric flask with citrate
buffer and Store at 4 ºC for a maximum of one week. DNS Reagent: Potassium
sodium tartrate solution: 300 grams of Potassium sodium tartrate were
dissolved in 500 ml of distilled water.
Dinitro salicylic acid reagent: 10 grams of Dinitro salicylic acid were dissolved
in 200 ml of 2.0 M sodium hydroxide.
Dinitro salicylic acid solution: This solution was prepared by mixing solution
1 and 2 and the volume was making up to 1 liter with distilled water. Procedure:
64
1ml of broth was added in 1.0 ml 1% Citrus pectin as a substrate in a test tube
and after mixing thoroughly, incubated in a water bath for 15 minutes at 60 ºC.
After incubation 1.0 ml DNS solution was added and the solution was heated in
a boiling water bath for 5 minutes, according to the Miller method (1959).
After 5 minutes the test tubes were cooled under tap water and color intensity
was observed against reagent blank at 540 nm. Reducing sugar concentration
was calculated from standard curve was calculated from galacturonic acid as
shown in Figure-4.1.
y = 0.3884x R2 = 0.9989
Figure-4.1: Standard graph for Galacturonic acid
ii- Protein Estimation
Total Protein estimation was carried out from the filtrate after harvesting the
biomass according to Lowry et al., method (1951). A brief description of the
method is given below:
0
0.1
0.2
0.3
0.4
0.5
0.6
0.2 0.4 0.6 0.8 1
5
4
0
nm
Concentration of Galacturonic acid
mg/mL
A
B
S
O
R
B
A
N
C
E
65
Reagents:
01. Alkaline sodium carbonate solution: 20.0 grams of sodium carbonate was
dissolved in one liter of 0.1M sodium hydroxide.
02. Copper sulphate- sodium potassium tartrate solution: 5.0 grams of copper
sulphate (CuSO4..5H2O) and 10.0 grams of sodium, potassium tartrate
(NaKC4H4O6. 4H2O) were dissolved in one liter distilled water.
03. Alkaline solution: Prepared on day of use by mixing 50 ml of reagent (1)
and 1.0 ml of reagent (2)
04. Folin‘s reagent (C10H5NaO5S): Commercially available by E. Merck, BD.H
Procedure: 0.5 ml test solution was added to 2.5 ml alkaline copper reagent.
Regents were mixed thoroughly and allow standing at room temperature for
10 minutes. 0.25 ml diluted Folin’s reagent (1:1 V/V with water) was added in
each tube and incubated for further 30 minutes. After 30 minutes, the
absorbance was noted against blank (blank containing water plus all reagents)
at 750 nm. Standard protein BSA (Bovine albumin) solutions were prepared in
100, 200, 300, 400 and 500 micrograms per ml for calibration curve. The graph of
absorption vs. concentration was plotted and the concentrations of test solutions
were calculated from protein calibration curve (Figure-4.2).
Figure-4.2 Standard Graph for total protein
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
100 200 300 400 500
Concentration of Albumin ug/ml
Ab
sorb
an
ce a
t 750n
m.
66
iii- Determination of Total Carbohydrate: The carbohydrate concentration in
the culture broth was carried out by phenol sulphuric acid method as described
by Montgomery (1961); a brief description of the method is given below:
Reagents: 01. Concentrated H2SO4.
02. 80% Phenol: 80.0 grams of phenol was dissolved in 100 ml of distilled water.
Procedure: 0.5 ml of test solution was added in 2.5 ml concentrated
sulphuric acid and 0.05 ml 80% phenol solution. After thoroughly mixing, it was
stand at room temperature for 15 minutes. The blank was prepared by
substituting distilled water for the test solution. The absorbance was monitored
against the blank at 485 nm. The concentration curve prepared by same manner
as test sample using glucose as standard (Figure-4.3).
Figure-4.3 Standard Graph for total carbohydrate
iv- Determination of Reducing Sugars: The concentration of reducing sugar
from culture broth was determined by dinitrosalicylic acid (DNS) method as
described by Miller (1959) and a brief description of the method is given below:
0
0.2
0.4
0.6
0.8
1
1.2
0.05 0.1 0.15 0.2 0.25 0.3
Concentration of Glucose mg/ml
Ab
sorb
an
ce a
t 485 n
m.
67
Reagents:
01. Potassium sodium tartrate (NaKC4H4O6. 4H2O) solution: 300 grams of
Potassium sodium tartrate was dissolved in 500 ml of distilled water.
02. Dinitrosalicylic acid reagent: 10 grams of Dinitrosalicylic acid was
dissolved in 200 ml of 2 M Sodium hydroxide.
03. Dinitrosalicylic acid solution: This solution was prepared by mixing
Solution 1 and 2 and the volume was making up to 1 liter with distilled
water. Procedure: 2.0 ml of test solution was added in 2.0 ml dinitrosalicylic
acid in a test tube and after mixing thoroughly heat it in a boiling water
bath for 5 minutes. After 5 minutes the test tubes were cooled under tap
water and color intensity was observed against reagent blank at 540 nm.
The concentration, reducing sugar was calculated from the standard curve
(Figure -4.4)
Figure-4.4 Standard curve for reducing sugar
0
0.2
0.4
0.6
0.8
1
1.2
0.06 0.12 0.18 0.24 0.3
Concentration of Glucose mg/ml
Ab
sorb
an
ce a
t 5
40
nm
.
68
v- Molecular Mass Determination:
Preparation of gel Solution
A. Acrylamide-bisacrylamide solution (30:0.8): 30.0 grams of acrylamide and 0.8
grams of bisacrylamide were dissolved in 100 ml distilled water. The stock of
acrylamide solution was then stored at 4 °C.
B. Resolving gel buffer (Tris- HCl pH 8.8): 36.6 gram of tris and 48.0 ml 1.0 M
HCl were mixed and volume was made up to 100 ml with distilled
water. This buffer solution was then stored at 4° C.
C. Stacking gel buffer (0.5M Tris–HCl pH 6.8): 6.0 gram of Tris was dissolved
in 40 ml distilled water and the pH was adjusted to 6.8 by titrating with 1.0 M
HCl. This buffer was stored in refrigerator at 4°C.
D. Reservoir buffer (0.25 M Tris and 1.92 M glycine pH 8.3): 3.03 grams of Tris,
14.4 grams of glycine and 1.0 gram of SDS were dissolved in 1 litre of distilled
water. The solution was stored at 4°C.
E. 1.0% SDS solution: 1.0 gram of sodium dodecyl sulphate was dissolved in 100
ml distilled water.
F. 1.5% Ammonium per sulphate solution: 1.5 gram of ammonium per sulphate
was dissolved in 100 ml distilled water. This solution was prepared freshly each
time.
G. Sample buffer: Tris - HCl buffer pH 6.8 (starching gel buffer) contains 1%
mercaptoethanol, 40% sucrose or 2% glycerol and 1% sodium dodecyl sulphate.
H. Staining solution: 0.1 gram of Coomassie blue R-250 was dissolved in water-
methanol- aceticacid (5:5:2 v/v/v/).
I. Destaining solution: Destaining solution contains methanol -acetic- water (30-
10-60 v/v/v).
69
Table–4.1 Composition of working resolving and stacking gels
Stock solutions Stacking gel ml
Resolving gel 10.0%
Acrylamide-bisacrylamide ml
Acrylamide-bisacrylamide 1.25 5.0
Stacking gel buffer - 2.0
Resolving gel buffer 2.50 -
1.0%SDS 1.00 1.5
1.5 Ammonium per sulphate 0.50 0.75
Distilled water 5.50 6.5
TEMED 0.005 0.005
Preparation of sample and their application: 20ul of tracking dye (0.05%
Bromophenol blue in water) 50 uL of test sample and 50ul of sample buffer were
mixed. The sample protein was denatured by heating at 100°C for 2 minutes
before applying to the gel. 50ul of denatured sample was applied on the surface
of gel without disturbing the buffer layer. Power supply was adjusted at 5 mA
current per plate for 180 minutes.
Development of protein bands: The gels after electrophoresis were stained
with Coomassie blue R 250 for 45 minutes. The gels were stained with
Coomassie blue R 250 for 45 minutes. The gels were then destained in methanol-
- water-acetic acid (30.60: 10 v/v/v) reported by (Hames and Rickwood, 1986)
Molecular weight determination: After purification the enzyme was subjected
to electrophoretic studies to confirm purity. Molecular mass was determined by
using SDS-PAGE electrophoresis (Vertical Gel Electrophoresis system Wealtec
Corp. USA). SDS-PAGE (10%) was performed as reported by Hames and
Rickwood (1986) using size markers SDS-6H (Pharmacia, Uppsala, Sweden)
comprising phosphorylase b (MW 94,000) bovine serum albumin (MW 67,000)
ovalbumin (MW 43,000) carbonic anhydrase (MW 30,000) soybean trypsin
inhibitor (MW 20,100) and lactalbumin (MW 14,400) were used as reference
proteins. Proteins were envisaged by staining with Coomassie brilliant blue.
70
CHAPTER NO.4
RESULTS AND DISCUSSION
In this study an agro-industrial waste and sugars were used as a
carbon source. New enzymes have been focused by researchers for low cost
production due to their commercial applications. The utilization of agro-
industrial wastes, on one hand, provides alternative substrates and, on the other
helps in solving pollution problems, which otherwise may cause their disposal
problems. Pectinases are industrially very important enzymes and are used in
different industries as processing aids for extraction, clarification and maceration
of fruits and vegetables. A number of fungal strains have shown great potential
to produce different types of pectinolytic enzymes (Junwei et al., 1992; Junwei et
al., 2000; Nitninkumar and Bhushan, 2010; Poonpairoj et al., 2001; Silva et al.,
1993; Solis et al., 2009).Pectinases can be produced by fermentation. Aspergillus
sp. are most frequently exploited filamentous fungi to produce pectinase
(Blandino et al., 2001; Patil and Dayanand, 2006 a and b; Rodríguez-Fernández et
al., 2011).
Microbial pectin degradation is important for the decomposition of the
plant material, digestion of plant food and the retting process. Pectin degrading
enzymes have been extensively used to improve the stability of fruit and
vegetable nectar and in the clarification of fruit juices and wines (Bailey and
Pessa, 1990, Fogarty and Kelly, 1983, Ros, et al., 1993, Sreekantian, et al., 1971;
Stressler and Joslyn, 1971). Currently, they are widely used in industries for
setting of natural fibers and the extraction of oils from vegetables and citrus peels
(Federici and Petruccioli, 1885, Fernandes-Salomão, et al., 1996). The enzyme
preparations used in the food industry are of fungal origin because fungi are
71
potent producers of pectic enzyme and the optimal pH of many fruit juices,
which ranges from pH 3–5.5 (Fonseca and Said, 1995). Furthermore, due to the
relatively low temperature stability of the fungal enzyme preparation maceration
needs to be carried out at a temperature not exceeding 45°C, necessitating the
incorporation of a pasteurization step to limit the growth of mesophillic
microorganisms (Silley, 1986).
In this study different concentration like 2.5% and 5% of the sugars were
used as a carbon source for the growth of fungi and production of pectinase
through the submerged fermentation process. Different nitrogen sources were
also used and optimized, optimization of temperature and pH was carried out to
acquire a maximum production of pectinase enzyme.
A- Growth conditions and enzyme production:
Culture conditions were optimized for optimum pectinase production by
fungi, changing one variable at a time while keeping the other constant.
i- Effect of size of the inoculum:
The effect of the size of the inoculum was studied by adding five days old culture
with different concentrations ranging from 0.5 to 2.5 ml in 50 ml of fermentation
medium and incubated in orbital shaker (120 rpm) at 30 ± 2 °C. Results are
shown in Table-5.1 to 5.5. 1ml inoculum size was optimum for maximum
enzyme production by different fungi as A. niger, A. fumigatus, and a mixed
culture of A. niger + A. fumigatus , M. geophillus and P. lilacinum. In the
subsequent 1.0 ml inoculum of micropropagation was used for the production of
pectinase.
72
Table-5.1 Effect of size of inoculums on growth and pectinase production by
A.fumigatus grown on mineral medium containing 1 % glucose as
carbon source at 30 ± 2 ºC pH was adjusted at 6.5.
Size of the inoculum Biomass Pectinase Activity
(ml) g/50 ml Broth U/ml
0.5 0.421±0.004 5.75±0.08 1.0 0.457±0.002 8.271±0.002 1.5 0.436±0.005 5.56±0.07
2.0 0.433±0.008 4.74±0.09
2.5 0.417±0.009 4.66±0.04
Table-5.2 Effect of size of inoculums on growth and pectinase production by
A.niger grown on mineral medium containing 1 % glucose as carbon
source at 30 ± 2 ºC pH was adjusted at 6.5.
Size of the inoculum Biomass Pectinase Activity
(ml) g/50 ml Broth U/ml
0.5 0.432±0.003 5.98±0.06 1.0 0.439±0.009 8.973±0.001 1.5 0.349±0.003 5.58±0.03
2.0 0.344±0.004 5.21±0.06 2.5 0.434±0.006 4.93±0.07
Table-5.3 Effect of size of inoculums on growth and pectinase production by a
mixed culture of A.fumigatus + A.niger grown on mineral medium containing
1% glucose as carbon source at 30 ± 2 ºC pH was adjusted at 6.5.
Size of the inoculum Biomass Pectinase Activity
(ml) g/50 ml Broth U/ml
0.5 0.332±0.003 2.02±0.01 1.0 0.419±0.003 7.37±0.09
1.5 0.449±0.004 5.98±0.03 2.0 0.423±0.002 5.21±0.02
2.5 0.434±0.006 5.93±0.06
73
Table-5.4 Effect of size of inoculums on growth and pectinase production by M. geophillus grown on mineral medium containing 1 % glucose
ascarbon source at 30 ± 2 ºC pH was adjusted at 6.5.
Size of the inoculum Biomass Pectinase Activity
(ml) g/50 ml Broth U/ml
0.5 0.564±0.001 6.19±0.04 1.0 0.412±0.01 7.54±0.03
1.5 0.451±0.003 6.58±0.03 2.0 0.456±0.008 6.21±0.02
2.5 0.434±0.009 6.13±0.05
Table-5.5 Effect of size of inoculums on growth and pectinase production by P. lilacinum grown on mineral medium containing 1 % glucose as
carbon source at 30 ± 2 ºC pH was adjusted at 6.5.
Size of the inoculum Biomass Pectinase Activity
(ml) g/50 ml Broth U/ml
0.5 0.432±0.005 5.98±0.01 1.0 0.469±0.005 8.79±0.02
1.5 0.449±0.002 5.58±0.06 2.0 0.440±0.001 6.21±0.06
2.5 0 .434±0.006 5.93±0.05
74
ii- Fermentation mode
Fermentation medium (Table-5.6) was inoculated with five days old 1 ml inoculum to each flask, incubated to optimize the
fermentation mode.
Table.5.6 Effect of fermentation mode for the growth and biosynthesis through different filamentous fungi
Fermentatin (Smf) after
72 hours
Biomass g/50 ml
Pectinase activity U/ml
Still culture
Semi shaking
Continuous
shaking
A.
fum
igat
us
A.
nig
er+
A.
fum
igat
us
A.
nig
er
M.g
eoph
illo
us
P. l
ilac
inu
m
A.
fum
igat
us
A.
nig
er+
A.
fum
igat
us
A.
nig
er
M.g
eoph
illo
us
P. l
ilac
inu
m
0.49±0.02
0.43±0.03
0.45±0.04
0.5±0.1
0.39±0.04
0.41±0.02
0.51±0.02
0.4±0.1
0.43±0.02
0.48±0.04
0.39±0.03
0.41±0.03
0.45±0.03
0.42±0.03
0.41±0.03
7.32±0.04
7.4±0.3
8.27±0.04
6.8±0.3
7.7±0.2
8.02±0.01
7.3±0.2
7.9±0.3
8.97±0.02
6.74±0.04
6.82±0.02
7.54±0.02
7.42±0.03
7.95±0.02
8.79±0.03
75
76
In this study submerged fermentation system was used as liquid culture
which was usually preferable to solid state culture not only due to it allowing
better aeration and proper agitation , but also the separation of the enzyme from
the solid substrate is more difficult than submerged fermentation (Alazard and
Raimbault, 1981). Submerged fermentation and solid state fermentation have
been used successfully in the production of pectinase by fungi (Dinu et al., 2007,
Pedrolli et al., 2008, Castilho et al., 2000). However, according to Sunnotel and
Nigam (2002) submerged fermentation is technically easier as compared to solid
state fermentation. It is a well developed system used in industrial scale to
synthesize a large variety of microbial metabolites. This system was strongly
developed from the 1940s onward to produce large scale of antibiotics. On the
other hand, despite the advantages, the application of SSF at industrial is hard to
imagine. There is difficult scale-up, the often unfeasible biomass determination
and complicated product purification by downstream processes resulting from
the use of heterogeneous organic growth substrates Sunnotel and Nigam (2002).
About 90% of all Industrially important enzymes are synthesized in the
submerged fermentation system Murad and Azzaz (2011). Table-5.6 shows that
continuous shaking was best for maximum growth and Pectinase production,
while result reveals that incubation period varies from organism to organism and
carbon to carbon sources but according to different experiments 72 hours were
optimum for the higher yield of pectinase enzyme. Acuna-Arguelles et al., (1995)
stated that the type of culture methods used influence the kinetic and
physiochemical properties of these enzymes. All the organisms have produced
maximum pectinase when continuous shaking was applied for the experiment.
iii- Effect of Incubation Period:
The optimum incubation time for the synthesis of pectinase was studied by A.
niger, A. fumigatus, and a mixed culture of A. niger + A. fumigatus , M. geophillus
and P. lilacinum grown on different carbon sources like 1% glucose and 2.5 and
5.0% date sugar, molasses, crude citrus pectin, and commercial citrus pectin as
77
shown in Fig- 5.1 to 5.9. The maximum pectinase production was achieved at at
72 hours. There was no fair activity for pectinase on the first day (up to 24 hours) .
After that Pectinase activity increased slowly and reached the peak on the 3rd day.
A decrease in activity was occurred just before and after the optimum period for
pectinase. The decline of pectinase production after reaching its maximum level
may be due to catabolic repression, cessation of enzyme synthesis, or to the
increased proteolysis in culture (Sakellaris et al., 1988). As the incubation period
for pectinase production by all the fungi used in this study were 72 hours, which
is against the results of pectinase production by other workers (Phutela et al.,
2005, Said et al., 1991). But these results are in agreement of Fujio and Eledago
(1993), who have reported 72 hours incubation time for polygalacturonase
production by Rhizopus oryzae. The incubation time less than 72 hours was
reported for the highest activity of pectinase enzyme from the cultures of
Aspergillus niger by Patil and Dayanand, (2006 b) and from Coriolus versicolor by
Freixo et al., (2008 a). It is obvious from the observation of the present study that
the addition of different carbon sources were enhanced the Pectinase production
and fungi produced highest enzyme amounts after 72 hours of fermentation
period. Results presented by other workers also reveal that incubation period
varies from organism to organism and carbon to carbon source (Botella et al.,
2007; Gummadi and Kumar, 2007; Hours et al., 1988; Jacob et al., 2008; Joshi et
al., 2006; Kashyap et al., 2000, 2003; Maria et al., 2002; Shivakumar and
Krishnanand (1995); Silva et al (2005; Pande, 1991; Patil and Dayanand, 2006b ;
Solis-Pereyra et al., 1996; Taragano et al., 1997; Teixeria, et al., (2000). Similar
results were also observed by Mrudula and Anitharai (2011) during pectinase
production by Penicillium sp.
78
Fig- 5.1: A. fumigatus, A. niger , A. niger + A. fumigatus , M. geophillus, P.
lilacinum were grown on mineral medium containing 1 % glucose
as carbon source at 30 ± 2 ºC pH was adjusted at 6.5.
Fig- 5.2 :A. fumigatus, A. niger , A. niger + A. fumigatus , M.geophillus, P. lilacinum
were grown on mineral medium containing 2.5 % date sugar as carbon source
at 30 ± 2 ºC pH was adjusted at 6.5.
0
1
2
3
4
5
6
7
8
24 48 72 96 120 144 168 192 216 240
Time (hours)
Pe
ctin
ase
Act
ivit
y U
/ml
A.fumigatus
A.niger
A.niger+A.fumigatus
M.geophllus
P.lilacinum
0
1
2
3
4
5
6
24 48 72 96 120 144 168 192 216 240
Time (hours)
Pe
ctin
ase
Act
ivit
y U
/mL
A.fumigatus
A.niger
A.niger+ A.fumigatus
M.geophillus
P.lilacinum
79
Fig- 5.3:A. fumigatus, A. niger , A. niger + A. fumigatus , M. geophillus,
P. lilacinum were grown on mineral medium containing 5 % date
sugar as carbon source at 30 ± 2 ºC pH was adjusted at 6.5.
Fig- 5.4: A. fumigatus, A. niger , A. niger + A. fumigatus , M.geophillus, P.lilacinum
were grown on mineral medium containing 2.5 % molasses as carbon
source at 30 ± 2 ºC pH was adjusted at 6.5.
0
1
2
3
4
5
6
7
8
9
24 48 72 96 120 144 168 192 216 240
Time (hours)
Pecti
nas
e A
cti
vit
y I
U/m
L
A.fumigatus
A.nigar
A.nigar+A.fumigatus
M.geophilus
P.lilacinum
0
2
4
6
8
10
12
24 48 72 96 120 144 168 192 216 240
Time
Pecti
nas
e A
cti
vit
y I
U/m
L
A.fumigatus
A.nigar
A.nigar+A.fumigatus
M.geophillus
P.lilacinum
80
Fig- 5.5:A. fumigatus, A. niger , A. niger + A. fumigatus , M.geophillus, P. lilacinum
were grown on mineral medium containing 5 % molasses as carbon
source at 30 ± 2 ºC pH was adjusted at 6.5.
Fig- 5.6: A. fumigatus, A. niger, A. niger + A. fumigatus , M.geophillus, P. lilacinum
were grown on mineral medium containing 2.5 % crude citrus pectin as
carbon source at 30 ± 2 ºC pH was adjusted at 6.5.
0
1
2
3
4
5
6
7
8
24 48 72 96 120 144 168 192 216 240
Time (hours)
Pe
ctin
ase
Act
ivit
y U
/mL
A.fumigatus
A.niger
A.niger+A.fumigatus
M.geophillus
P.lilacinum
0
1
2
3
4
5
6
7
8
24 48 72 96 120 144 168 192 216 240
Time (hours)
Pect
inase
Act
ivit
y U
/mL
A.fumigatus
A.niger
A.niger + A.fumigatus M.geophillus
P.lilacinum
81
Fig -5.7: A. fumigatus, A. niger , A. niger + A. fumigatus , M. geophillus, P. lilacinum
were grown on mineral medium containing 5 % crude citrus pectin
as carbon source at 30 ± 2 ºC pH was adjusted at 6.5.
Fig-5.8:A. fumigatus, A. niger , A. niger + A. fumigatus , M.geophillus, P. lilacinum
were grown on mineral medium containing 5 % commercial citrus pectin
as carbon source at 30 ± 2 ºC pH was adjusted at 6.5.
0
1
2
3
4
5
6
7
8
24 48 72 96 120 144 168 192 216 240
Time (hours)
Pecti
nas
e A
cti
vit
y I
U/m
L
A.fumigatus
A.niger )
A.niger+ A.fumigatus , M.geophillus
P.lilacinum
0
1
2
3
4
5
6
7
8
9
24 48 72 96 120 144 168 192 216 240
Time (hours)
Pect
inase
Act
ivit
y U
/mL
A.fumigatus A.niger A.niger+ A.fumigatus M.geophillus P.lilacinum
82
Fig -5.9: A. fumigatus, A. niger , A. niger + A. fumigatus , M. geophillus, P.lilacinum
were grown on mineral medium containing 5% commercial citrus
pectin as carbon source at 30 ± 2 º C pH was adjusted at 6.5.
iv- Effect of agro-industrial waste as carbon sources:
The use of low cost substrates for the production of industrial enzymes is one of
the ways to reduce production costs significantly. This can be achieved using
solid agricultural waste materials after treatment as substrates, Agricultural
based carbon sources are more appropriate and these are very cost effective,
renewable and available in huge quantities Yugandhar et al., (2008).
Table.5.7-5.11. Shows the results of pectinase produced by A. fumigatus, mixed
culture of A. niger + A. fumigatus, A. niger, M. geophillus and P. lilacinum when
grown on medium without sugar. The maximum production of pectinase 0.87
U/ml was achieved by A.fumigatus at 120 hours and then pectinase was
decreased with the increase of the time period. The concentration of total sugar
and reducing sugar was absent at the initial period Aspergillus niger produced
0.92 U/ml at 120 and then pectinase was decreased with the increase of the time
period. The fluctuation was noted in the final pH of the culture broth of A. niger,
M. geophillus and P. lilacinum produced 0.91 U/ml, 0.95 U / ml and 0.89 U/ml
respectively after 120 hours. The increase of the time period, pectinase
0
2
4
6
8
10
12
24 48 72 96 120 144 168 192 216 240
Time (hours)
Pe
ctin
ase
Act
ivit
y U
/mL
A.fumigatus
A.niger
A.niger+ A.fumigatus
M.geophillus
P.lilacinum
83
production was decreased in each case. From above results it can be understood
that in the absence of any carbon source the filamentous fungi are capable to
produce very low or negligible amount of pectinase enzyme but it took more
time to produce enzymes.
Table- 5.7: A. fumigatus was grown on mineral medium without glucose at
30± 2º C pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml Broth
Total Proteins (mg/ml)
Total Sugar (mg/ml)
Reducing Sugars (mg/ml)
Pectinase Activity (U/ml)
24 6.30±0.03 0.005±0.003 0.0013±0.003 - - -
48 6.21±0.03 0.01±0.001 0.0013±0.001 - - -
72 6.26±0.05 0.01±0.005 0.017±0.005 0.045±0.004 0.039±0.003 0.26±0.03
96 6.12±0.02 0.01±0.006 0.018±0006 0.067±0.005 0.059±0.002 0.43±0.05
120 6.22±0.05 0.01±0.001 0.018±0.001 0.093±0.005 0.08±0.05 0.87±0.05
144 6.35±0.04 0.03±0.02 0.014±0.02 0.098±0.003 0.08±0.02 0.77±0.07
168 5.94±0.05 0.03±0.01 0.012±0.01 0.12±0.05 0.10±0.04 0.74±0.06
192 5.89±0.02 0.03±0.02 0.019±0.02 0.15±0.04 0.12±0.06 0.38±0.03
216 5.64±0.02 0.02±0.01 0.018±0.01 0.19±0.06 0.14±0.02 0.36±0.05
240 6.51±0.06 0.02±0.01 0.01±0.01 0.2±0.1 0.18±0.05 0.23±0.04
Table-5.8: A mixed culture of A. fumigatus + A. niger was grown on mineral
medium without glucose at 30 ± 2 ºC pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.26±0.07 0.005±0.003 0.005±0.002 - - -
48 6.15±0.05 0.01±0.005 0.008±0.003 0.058±0.003 0.050±0.003 0.18±0.04
72 6.17±0.05 0.01±0.002 0.03±0.02 0.069±0.005 0.061±0.004 0.32±0.03
96 6.19±0.04 0.01±0.008 0.03±0.01 0.093±0.005 0.082±0.003 0.53±0.03
120 6.17±0.06 0.01±0.005 0.005±0.004 0.105±0.003 0.095±0.004 0.92±0.06
144 6.32±0.03 0.03±0.02 0.015±0.007 0.11±0.04 0.098±0.003 0.81±0.04
168 6.22±0.04 0.03±0.01 0.035±0.004 0.113±0.005 0.10±0.04 0.73±0.03
192 6.28±0.04 0.06±0.02 0.037±0.004 0.12±0.03 0.10±0.06 0.70±0.04
216 6.41±0.05 0.03±0.02 0.04±0.02 0.12±0.06 0.10±0.07 0.36±0.04
240 6.41±0.01 0.02±0.01 0.041±0.002 0.135±0.004 0.12±0.06 0.29±0.02
84
Table-5.9: A. niger was grown on mineral medium without glucose at 30 ± 2 ºC pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
mg/ml
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 6.23±0.06 0.01±0.003 0.005±0.002 - - -
48 6.21±0.06 0.01±0.001 0.015±0.008 - - -
72 6.11±0.02 0.01±0.006 0.015±0.006 0.08±0.03 0.05±0.04 0.28±0.01
96 6.25±0.03 0.01±0.002 0.1±0.01 0.091±0.004 0.088±0.003 0.49±0.02
120 6.22±0.03 0.01±0.005 0.12±0.04 0.13±0.05 0.12±0.03 0.91±0.02
144 6.32±0.01 0.03±0.02 0.14±0.02 0.135±0.006 0.133±0.008 0.87±0.04
168 6.17±0.05 0.04±0.02 0.11±0.04 0.154±0.004 0.12±0.03 0.77±0.05
192 5.70±0.04 0.03±0.02 0.01±0.002 0.167±0.002 0.139±0.005 0.70±0.03
216 6.05±0.04 0.02±0.01 0.004±0.003 0.16±0.02 0.15±0.04 0.40±0.06
240 6.50±0.03 0.02±0.01 0.05±0.03 0.21±0.06 0.15±0.03 0.29±0.03
Table- 5.10: M. geophillus was grown on mineral medium without glucose at 30 ± 2 ºC pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.27±0.04 0.01±0.006 0.025±0.1333 0.05±0.04 0.045±0.003 -
48 6.25±0.04 0.01±0.004 0.043±0.004 0.051±0.005 0.046±0.003 0.19±0.0
72 6.27±0.02 0.01±0.007 0.04±0.0213 0.069±0.002 0.061±0.005 0.37±0.0
96 6.25±0.06 0.01±0.003 0.04±0.03 0.071±0.006 0.069±0.004 0.51±0.0
120 6.21±0.06 0.01±0.006 0.045±0.004 0.093±0.006 0.08±0.04 0.95±0.02
144 6.04±0.03 0.03±0.01 0.075±0.002 0.099±0.004 0.085±0.007 0.84±0.03
168 6.18±0.04 0.03±0.02 0.084±0.008 0.12±0.08 0.10±0.05 0. 81±0.084
192 5.73±0.02 0.05±0.03 0.078±0.004 0.154±0.003 0.14±0.03 0.55±0.04
216 6.13±0.05 0.10±0.03 0.011±0.005 0.164±0.004 0.151±0.006 0.31±0.04
240 6.53±0.0 0.02±0.01 0.006±0.003 0.187±0.0062 0.166±0.003 0.27±0.04
85
Table 5. 11. P. lilacinum was grown on mineral medium without glucose at 30± 2 ºC pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 6.28±0.03 0.005±0.003 0.00013±0.00002 - - -
48 6.23±0.04 0.01±0.007 0.00014±0.00002 - - -
72 6.25±0.02 0.01±0.006 0.0112±0.0003 0.04±0.03 0.031±0.004 0.31±0.0
96 6.22±0.03 0.01±0.002 0.0114±0.0007 0.061±0.004 0.053±0.006 0.54±0.0
120 6.33±0.02 0.01±0.006 0.013±0.004 0.09±0.06 0.079±0.007 0.89±0.07
144 6.10±0.04 0.01±0.005 0.0014±0.0004 0.11±0.05 0.087±0.004 0.81±0.04
168 5.71±0.03 0.02±0.01 0.009±0.002 0.11±0.02 0.09±0.05 0.57±0.06
192 0.65±0.03 0.03±0.01 0.019±0.006 0.125±0.006 0.099±0.003 0.39±0.08
216 5.20±0.04 0.02±0.01 0.018±0.003 0.14±0.05 0.12±0.04 0.35±0.06
240 5.21±0.02 0.03±0.02 0.01±0.002 0.152±0.005 0.126±0.003 0.32±0.02
The enzyme synthesis has been greatly influenced by the addition of different
carbon sources. The carbon sources affect not only the mode of enzyme formation,
but also the rate by which carbohydrates are metabolized (Dubey, 2000; Abdullah
et al., 2003). It is reported by Teixeira et al., (2000) that concentration of carbon
sources affects the pectinase enzyme production.
Table-5.12-5.16 shows the results of pectinase biosynthesis by different
filamentous fungi as A. fumigatus, mixed culture of A. niger + A. fumigatus , A.
niger, M. geophillus and P. lilacinum when grown on a culture medium
supplemented with 1% glucose as a carbon source. The maximum production of
pectinase 5.89 U/ml by A. fumigatus was achieved at 72 hours and then
production was decreased with the increase of the time period. The concentration
of total sugar and reducing sugar decreases with increase of the time period. The
fluctuation was noted in the final pH of the culture broth. Mixed culture of A.
niger + A. fumigatus produced 5.52 U/ml of pectinase produced in 72 hours when
cultured on the same medium. A. niger when inoculated on a culture medium
containing 1% glucose as carbon source produced 7.16 U / ml of pectinase at 72
hours. M. geophillus and P. lilacinum when inoculated on the same medium,
86
synthesized 5.76 U / ml and 6.79 U/ml of pectinase respectively at 72 hours. It is
observed that pectinase production was higher at 72 hours of incubation and
later it was decreased with the passage of time. In most of fungi glucose shows a
catabolic repression when used as a carbon source and many genes turned off in
its presence and metabolize other carbon sources (Ronne, 1995, Apel et al., 1993,
Tonukari et al., 2002).In this study glucose has exhibited repression, which is supported
by many early researchers , in their opinion, this may be due to lowering of pH
during incubation time period . Another cause may be catabolic repression of pectinase
production due to high initial glucose/sugar concentration. The lower amount of
enzyme produced when glucose was used as a carbon source as reported by
Zeilinger et al., (1996), Wang et al., (1992) and Southerton et al., (1993). All the
above researchers carried out studies on application of various carbon sources for different
microbes and strains are in agreement with Kunte and Shastri, (1980) who worked with
Atlemaria altemata, Sakellaris et al., (1988) in Lactobacillus plantarum. Macfarlane et al.,
(1990) in Bacteroides ovatus, Said et al., 1991) in Penicillium frequentans, Solis- Pereyra et
al., (1993) in Aspergillus niger, Bahkali , (1995) in verticillium tricorpus, Acuna-Arguelles et
al., (1995) in production was and Kapoor et al., (2000) in Bacillus species , have reported
that pectinase activities were inhibited by the presence of glucose and other sugars .
Catabolic repression has been expressed in many microorganisms as reported by
Fraissinet and Fevre (1996), Runco, et al., (2001) and Panda et al., (2004).
Pedrolli et al., (2008) pointed out that A. giganteus did not produce
polygalacturonase in a media supplemented with only glucose as a substrate
probably be due to catabolic repression, same findings were also presented by
Runco et al., (2001) and Fawole and Odunfa 2003) in Aspergillus terreus and
Aspergillus niger, respectively.
87
Table-5.12: A. fumigatus was grown on mineral medium supplemented with 1 %
glucose at 30 ± 2 ºC pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.7±0.2 0.052±0.001 0.13±0.02 13.735±0.004 12.485±0.004 3.91±0.08
48 5.52±0.20 0.251±0.003 0.09±0.02 19.225±0.003 18.8±0.1 4.77±0.02
72 4.9±0.3 0.285±0.004 0.103±0.002 12.8±0.3 10.5±0.3 5.89±0.02
96 4.6±0.3 0.325±0.003 0.225±0.002 4.4±0.3 3.15±0.02 4.46±0.02
120 4.35±0.03 0.25±0.02 0.09±0.03 1.2±0.1 0.98±0.03 3.97±0.03
144 4.2±0.2 0.26±0.02 0.208±0.001 1.2±0.2 0.86±0.01 3.9±0.2
168 3.7±0.2 0.345±0.003 0.2±0.1 1.1±0.01 0.81±0.03 1.8±0.1
192 3.6±0.2 0.28±0.01 0.1±0.03 1.1±0.03 0.75±0.02 1.63±0.04
216 4.4±0.3 0.295±0.004 0.078±0.002 0.96±0.03 0.72±0.03 1.5±0.2
240 4.47±0.01 0.27±0.02 0.068±0.003 0.89±0.02 0.71±0.01 0.8±0.3
Table -5.13: A mixed culture of A. fumigatus + A. niger was grown on mineral
medium supplemented with 1 % glucose at 30± 2 ºC pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 2.9±0.2 0.245±0.004 0.385±0.003 10.1±0.1 9.79±0.02 4.1±0.1
48 2.40±0.03 0.315±0.003 0.397±0.002 8.45±0.02 8.12±0.03 4.9±0.3
72 1.77±0.02 0.385±0.004 0.418±0.004 6.94±0.03 6.91±0.03 5.52±0.03
96 1.69±0.03 0.458±0.003 0.429±0.003 5.86±0.03 5.7±0.2 5.3±0.3
120 4.17±0.01 0..499±0.002 0.434±0.004 4.31±0.03 4.29±0.02 4.21±0.02
144 2.18±0.03 0.516±0.003 0.417±0.003 3.85±0.03 3.79±0.05 3.6±0.3
168 1.89±0.03 0.525±0.004 0.412±0.003 2.59±0.04 2.53±0.02 3.2±0.2
192 0.14±0.03 0.524±0.001 0.41±0.02 1.96±0.04 1.76±0.03 2.9±0.4
216 4.42±0.03 0.635±0.003 0.412±0.003 1.79±0.04 1.7±0.2 1.11±0.02
240 6.05±0.04 0.722±0.004 0.403±0.002 0.52±0.02 0.49±0.03 0.95±0.05
88
Table -5.14 : A. niger was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 3.8±0.2 0.32±0.01 0.18±0.04 11.2±0.2 10.9±0.2 4.92±0.01
48 4.4±0.3 0.47±0.03 0.275±0.004 9.52±0.03 9.41±0.03 5.11±0.03
72 4.7±0.2 0.69±0.02 0.297±0.002 8.25±0.04 8.1±0.1 7.16±0.03
96 4.7±0.5 0.71±0.05 0.205±0.004 6.65±0.03 6.43±0.04 4.95±0.04
120 4.5±0.2 0.73±0.02 0.252±0.004 4.7±0.4 4.6±0.4 4.50±0.5
144 4.1±0.1 0.75±0.02 0.197±0.004 3.8±0.4 3.5±0.2 3.29±0.04
168 4.2±0.2 0.74±0.02 0.155±0.003 2.5±0.3 2.44±0.05 2.57±0.05
192 3.9±0.3 0.73±0.04 0.171±0.004 1.34±0.06 1.22±0.05 2.3±0.2
216 3.8±0.2 0.73±0.06 0.179±0.002 0.85±0.04 0.8±0.4 1.1±0.1
240 4.1±0.1 0.7±0.2 0.177±0.005 0.7±0.2 0.68±0.02 0.93±0.05
Table -5.15:M. geophillus was grown on mineral medium supplemented with 1 %
glucose at 30 ± 2 ºC pH was adjusted at 6.5.
Time Hours
Final pH
Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar (mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.8±0.4 0.706±0.003 0.915±0.002 12.485±0.004 11.525±0.004 4.1±0.1
48 5.4±0.2 0.812±0.003 0.995±0.002 9.913±0.006 9.625±0.002 4.34±0.03
72 5.7±0.3 0.842±0.005 1.237±0.004 7.642±0.005 7.635±0.004 5.76±0.03
96 6.1±0.1 0.878±0.001 1.242±0.005 4.1±0.1 3.9±0.2 5.4±0.2
120 5.6±0.3 0.885±0.004 1.397±0.005 3.6±0.3 3.1±0.1 4.7±0.2
144 5.5±0.4 0.965±0.003 1.178±0.005 2.37±0.05 2.085±0.002 4.5±0.2
168 5.3±0.3 0.974±0.002 1.159±0.003 1.72±0.02 1.629±0.006 3.13±0.02
192 5.2±0.1 0.98±0.01 1.19±0.02 1.126±0.002 1.048±0.004 2.8±0.2
216 4.9±0.2 1.011±0.003 1.184±0.003 0.845±0.002 0.699±0.002 1.2±0.1
240 4.8±0.5 1.135±0.002 1.176±0.003 0.615±0.004 0.591±0.003 1.1±0.1
89
Table 5.16: P.lilicinum was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5.
Time Hours
Final pH
Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar (mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.8±0.2 0.276±0.005 0.17±0.04 13.882±0.002 12.96±0.03 3.87±0.04
48 5.1±0.3 0.369±0.002 0.13±0.03 9.977±0.002 9.91±0.01 4.92±0.02
72 5.6±0.3 0.377±0.005 0.21±0.01 6.5±0.3 6.49±0.04 6.79±0.03
96 5.2±0.2 0.392±0.002 0.28±0.02 5.621±0.001 5.558±0.002 5.16±0.02
120 5.1±0.1 0.416±0.003 0.19±0.03 4.431±0.003 4.35±0.02 3.82±0.02
144 4.9±0.3 0.421±0.001 0.23±0.03 2.3±0.1 2.28±0.05 3.6±0.3
168 4.6±0.2 0.438±0.003 0.17±0.03 2.25±0.02 2.168±0.004 2.92±0.01
192 4.3±0.2 0.317±0.004 0.23±0.02 1.75±0.02 1.629±0.003 2.3±0.3
216 4.1±0.1 0.36±0.02 0.23±0.02 1.72±0.02 1.24±0.03 1.86±0.02
240 3.9±0.6 0.308±0.002 0.29±0.03 0.948±0.005 0.82±0.01 1.2±0.1
Table.5.17 shows the results of pectinase synthesis by A. fumigatus, ( A. niger + A.
fumigatus), A. niger, M. geophillus and P. lilacinum when grown in mineral
medium without glucose in comparison to a medium contains 1% glucose as a
carbon source. The maximum production of pectinase 0.87/ml, 0.91 was
achieved at 120 hours by A. fumigatus and mixed culture of A. niger +A. fumigatus
respectively, then decreased with increase of time period. On the other hand A.
fumigatus and mixed culture of A. niger + A. fumigatus produced 5.89 U/ml and
5.52 U/ml, respectively at 72 hours, when it was inoculated on a culture medium
supplemented with 1% glucose as a carbon source. The maximum production of
pectinase 0.97 U /ml was achieved at 120 hours on medium with and without
glucose while pectinase 7.16 U/ml was produced by A.niger in 72 hours, the
medium supplemented with 1% glucose. (Table.5.17). The maximum production
of pectinase 0.95 U/ml by M. geophillus was achieved at 120 hours in medium
without glucose and then decreased with the increase of the time period, but it
produced an amount of pectinase 5.76 U/ ml in 1% glucose medium at 72 hours
and then decreased with the increase of the time period. The maximum
production of pectinase 0.89U/ml was achieved at 120 hours by P.lilacinum in
90
without glucose medium. The maximum production of pectinase 6.79 U/ml was
achieved at 72 hours in medium containing 1% glucose and with the increase of
the time period pectinase production decreased. The concentration of total sugar
and reducing sugar decreased with the increase of the growth period after 72
hours. Teixeira et al., (2000) reported that high concentrations of carbon sources
have an inhibition effect on enzyme production.
Table-5.17: Effect on growth and pectinase production by different filamentous
fungi when grown on mineral medium supplemented with 1% glucose and
without glucose at 30± 2 ºC and pH was adjusted at 6.5.
Filamentous fungi Biomass g/50 ml Broth
Pectinase Activity U/ml
Biomass g/ 50 ml Broth
Pectinase Activity U/ml
Without Glucose With 1% Glucose
A. fumigates 0.04±0.02 0.87±0.02 0.352±0.001 5.89±0.02
A. niger + A. fumigates 0.03±0.02 0.91±0.01 0.415±0.001 5.52±0.02
A. niger 0.03±0.01 0.97±0.03 0.43±0.03 7.16±0.03
M.geophillus 0.02±0.01 0.95±0.01 0.65±0.03 5.76±0.02
P. lilacinum 0.01± 0.89±0.03 0.398±0.001 6.79±0.03
Table-5.18 to 5.22 shows the results of Pectinase production when A. fumigatus
(A. niger + A. fumigatus), A. niger, M. geophillus and P. lilacinum grown on a
culture medium supplemented with 2.5% date sugar as a carbon source. The date
syrup is a liquid by-product of date palm industry containing 75% carbohydrates
w/w small amount of fats and proteins along with other micro and macro
elements (Al-Farsi et al., 2007; Al-Hooti et al., 2002).The maximum production of
Pectinase 3.82 U/ml by A. fumigatus was achieved at 72 hours and then
decreased with the increase of the time period. (Table-5.18)
Table-5.19 to 5.21 shows the results of pectinase production by, A. niger ,
mixed culture of A. niger + A. fumigatus and M. geophillus as 3.47 U/ml, 4.67
U/ml and 3. 9 U/ ml, respectively at 72 hours. Table-5.22 shows the results of
Pectinase production when P. lilacinum was grown on a culture medium
91
supplemented with date sugar at 2.5 % at 30 ± 2 ºC and pH was maintained as
6.5. The highest production 4.32 U /ml was recorded at 72 hours fermentation
period. The decrease in pectinase production after 72 hours may be due to
change in pH during the incubation period (Sampriya et al., 2012) or may be due
to denaturation of enzyme inhibition or interaction with other components of
medium (Soares et al., 1999). The low level of production could be also due to
depletion of nutrients in the medium (Palaniyappan et al., 2009).
Table-5.18: A. fumigatus was grown on mineral medium supplemented with
2.5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.35±0.03 0.609±0.003 1.0978±0.0002 7.248±0.004 6.32±0.02 2.23±0.02
48 5.55±0.04 0.642±0.001 0.9448±0.0003 4.858±0.002 4.758±0.006 3.36±0.03
72 5.35±0.03 0.717±0.004 0.894±0.003 3.830±0.01 3.792±0.002 3.82±0.01
96 4.9±0.3 0.732±0.001 0.726±0.003 2.99±0.06 2.57±0.04 2.51±0.01
120 4.8±0.3 0.802±0.002 0.679±0.004 2.54±0.03 2.43±0.02 2.19±0.03
144 4.5±0.3 0.837±0.003 0.687±0.005 1.967±0.002 1.95±0.03 1.53±0.02
168 4.8±0.6 0.874±0.002 0.666±0.005 1.863±0.003 1.81±0.01 1.32±0.01
192 4.2±0.2 0.926±0.001 0.638±0.003 1.616±0.003 1.63±0.02 1.3±0.3
216 4.1±0.1 0.942±0.002 0.628±0.005 1.516±0.001 1.41±0.01 0.82±0.05
240 3.8±0.3 0.979±0.003 0.619±0.001 1.120±0.004 0.99±0.06 0.74±0.03
92
Table- 5.19: A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2 ºC and pH was
adjusted to 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.4±0.2 0.530±0.002 1.04±0.03 13.66±0.05 6.167±0.003 2.74±0.03
48 5.9±0.2 0.611±0.001 1.06±0.03 7.53±0.02 4.07±0.05 3.18±0.05
72 6.1±0.1 0.624±0.003 1.04±0.02 5.212±0.02 3.112±0.002 3.47±0.04
96 6.45±0.04 0.63±0.02 0.99±0.04 4.069±0.06 1.891±0.001 2.98±0.05
120 6.15±0.02 0.65±0.03 0.92±0.02 3.75±0.03 1.567±0.002 2. 78±0.056
144 6.35±0.03 0.65±0.02 0.90±0.03 3.06±0.02 1.367±0.004 1.82±0.02
168 7.25±0.01 0.66±0.04 0.89±0.03 0.84±0.03 1.0728±0.0005 1.4±0.2
192 6.35±0.02 0.67±0.06 0.87±0.03 2.22±0.02 1.0592±0.0002 0.97±0.04
216 7.05±0.04 0.66±0.01 0.7±0.4 1.9±0.2 0.609±0.002 0.86±0.02
240 7.2±0.2 0.65±0.03 0.7±0.2 1.98±0.03 0.551±0.001 0.58±0.04
T able-5.20: A. niger was grown on mineral medium supplemented with 2.5%
date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 3.9±0.3 0.542±0.001 1.026±0.002 8.229±0.006 7.973±0.002 2.94±0.02
48 3.6±0.2 0.627±0.002 1.022±0.001 5.985±0.002 5.329±0.003 3.243±0.002
72 3.75±0.04 0.637±0.005 0.855±0.004 4.950±0.002 4.77±0.03 4.67±0.03
96 4.00±0.3 0.667±0.004 0.776±0.004 2.826±0.003 2.719±0.005 3.37±0.04
120 4.2±0.2 0.674±0.001 0.663±0.002 2.184±0.003 1.461±0.001 2.98±0.04
144 4.5±0.3 0.707±0.003 0.547±0.004 1.727±0.004 1.029±0.003 1.82±0.02
168 4.8±0.3 0.752±0.001 0.425±0.004 1.560±0.02 0.965±0.004 1.78±0.04
192 5.4±0.2 0.773±0.003 0.493±0.002 1.500±0.2 0.675±0.002 1.64±0.03
216 5.5±0.3 0.907±0.003 0.535±0.002 1.301±0.001 0.569±0.003 0.57±0.03
240 5.7±0.4 0.887±0.005 0.582±0.002 0.723±0.002 0.391±0.001 0.5±0.01
93
Table-5.21:M. geophillus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.4±0.2 0.51±0.04 0.81±0.03 9.4±0.3 8.76±0.03 2.44±0.04
48 5.65±0.4 0.60±0.05 0.85±0.03 7.18±0.06 6.94±0.07 3.19±0.06
72 5.5±0.6 0.61±0.04 0.82±0.07 6.54±0.06 6.21±0.07 3.9±0.4
96 6.25±0.03 0.74±0.07 0.83±0.02 5.98±0.06 5.73±0.07 2.82±0.06
120 6.25±0.04 0.78±0.04 0.84±0.07 4.76±0.03 4.49±0.05 1.77±0.04
144 6.55±0.06 0.81±0.05 0.83±0.04 3.72±0.04 3.59±0.04 1.46±0.03
168 6.8±0.5 0.81±0.02 0.82±0.02 2.21±0.04 2.16±0.05 0.78±0.03
192 6.5±0.4 0.82±0.01 0.81±0.04 1.92±0.04 1.86±0.08 0.69±0.04
216 6.6±0.4 0.83±0.04 0.75±0.02 1.18±0.03 1.15±0.03 0.66±0.02
240 7.1±0.2 0.84±0.07 0.71±0.03 0.91±0.04 0.76±0.04 0.56±0.05
Table-5.22: P. lilacinum was grown on mineral medium supplemented with
2.5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.55±0.06 0.749±0.006 0.7512±0.0005 8.69±0.07 7.997±0.006 2.18±0.04
48 5.57±0.06 0.822±0.009 0.747±0.003 6.8±0.3 5.76±0.03 2.67±0.02
72 5.95±0.03 0.857±0.002 0.679±0.004 5.77±0.09 4.63±0.06 4.32±0.03
96 5.25±0.02 0.873±0.007 0.692±0.007 4.6±0.3 4.34±0.07 2.73±0.08
120 5.45±0.06 0.889±0.006 0.632±0.004 3.942±0.003 3.649±0.002 1.84±0.08
144 5.7±0.4 0.922±0.006 0.598±0.003 2.983±0.008 2.86±0.04 1.65±0.08
168 6.35±0.04 0.981±0.005 0.567±0.004 2.84±0.09 2.67±0.04 1.44±0.07
192 6.5±0.3 1.021±0.002 0.546±0.008 2.25±0.02 2.167±0.008 0.97±0.05
216 7.15±0.06 1.1±0.4 0.518±0.005 1.86±0.11 1.78±0.06 0.78±0.09
240 7.45±0.04 1.127±0.006 0.513±0.005 0.84±0.03 0.63±0.05 0.71±0.04
94
Table-5.23-5.27 show the results of pectinase production when filamentous fungi
like A. fumigatus, A. niger + A. fumigatus, A. niger, M. geophillus and P. lilacinum
were grown on a culture medium supplemented with 5% date syrup as a
carbon source. A. fumigatus and a mixed culture of A. niger+A. fumigatus,
produced pectinase 4.87U/ml and 4.21 U/ml respectively, and production
decreased with the passage of time. Table - 5.25 shows the results of highest
(5.68 U/ml) of [ectinase production by A. niger when grown on mineral medium
supplemented with 5 % date syrup as carbon source. The increase of time
period decreased the rate of enzyme production and concentration of sugar
and proteins. Tables-5.26 and 5.27 shows the results of Pectinase production by
M. geophillus and P. lilacinum.The highest Pectinase production was recorded as
4.51U/ml and 5.16 U/ml respectively, while with the passage of time period
decreased Pectinase production, concentration of total sugar, reducing sugar and
total protein. The pH of the medium also changed gradually from acidic to
alkaline except in A. niger .
Table-5.23:.A. fumigatus was grown on mineral medium supplemented with 5%
date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.4±0.3 1.542±0.005 1.17±0.03 9.67±0.04 9.17±0.04 2.53±0.04
48 5.9±0.4 1.442±0.006 1.23±0.03 7.56±0.04 6.14±0.08 3.97±0.05
72 6.1±0.4 1.367±0.005 1.23±0.04 5.97±0.05 5.46±0.07 4.87±0.03
96 6.45±0.08 1.817±0.004 1.24±0.06 5.69±0.06 4.93±0.04 3.96±0.04
120 6.15±0.03 1.817±0.006 1.24±0.04 3.75±0.07 3.67±0.05 2.84±0.03
144 6.85±0.04 1.732±0.005 1.24±0.05 3.56±0.04 3.51±0.04 2.97±0.04
168 7.35±0.06 1.852±0.009 1.36±0.04 2.84±0.03 2.71±0.05 1.95±0.04
192 7.3±0.4 1.817±0.005 1.39±0.04 2.22±0.03 2.17±0.06 1.69±0.04
216 7.1±0.3 1.792±0.003 1.47±0.04 1.19±0.04 0.96±0.03 1.26±0.08
240 7.8±0.6 1.057±0.003 1.48±0.04 0.61±0.07 0.57±0.04 0.8±0.04
95
Table-5.24: A mixed culture of A. niger + A. fumigatus was grown on mineral
medium supplemented with 5% date syrup at 30 ± 2 ºC and pH was
adjusted to 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.65±0.04 1.522±0.005 1.085±0.007 11.946±0.004 11.188±0.003 2.98±0.07
48 5.7±0.4 1.857±0.006 1.206±0.003 7.47±0.06 7.091±0.005 3.22±0.05
72 5.8±0.3 1.667±0.004 1.138±0.003 5.125±0.07 4.969±0.004 4.21±0.02
96 6.45±0.4 1.737±0.03 0.995±0.04 4.252±0.004 4.012±0.007 3.98±0.04
120 6.8±0.5 1.847±0.05 0.991±0.04 3.25125±0.0001 3.180±0.003 2.64±0.03
144 6.75±0.4 1.957±0.07 0.987±0.04 3.157±0.005 3.079±0.003 1.54±0.03
168 7±0.2 1.957±0.008 0.979±0.002 2.467±0.002 2.415±0.006 1.24±0.07
192 7.7±050 1.542±0.006 0.974±0.004 1.962±0.005 1.853±0.003 0.95±0.08
216 7.95±0.02 1.307±0.005 0.973±0.005 1.763±0.004 1.587±0.002 0.72±0.03
240 8.2±0.2 1.387±0.006 0.974±0.003 0.942±0.005 0.837±0.006 0.54±0.03
Table-5.25: A. niger was grown on mineral medium supplemented with 5% date
syrup at 30 ± 2 ºC and pH was adjusted to 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.2±0.2 1.113±0.006 1.348±0.003 9.246±0.003 8.719±0.006 2.89±0.04
48 4.45±0.03 1.2902±0.0002 1.382±0.006 7.973±0.004 7.815±0.006 3.46±0.08
72 4.55±0.04 1.342±0.003 1.55±0.03 5.547±0.004 4.296±0.008 5.68±0.04
96 4.7±0.4 1.357±0.005 1.673±0.003 3.951±0.008 3.598±0.004 3.65±0.04
120 4.45±0.03 1.352±0.006 1.567±0.006 2.29±0.08 2.181±0.005 2.3±0.2
144 6.35±0.03 1.355±0.004 1.584±0.002 2.212±0.004 1.174±0.003 1.99±0.05
168 6.4±0.3 1.392±0.006 1.256±0.005 1.987±0.006 1.859±0.004 1.32±0.03
192 6.6±0.4 1.417±0.004 1.189±0.007 1.546±0.004 1.424±0.007 1.2±0.2
216 6.65±0.07 1.547±0.004 1.161±0.004 1. 3±0.2828 0.98±0.04 0.9±0.3
240 6.85±0.02 1.52±0.03 0.998±0.005 0.87±0.06 0.81±0.03 0.62±0.03
96
Table-5.26: M. geophillus was grown on mineral medium supplemented with 5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.65±0.06 1.113±0.006 1.018±0.005 8.59±0.06 7.87±0.04 2.54±0.03
48 5.6±0.3 1.117±0.003 0.997±0.005 8.17±0.08 7.53±0.06 3.14±0.07
72 6.05±0.05 1.122±0.004 0.988±0.004 6.8±0.4 5.95±0.08 4.51±0.03
96 6.6±0.2 1.072±0.003 0.986±0.003 6.5±0.3 5.38±0.06 3.29±0.02
120 6.5±0.2 1.812±0.006 0.981±0.004 5.51±0.04 5.14±0.02 2.34±0.07
144 6.95±0.06 1.452±0.005 0.979±0.006 4.81±0.05 4.19±0.04 2.16±0.08
168 6.35±0.03 1.567±0.004 0.893±0.005 3.9±0.2 3.37±0.04 1.85±0.04
192 7.15±0.03 1.692±0.004 0.884±0.003 1.43±0.04 1.23±0.06 1.03±0.02
216 7.55±0.04 1.427±0.004 0.835±0.003 1.2±0.2 0.97±0.04 0.87±0.03
240 7.7±0.4 0.777±0.005 0.825±0.008 0.7±0.5 0.58±0.03 0.81±0.04
Table -5.27: P. lilacinum was grown on mineral medium supplemented with 5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.6±0.2 0.857±0.004 1.123±0.006 13.404±0.003 8.39±0.06 2.86±0.03
48 6.15±0.06 0.987±0.006 1.931±0.004 9.879±0.006 6.038±0.003 3.7±0.3
72 6.05±0.01 1.122±0.003 1.905±0.002 7.549±0.005 5.209±0.005 5.16±0.03
96 6.05±0.03 1.129±0.007 1.722±0.007 5.729±0.003 3.766±0.004 4.18±0.06
120 6.5±0.2 1.134±0.003 1.698±0.005 3.623±0.006 2.13±0.04 2.9±0.2
144 6.25±0.06 1.259±0.003 1.569±0.006 2.640±0.004 2.021±0.004 2.21±0.03
168 7.15±0.07 1.27±0.04 1.537±0.006 1.775±0.003 1.181±0.002 1.76±0.04
192 7.45±0.04 1.331±0.002 1.013±0.004 1.073±0.006 0.819±0.006 1.33±0.04
216 7.35±0.02 1.34±0.01 0.982±0.005 0.9667±0.0002 0.959±0.004 1.3±0.2
240 8.3±0.4 1.37±0.03 0.916±0.001 0.888±0.007 0.831±0.004 0.49±0.06
97
Table-5.28 shows the comparative results of pectinase synthesis by different
filamentous fungi like A. fumigatus, (A. niger + A. fumigatus) , A. niger, M.
geophillus and P. lilacinum when grown on medium supplemented (2.5% and 5%)
of date sugar as carbon source. Results of pectinase production indicate that A.
niger produces a higher level of pectinase when the culture medium containing
2.5% and 5% date syrup as a carbon source in comparison to other fungi.
Table-5.28: Effect on growth and pectinase production by different fungi when
grown on mineral medium supplemented with 2.5 and 5% date syrup at 30 ± 2
ºC and pH was adjusted 6.5.
Filamentous fungi Biomass g/50 ml Broth
Pectinase Activity U/ml
Biomass g/ 50 ml Broth
Pectinase Activity U/ml
2.5% Date syrup 5% Date syrup
A. fumigatus 0.717±0.006 3.82±0.02 1.167±0.002 4.87±0.02
A.niger+
A. fumigatus 0.624±0.001 3.47±0.06 1.208±0.003 4.21±0.04
A. niger 0. 637±0.002 4.67±0.01 1.667±0.005 5.68±0.05
M. geophillus 0.612±0.001 3.96±0.04 1.122±0.001 4.5±0.2
P. lilacinum 0.857±0.002 4.32±0.03 1.2±0.1 5.16±0.01
The results of pectinase synthesis by A. fumigatus and a mixed culture of A. niger
+ A. fumigatus are shown in Table-5.29 and 5.30, when these fungi were
inoculated on a fermentation culture medium supplemented with 2.5 % molasses
as a carbon source and incubated at 30 + 2 ºC and initial pH was adjusted at 6.5.
The maximum production of pectinase 6.69 U/ml and 6.16 U/ml respectively,
were recorded at 72 hours and with the passage of time sugars concentration
decreased. This indicates an enzymatic repression may be due to some kind of
metabolites which act as enzyme inhibitors.
98
Tables-5.31 and 5.33 shows the results of pectinase synthesis by A. niger,
M. geophillus and P.lilacinum when inoculated on a culture medium incorporated
with 2.5% molasses as a carbon source. The maximum production of Pectinase
7.76 U/ml and 6.58 U/ ml and 6.98 U/ml by above reported fungi respectively
was found respectively at 72 hours and then pectinase activity was decreased
with the increase of the time period. The concentration of total sugars, reducing
sugars and protein in culture broth was decreased with the increase of the
fermentation time period. The pH value was also changed with increase of
fermentation time and becomes basic after 192 and 216 hours in case of mixed
culture and M. geophillus .
Table-5.29: A. fumigatus was grown on mineral medium supplemented with
2.5 % molasses at 30 + 2 ºC and the pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.4±0.3 0.985±0.007 1.01±0.001 8.67±0.06 8.1±0.2 4.8±0.4
48 5.9±0.4 0.990±0.004 1.11±0.03 7.95±0.04 6.96±0.05 4.58±0.04
72 6.1±0.2 1.089±0.004 1.13±0.04 7.2±0.3 6.12±0.03 6.69±0.04
96 6.45±0.03 1.033±0.002 1.14±0.03 6.61±0.04 5.92±0.06 5.9±0.2
120 6.15±0.04 1.045±0.008 1.2±0.2 5.45±0.04 4.68±0.05 4.63±0.07
144 6.2±0.2 1.042±0.005 1.29±0.06 3.85±0.02 3.27±0.04 3.37±0.04
168 6.25±0.02 1.205±0.002 1.2±0.1 2.8±0.4 2.28±0.04 3.2±0.4
192 6.35±0.03 1.206±0.006 1.17±0.02 1.62±0.02 1.49±0.04 2.21±0.03
216 6.5±0.4 1.203±0.002 1.16±0.02 1.25±0.07 0.99±0.03 1.81±0.04
240 6.75±0.04 1.165±0.003 1.15±0.06 0.98±0.05 0.75±0.07 0.789±0.006
99
Table-5.30: A mixed culture of A. niger +A. fumigatus was grown on mineral medium supplemented with 2.5 % molasses when incubated at 30 + 2ºC
and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.4±0.2 0.819±0.005 0.918±0.005 9.1±0.01 8. 32±0.01 3.942±0.002
48 5.65±0.04 0.922±0.002 0.925±0.004 8. 18±0.05 7.74±0.02 4.523±0.002
72 5.5±0.2 0.962±0.001 0.898±0.004 7.9±0.3 6.87±0.02 6.16±0.03
96 6.25±0.03 1.057±0.003 0.872±0.002 6.74±0.03 6.23±0.02 5.82±0.01
120 6.25±0.02 1.122±0.002 0.853±0.002 5.7±0.3 5.17±0.03 3.98±0.04
144 6.55±0.01 1.189±0.004 0.842±0.001 4.72±0.01 3.8±0.3 3.16±0.02
168 6.8±0.4 1.197±0.003 0.831±0.001 2.97±0.03 2.5±0.2 2.76±0.03
192 7.6±0.3 1.242±0.001 0.818±0.005 2.37±0.05 1.85±0.04 2.43±0.02
216 8.15±0.04 1.32±0.02 0.811±0.001 1.27±0.03 1.16±0.03 1.76±0.04
240 7.45±0.02 1.447±0.004 0.802±0.001 0.95±0.03 0.73±0.03 1.22±0.02
Table-5.31: A. niger was grown on mineral medium supplemented with 2.5 %
molasses at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
U/ml)
24 4.57±0.05 1.113±0.002 0.995±0.002 8.87±0.04 7.93±0.07 4.67±0.04
48 5.52±0.02 1.124±0.004 1.192±0.006 8.62±0.04 7.21±0.04 5.36±0.04
72 5.13±0.04 1.276±0.003 1.253±0.005 7.8±0.3 7.1±0.2 7.76±0.03
96 6.95±0.04 1.325±0.006 1.25±0.04 6. 7±0.4243 5.915±0.006 5.71±0.03
120 6.75±0.03 1.25±0.08 1.219±0.004 5.53±0.04 4.98±0.03 5.1±0.3
144 5.2±0.3 1.26±0.08 1.212±0.006 4.2±0.3 3.71±0.04 4.28±0.03
168 5.8±0.3 1.345±0.004 1.195±0.007 3.6±0.4 2.82±0.04 3.13±0.03
192 3.62±0.04 0.28±0.03 1.189±0.006 2.31±0.04 1.75±0.04 2.5±0.2
216 4.405±0.003 0.295±0.007 1.167±0.004 1.36±0.03 1.1±0.2 2.25±0.08
240 4.475±0.004 0.27±0.04 1.155±0.003 0.93±0.06 0.64±0.07 1.4±0.2
100
Table-5.32:M. geophillus was grown on mineral medium supplemented with 2.5 % molasses at 30 + 2ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.55±0.04 0.87±0.02 1.119±0.006 8.248±0.003 7.67±0.03 3.298±0.004
48 5.57±0.03 0.89±0.05 1.142±0.001 7.5687±0.0005 7.17±0.03 4.967±0.003
72 5.95±0.03 0.97±0.01 1.218±0.004 6.98±0.02 6.21±0.01 6.58±0.03
96 5.45±0.02 1.1±0.1 1.225±0.003 5.91±0.01 4.86±0.02 5.673±0.002
120 5.45±0.01 1.11±0.04 1.228±0.004 3.85±0.04 3.44±0.03 3.576±0.004
144 6.35±0.02 1.14±0.03 1.239±0.004 3.49±0.05 3.12±0.01 3.535±0.002
168 6.35±0.03 1.17±0.02 1.241±0.001 2.78±0.04 2.21±0.01 3.446±0.003
192 6.5±0.1 1.2±0.1 1.249±0.003 1.63±0.02 1.26±0.03 2.765±0.003
216 7.15±0.04 1.21±0.01 1.232±0.001 1.98±0.02 0.89±0.03 1.869±0.003
240 7.45±0.03 1.22±0.01 1.116±0.002 0.89±0.04 0.653±0.002 0.783±0.003
Table-5.33: P. lilacinum was grown on mineral medium supplemented with 2.5 % molasses at 30 + 2ºC and pH was adjusted at 6.5.
Time
Hours
Final
pH
Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 3.8±0.2 0.967±0.002 0.915±0.002 8.46±0.002 7.59±0.04 4.154±0.002
48 3.4±0.2 0.988±0.005 1.198±0.003 8.17±0.003 6.84±0.03 5.54±0.03
72 2.7±0.2 1.029±0.003 1.212±0.001 7.85±0.001 6.3±0.02 6.98±0.05
96 2.8±0.4 1.078±0.003 1.317±0.004 6.1±0.004 5.94±0.1 5.76±0.05
120 2.6±0.2 1.132±0.001 1.32±0.02 5.98±0.02 5.51±0.04 5.13±0.02
144 3.1±0.1 1.137±0.003 1.336±0.003 5.3±0.003 4.76±0.2 4.35±0.03
168 3.8±0.6 1.145±0.004 1.342±0.001 4.72±0.001 4. 25±0.02 3.3±0.1
192 3.7±0.3 1.205±0.003 1.415±0.004 3.54±0.004 3.2±0.01 2.82±0.01
216 4.8±0.3 1.255±0.004 1.245±0.002 2.45±0.002 2.39±0.05 1.4±0.3
240 4.9±0.3 1.358±0.002 1.225±0.003 1.3±0.003 0.62±0.2 1.2±0.2
Table - 5.34-5.36. reveal the results of A. fumigatus , (A. niger + A. fumigatus ) and
A. niger after 72 hours of incubation pectinase production 8.62 U/ml, 8.54 U/ml
and 10.35 U/ml respectively. The same trend has been noticed in the presence of
the same substrate i-e when the concentration of 2.5% molasses was used. With
the passage of time sugars concentration decreased and results were also
indicative of an enzymatic repression, may be due to some kind of metabolites
101
which act as enzyme inhibitors. pH values fluctuated as incubation time was
increased. The change of pH towards the acidic range during 72-96 hours of
fermentation, Probably due to microbial production of organic acids, when the
concentration of reducing sugars was very low, the pH increased, may be due to
microbial assimilation of organic acids (Botella et al., 2007). Similar pH trends
have been reported by many other researchers (Blandino et al., (2001); Roque
and Takuo (1994); Yoshikawa et al., (1995); Domenguez, (2002).
Tables 5.37 and 5.38 show that best amount of pectinase as 8.14 U/ml and 9.89
U /ml respectively, was obtained at 72 hours when M. geophillus and P.
lilacinum were grown on a medium supplemented with 5% molasses and
incubated at 30+2 ºC and the pH was adjusted to 6.5. With the passage of time
sugars decreased in amount, may be due to some kind of metabolites which
perform as enzyme inhibitors (Teixeira et al., 2000).
Table-5.34: A. fumigatus was grown on mineral medium supplemented with 5%
molasses at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.9±0.3 0.997±0.004 2.18±0.05 8.145±0.002 7.98±0.05 5.89±0.04
48 4.4±0.2 1.206±0.003 2.25±0.03 7.78±0.06 6.74±0.04 6.76±0.04
72 4.3±0.3 1.295±0.004 1.8±0.3 6.98±0.07 5.87±0.04 8.62±0.02
96 4.7±0.3 1.285±0.003 1.98±0.04 6.32±0.01 5.31±0.01 6.78±0.03
120 4.9±0.5 1.338±0.003 1.97±0.04 5.38±0.04 4.78±0.02 6.27±0.05
144 5.3±0.2 1.360±0.003 1.96±0.03 4.89±0.03 4.24±0.03 5.785±0.004
168 5.4±0.2 1.378±0.006 1.95±0.03 3.6±0.2 3.29±0.06 4.38±0.06
192 5.45±0.04 1.391±0.001 1.9±0.5 2.62±0.02 2.35±0.02 3.58±0.01
216 5.85±0.03 1.316±0.004 1.88±0.05 1.94±0.03 1.31±0.01 2.16±0.03
240 6.4±0.3 1.408±0.002 1.86±0.05 0.78±0.03 0.62±0.01 1.71±0.01
102
Table-5.35: A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 5 % molasses at 30 + 2 ºC
and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.2±0.2 1.165±0.003 1.476±0.005 8.96±0.04 7.94±0.03 4.43±0.02
48 4.75±0.04 1.656±0.002 1.97±0.05 8.49±0.02 7.32±0.02 6.1±0.1
72 5.4±0.2 1.684±0.003 1.97±0.02 7.96±0.01 6.83±0.03 8.54±0.01
96 6.15±0.02 1.68±0.06 1.84±0.04 6.88±0.03 6.15±0.04 7.87±0.05
120 6.25±0.02 1.875±0.002 1.83±0.02 5.97±0.02 4.96±0.03 6.49±0.05
144 6.45±0.03 1.78±0.03 1.79±0.06 4.61±0.01 3.52±0.01 5.44±0.03
168 6.6±0.5 1.78±0.06 1.75±0.03 3.95±0.03 3.27±0.05 4.77±0.04
192 6.7±0.4 1.75±0.04 1.7±0.3 2.9±0.2 2.47±0.04 2.26±0.03
216 6.5±0.5 1.77±0.05 1.65±0.05 1.68±0.05 1.43±0.03 1.19±0.02
240 6.1±0.1 1.77±0.01 1.6±0.3 1.23±0.02 0.84±0.02 0.99±0.03
Table-5.36:A. niger was grown on mineral medium supplemented with 5 %
molasses at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.4±0.3 1.142±0.002 1.19±0.06 9.66±0.05 8.13±0.03 5.53±0.02
48 5.9±0.3 1.19±0.05 1.27±0.03 8.53±0.02 7.1±0.1 6.2±0.1
72 6.1±0.1 1.22±0.01 1.295±0.002 8.12±0.02 6.94±0.03 10.35±0.03
96 6.4±0.2 1.23±0.03 1.29±0.03 7.9±0.4 6.23±0.01 8.16±0.03
120 6.1±0.2 1.25±0.03 1.31±0.01 5.75±0.03 4.98±0.01 6.45±0.02
144 6.8±0.3 1.3±0.2 1.33±0.02 4.18±0.05 3.43±0.03 5.19±0.05
168 7.35±0.02 1.33±0.01 1.33±0.03 3.84±0.02 2.95±0.05 4.37±0.02
192 7.3±0.2 1.41±0.01 1.39±0.07 2.23±0.02 1.76±0.03 2.61±0.01
216 7.1±0.1 1.42±0.01 1.44±0.03 1.91±0.01 1.69±0.04 1.76±0.05
240 7.8±0.2 1.45±0.03 1.45±0.05 0.98±0.02 0.57±0.02 1.18±0.02
103
Table-5.37:M. geophillus was grown on mineral medium supplemented with 5 % molasses at 30 + 2 ºC and pH was adjusted at 6.5
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.65±0.03 1.37±0.04 1.921±0.001 9.9±0.4 8. 97±0.02 4.54±0.03
48 5.6±0.2 1.452±0.002 1.929±0.003 9.29±0.07 7.8±0.3 6.54±0.02
72 6.05±0.02 1.512±0.001 1.922±0.001 7.861±0.001 7.59±0.03 8.14±0.03
96 6.6±0.3 1.677±0.004 1.876±0.004 6.28±0.04 5.83±0.03 6.9±0.7
120 6.5±0.3 1.681±0.001 1.865±0.004 4.97±0.04 4.74±0.04 5.34±0.03
144 6.95±0.05 1.705±0.002 1.857±0.003 3.9±0.3 3.48±0.05 4.63±0.02
168 6.35±0.04 1.737±0.005 1.854±0.002 3.87±0.04 2.93±0.02 3.7±0.5
192 7.15±0.03 1.782±0.002 1.8±0.4 3.3±0.2 2.8±0.4 2.67±0.04
216 7.55±0.05 1.79±0.06 1.85±0.04 1.9±0.4 1.5±0.1 2. 9±0.5
240 7.7±0.5 1.823±0.002 1.88±0.05 1.16±0.03 0.831±0.001 1.15±0.04
Table-5.38: P. lilacinum was grown on mineral medium supplemented with 5 %
molasses at 30 + 2 ºC and pH was adjusted at 6.5
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.8±0.5 0.970±0.002 2.013±0.002 8.52±0.02 7.93±0.03 5.13±0.02
48 6.1±0.1 0.975±0.002 2.59±0.03 8.13±0.02 7.38±0.03 6.83±0.03
72 6.15±0.04 1.083±0.003 1.96±0.01 7.86±0.03 6.89±0.02 9.89±0.04
96 6.0±0.3 1.04±0.02 1.86±0.03 6.97±0.04 5.57±0.04 7.14±0.03
120 5.8±0.6 1.151±0.001 1.86±0.04 5.65±0.02 4.55±0.01 5.84±0.02
144 5.4±0.2 1.17±0.03 1.77±0.04 4.6±0.2 3.97±0.02 4.12±0.02
168 5.75±0.04 1.22±0.01 1.84±0.04 3.69±0.06 2.94±0.03 3.2±0.1
192 6.4±0.1 1.25±0.03 1.85±0.03 2.48±0.04 2.13±0.02 2.74±0.02
216 6.65±0.02 1.3±0.2 1.82±0.02 1.86±0.03 1.7±0.5 2. 5±0.3
240 6.8±0.3 1.32±0.01 1.88±0.03 0.97±0.03 0.69±0.05 1.12±0.02
Table- 5.39 shows the results of pectinase synthesis by A. fumigatus, (A. fumigatus
+ A. niger ), A. niger, M. geophillus, P. lilacinum.When these fungi were inoculated
on a fermentation culture medium supplemented with 2.5 % and 5% molasses as
a carbon source produces pectinase 8.62 U/ml, biomass 1.295 g/50 ml, 5.97
U/ml, biomass 1.68 g/50 ml, 10.35 U/ml, biomass 1.226 g /50 ml, 8.14 U/ml,
104
biomass 1.512 g/50ml and 9.89 U/ml, biomass 1.083 g/50 ml respectively was
recorded at 72 hours.These results reveal that maximum amount of pectinase
was achieved by A. niger and P. lilacinum as 10.132 U/ml and 9.89 U/ml
respectively, when the same amount of molasses was used in a medium as a
carbon source.
Table-5.39: Effect on growth and pectinase production by different fungi when
grown on mineral medium supplemented with 2.5 and 5% molasses at 30 ± 2 ºC
and pH was adjusted 6.5.
Filamentous Fungi
Biomass g/50 ml Broth
Pectinase Activity U/ml
Biomass g/ 50 ml Broth
Pectinase Activity U/ml
2.5% Molasses 5% Molasses
A. fumigates 1.089±0.005 6.69±0.03 1.295±0.004 8.62± 0.02
A. niger and A.
fumigates 0.962±0.002 6.16±0.03 1.68± 0.06 5.97±0.03
A. niger 1.27±0.04 7.76±0.01 1.226 ±0.003 10.35±0.03
M. geophillus 0.998±0.005 6.58±0.02 1.512±0.003 8.14±0.03
P. lilacinum 1.029±0.002 6.98±0.02 1.083±0.002 9.89±0.03
Table- 5.40 indicated that the production of pectinase 5.4 U/ml was obtained by
A. fumigatus after 72 incubation hours when grown on 2.5% citrus pectin where
as mixed culture of A. niger + A. fumigatus and A. niger produced 5.17 U/ml and
6.59 U/ml respectively after 72 hours (Table-5.41-5.42). The pH value shown
fluctuation as incubation time was increased. The results represented in Table-
5.43 and 5.44 show that production of pectinase 4.65 U/ml and 6.35 U/ml
respectively, at 72 hours incubation when M. geophillus and P. lilacinum were
grown on a medium supplemented with 2.5 % citrus pectin. The activity was
reduced after 96 hours of incubation period that may be due to some metabolites
which act as enzyme inhibitors. Stutzenberger, (1992) investigated that limitations of
105
the microorganisms to use pectin as the carbon and energy source is might be due to their
inability to make pectin methyl esterase. Tsuymu, (1979) reported that this type of
phenomenon may be a catabolic repression of galacturonic acid or one of the metabolite
produced undergoing self catabolite repression. Palaniyappan et al., (2009) has reported
similar findings of enzyme inhibition when higher concentrations of pectin was used as a
substrate.
Table-5.40: A. fumigatus was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.1±0.1 0.99±0.06 1.43±0.03 9.68±0.04 8.23±0.02 3.75±0.04
48 3.35±0.04 1.06±0.03 1.56±0.03 8.82±0.02 7.54±0.03 4.19±0.01
72 3.55±0.02 1.31±0.01 1.83±0.02 7.55±0.02 7.13±0.03 5.4±0.3
96 3.05±0.03 1.53±0.02 1.87±0.03 7.12±0.01 6.93±0.03 4.32±0.01
120 3.5±0.3 1.57±0.05 1.69±0.04 5.54±0.02 4.66±0.04 3.97±0.02
144 3.8±0.4 1.67±0.03 1.682±0.002 4.7±0.1 3.54±0.02 3.28±0.05
168 3.95±0.04 1.69±0.04 1.587±0.006 3.98±0.05 2.9±0.3 2.95±0.04
192 4.55±0.02 1.82±0.01 1.565±0.003 2.89±0.02 2.4±0.2 2.6±0.2
216 4.15±0.01 1.89±0.03 1.517±0.006 2.7±0.1 2.1±0.1 1.42±0.02
240 4.2±0.1 2.26±0.02 1.466±0.003 1.6±0.3 1.29±0.04 1.34±0.03
Table-5.41: A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 + 2ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.15±0.04 0.985±0.002 2.176±0.003 12.637±0.005 9.784±0.003 3.67±0.02
48 3.6±0.2 0.991±0.001 2.3779±0.0004 101.249±0.006 9.394±0.002 3.97±0.03
72 3.45±0.03 1.112±0.002 2.426±0.003 90.622±0.001 8.734±0.001 5.17±0.06
96 3.65±0.01 1.136±0.003 2.476±0.004 9.183±0.003 7.556±0.003 3.94±0.01
120 3.55±0.05 1.243±0.002 2.396±0.005 7.159±0.002 6.498±0.003 3.2±0.1
144 3.85±0.04 1.383±0.002 2.297±0.003 5.996±0.003 4.454±0.004 2.53±0.03
168 4.00±0.3 1.446±0.004 2.278±0.004 4.986±0.004 3.975±0.002 1.97±0.03
192 4.15±0.03 1.651±0.002 2.259±0.003 3.739±0.003 2.928±0.004 1.53±0.05
216 4.15±0.02 1.682±0.002 2.208±0.004 2.185±0.004 1.987±0.005 1.327±0.0089
240 4.2±0.1 1.921±0.003 1.979±0.004 0.959±0.007 0.670±0.003 1.3±0.2
106
Table-5.42: A. niger was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.15±0.03 0.886±0.003 1.376±0.005 9.237±0.004 8.63±0.02 3.97±0.05
48 3.6±0.2 1.219±0.006 1.779±0.004 8.497±0.001 8.1±0.1 4.47±0.03
72 3.45±0.04 1.223±0.001 1.826±0.003 8.64±0.04 7.734±0.002 6.57±0.01
96 3.65±0.05 1.237±0.005 1.877±0.006 7.844±0.006 7.13±0.02 4.6±0.2
120 3.55±0.01 1.346±0.003 1.899±0.001 6.817±0.006 5.49±0.04 4.13±0.02
144 3.85±0.02 1.383±0.002 1.687±0.004 4.69±0.05 4.15±0.04 3.76±0.04
168 4.00±0.3 1.4465±0.0003 1.649±0.007 3.98±0.02 3.75±0.03 3.15±0.02
192 4.15±0.06 1.551±0.001 1.597±0.002 3.83±0.03 3.28±0.05 2.74±0.03
216 4.15±0.03 1.682±0.002 14208±0.0004 2.58±0.04 2.46±0.03 2.18±0.03
240 4.2±0.2 1.8215±0.0004 1.1055±0.0002 1.39±0.03 1. 2±0.2 1.89±0.01
Table-5.43:M. geophillus was grown on mineral medium supplemented with 2.5%
citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.55±0.03 0.978±0.004 2.5397±0.003 13.007±0.003 8.101±0.001 3.15±0.03
48 3.55±0.05 1.036±0.001 2.606±0.003 10.859±0.003 8.979±0.002 4.13±0.02
72 3.35±0.01 1.27±0.04 2.435±0.002 9.317±0.006 7.912±0.002 4.65±0.02
96 3.65±0.02 1.379±0.001 2.449±0.001 8.887±0.001 6.398±0.003 3.58±0.02
120 3.75±0.04 1.4395±0.0004 2.857±0.002 6.338±0.006 5.497±0.002 3.18±0.03
144 3.95±0.06 1.5885±0.0003 2.758±0.003 5.315±0.002 5.308±0.001 2.62±0.01
168 4.15±0.03 1.4865±0.0003 2.489±0.004 4.033±0.002 3.71±0.01 1.98±0.03
192 4.25±0.01 1.4866±0.0003 2.357±0.004 2.785±0.002 2.196±0.003 1.57±0.02
216 4.25±0.05 1.7705±0.0004 2.322±0.002 2.178±0.004 1.679±0.001 1.19±0.03
240 4.15±0.02 1.957±0.003 2.309±0.001 1.301±0.001 0.7225±0.0004 1.17±0.06
107
Table-5.44: P. lilacinum was grown on mineral medium supplemented with 2. 5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.55±0.04 0.978±0.004 1.26±0.03 9.97±0.02 7.819±0.003 3.75±0.02
48 3.55±0.03 1.036±0.003 1.507±0.001 9.766±0.004 7.097±0.005 4.81±0.01
72 3.35±0.01 1.27±0.05 1.968±0.003 8.527±0.006 6.912±0.002 6. 35±0.03
96 3.65±0.05 1.379±0.001 1.893±0.002 7.489±0.001 6.76±0.04 4.66±0.03
120 3.75±0.02 1.439±0.004 1.878±0.006 6.857±0.003 5.49±0.05 3.48±0.07
144 3.95±0.01 1.588±0.002 1.768±0.002 4.752±0.002 3.35±0.04 3.22±0.01
168 4.15±0.03 1.486±0.002 1.748±0.004 3.859±0.003 2.71±0.01 2.8±0.2
192 4.25±0.06 1.486±0.001 1.658±0.003 1.78±0.05 2.19±0.01 2.17±0.03
216 4.25±0.08 1.775±0.003 1.596±0.002 1.62±0.01 1.34±0.03 1.45±0.03
240 4.15±0.06 1.957±0.003 1.603±0.002 1.43±0.02 1.2±0.1 1.3±0.2
The higher amount of pectinase 6.15 U/ml was obtained after 72 incubation
hours when a culture of A. fumigatus was grown on a medium supplemented
with 5 % citrus pectin as carbon source and incubated at 30 + 2 ºC when pH was
adjusted at 6.5 ( Table-5.45). Pectinase activity started to reduce after 96 hours of
incubation period. A mixed culture of A. niger + A. fumigatus produced the
higher amount of pectinase 5.97 U/ml after 72 hours of incubation when a
mixed culture was grown on a medium supplemented with 2.5 % citrus pectin
Table-5.46).
Tables 5.47and 5.48 represent the highest amount of pectinase 6.86 U/ml and
5.81 U/ml respectively produced by A. niger and M. geophillus when grown on
a medium supplemented with 5 % citrus pectin .Table - 5.49. shows the highest
amount of pectinase 6.71 U/ml obtained by P. lilacinum after 72 hours, grown on
a medium supplemented with 5 % citrus pectin. Teixeira et al., (2000) reported
the best production of pectic enzymes in the presence of pectin. With the
passage of time sugars concentration decreased, which is showing an enzymatic
suppression and the reason of some kind of metabolites which act as enzyme
inhibitors, reduction in pectinase activity by pectin may be due to the presence of
108
catabolic repression formed by the high galacturonic acid concentration by pectin
degradation. The present t results are in agreement with observations reported by
Aguilar and Huitron, (1987); Maldonado et al., (1989) and Teixeira et al., (2000).
Catabolic repression can occur not only due to pectin degradation, but also due to the
neutral sugars attached to the pectin molecule.
Table-5.45: A. fumigatus was grown on mineral medium supplemented with 5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.1±0.1 0.98±0.04 2.06±0.03 11.61±0.01 8.23±0.02 4.75±0.04
48 3.35±0.02 1.06±0.03 2.56±0.05 11.21±0.01 7. 63±0.03 5.89±0.03
72 3.55±0.04 1.31±0.01 2.29±0.05 8.58±0.04 6.93±0.02 6.15±0.02
96 3.05±0.03 1.53±0.02 2.27±0.05 8.12±0.01 5.93±0.01 5.5±0.1
120 3.5±0.1 1.57±0.03 2.256±0.002 6.54±0.04 4.66±0.03 5.177±0.003
144 3.8±0.3 1.67±0.05 2.25±0.03 4.713±0.002 4.14±0.03 4.85±0.04
168 3.65±0.03 1.69±0.04 2.233±0.002 3.798±0.004 2.77±0.03 3.345±0.003
192 3.25±0.05 1.82±0.01 2.225±0.002 3.351±0.001 2.29±0.05 2.955±0.001
216 3.15±0.04 1.89±0.04 2.179±0.005 2.83±0.02 1.98±0.03 2.65±0.05
240 3.2±0.1 2.26±0.02 2.116±0.003 1.11±0.01 0.993±0.002 1.425±0.004
Table-5.46:A mixed culture of A. niger + A. fumigatus was grown on mineral
medium supplemented with 5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 3.8±0.4 0.995±0.003 1.08±0.04 9.152±0.001 8.12±0.01 3.31±0.01
48 3.55±0.04 1.107±0.004 1.19±0.05 8.47±0.02 7.38±0.04 4.46±0.03
72 3.15±0.02 1.26±0.03 1. 2±0.1 7.961±0.001 6.74±0.02 5.97±0.03
96 3.1±0.1 1.215±0.004 1.27±0.04 7.78±0.04 6.15±0.04 4.48±0.02
120 2.8±0.3 1.273±0.002 1.31±0.01 5.987±0.004 4.25±0.03 3.84±0.03
144 2.4±0.2 1.366±0.002 1.34±0.02 4.796±0.003 3.99±0.06 3.49±0.04
168 2.75±0.04 1.398±0.003 1.32±0.01 3.884±0.002 3.39±0.01 2.42±0.01
192 3.45±0.02 1.418±0.002 1.3±0.2 2.481±0.001 2.13±0.02 2.14±0.03
216 3.65±0.03 1.425±0.004 1.3±0.1 1.868±0.003 1.9±0.3 1.78±0.04
240 3.8±0.5 1.539±0.003 1.2±0.2 1.592±0.001 0.98±0.02 1.2±0.1
109
Table-5.47: A. niger was grown on mineral medium supplemented with 5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.2±0.1 0.989±0.001 2.101±0.001 11.675±0.003 9.792±0.001 3.913±0.002
48 3.75±0.03 1.022±0.001 2.187±0.002 9.98±0.03 7.469±0.004 5.235±0.004
72 3.4±0.2 1.19±0.03 2.235±0.004 8.69±0.03 6.762±0.001 6.86±0.05
96 3.15±0.02 1.125±0.004 2.243±0.002 7.93±0.02 6.019±0.003 5.14±0.03
120 2.95±0.01 1.242±0.001 1.998±0.002 5.71±0.01 4.198±0.004 4.29±0.05
144 3.15±0.02 1.129±0.005 1.987±0.002 4.93±0.02 3.998±0.003 3.9±0.3
168 3.45±0.03 1.231±0.001 1.982±0.001 3.69±0.03 2.712±0.001 3.2±0.1
192 3.8±0.3 1.337±0.003 1.978±0.002 2.98±0.02 2.259±0.003 2.9±0.6
216 3.85±0.04 1.392±0.001 1.867±0.002 1.68±0.03 1.131±0.001 2.1±0.1
240 4.0±0.03 1.4±0.2 1.869±0.005 1.11±0.01 0.998±0.004 1.97±0.02
Table-5.48: M. geophillus was grown on mineral medium supplemented with
5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.15±0.03 1.09±0.04 2.14±0.03 11.99±0.01 9.256±0.003 3.3±0.2
48 5.0±0.4 1.16±0.03 2.15±0.01 11.69±0.05 9.667±0.005 4.45±0.04
72 4.85±0.04 1.22±0.01 2.27±0.05 10.31±0.01 9.36±0.02 5.81±0.01
96 4.3±0.2 1.23±0.01 2.27±0.04 9.17±0.02 7.85±0.03 5.32±0.01
120 4.05±0.03 1.24±0.03 2.27±0.01 6.98±0.05 6.19±0.05 4.25±0.03
144 4.3±0.2 1.28±0.03 1.22±0.01 4.88±0.01 4.24±0.03 3.45±0.01
168 4.4±0.2 1.39±0.03 2.1±0.1 3.32±0.02 3.14±0.01 3. 15±0.02
192 4.45±0.02 1.54±0.02 2.1±0.2 2.69±0.08 2.16±0.03 2.85±0.05
216 4.55±0.05 1.57±0.02 1.99±0.04 1.82±0.01 1.13±0.02 1.97±0.01
240 5.5±0.1 1.58±0.04 1.98±0.02 1.1±0.1 0.98±0.03 1.59±0.06
110
Table-5.49: P. lilacinum was grown on mineral medium supplemented with 5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.92±0.01 0.998±0.004 2.275±0.004 9.97±0.01 8.149±0.004 3.66±0.03
48 3.75±0.01 1.113±0.002 2.592±0.001 8.13±0.03 7.24±0.03 4.96±0.03
72 3.55±0.04 1.24±0.02 2.603±0.002 7.56±0.03 6.86±0.02 6.71±0.01
96 3.35±0.03 1.417±0.003 2.325±0.004 7. 26±0.02 6.75±0.03 5.45±0.01
120 3.0±0.2 1.498±0.002 2.291±0.001 6.87±0.01 5.48±0.02 4.81±0.01
144 2.8±0.3 1.518±0.002 2.264±0.002 4.89±0.04 3.353±0.002 3.21±0.03
168 2.95±0.02 1.632±0.002 2.249±0.002 4.16±0.02 3.167±0.002 2.84±0.03
192 3.3±0.2 1.819±0.002 2.437±0.005 3.6±0.3 2.13±0.02 2.23±0.02
216 3.55±0.03 1.861±0.001 2.218±0.004 1.72±0.01 1.32±0.02 1.98±0.02
240 3.8±0.1 1.911±0.003 2.125±0.002 1.32±0.02 1.1±0.1 1.27±0.01
Table - 5.50 shows the results of pectinase synthesis by different filamentous
fungi like A. fumigatus, (A. niger + A. fumigatus), A. niger, M. geophillus and P.
lilacinum.when grown on 2.5% and 5% of crude citrus pectin . Results show that
highest pectinase production (6.86 U/ml) and growth ( biomass 1.22 g/50ml )
was obtained after 72 of incubation period with A. niger and on a medium
supplemented with 5% crude citrus pectin. P. lilacinum produced pectinase 6.71
U/ml and biomass 1.24 g/50ml when grown on with 5% crude citrus pectin as
a sole carbon source at 30 + 2 ºC and pH was adjusted at 6.5. Pectinase activity
was reduced after 120 hours of incubation period, with the passage of time.
Naidu and Panda (1998) and Pedrolli et al., (2009) have reported that high
concentrations of carbon source create repression in the biosynthesis of enzymes.
111
Table-5.50: Effect on growth and pectinase production by different fungi when grown on mineral medium supplemented with 2.5 and 5% citrus pectin at
30 ± 2 ºC and pH was adjusted 6.5.
Filamentous Fungi
Biomass g/50 ml Broth
Pectinase Activity U/ml
Biomass g/ 50 ml Broth
Pectinase Activity U/ml
2.5% Crude citrus pectin 5% Crude citrus pectin
A. fumigatus 1.31±0.01 5.4±0.2 1.31±0.02 6.15±0.02
A. niger and
A. fumigatus 1.11±0.01 5.17±0.03 1.26±0.01 5.97±0.01
A. niger 1.225±0.002 6.57±0.03 1.19±0.03 6.86±0.02
M. geophillus 1.28±0.07 4.65±0.03 1.22±0.01 5.81±0.01
P. lilacinum 1.27±0.01 6.35±0.02 1.24±0.03 6.71±0.03
Table- 5.51 to 5.53 reveal the activity of pectinase obtained after 72 hours 6.47
U/ml, 6.12 U /ml , 7.36 U/ml respectively by A. fumigatus, mixed culture of A.
niger + A. fumigatus and A. niger when grown on a medium supplemented with
2.5 % pure citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5. Tables 5.54 and
5.55 shows the results of pectinase synthesis by M. geophillus and P. lilacinum
when inoculated on a culture medium incorporated with 2.5% citrus pectin as a
carbon source. The maximum production of pectinase was achieved 6.27 U/ml
; 7.14 U / ml after 72 hours respectively, and then started to decrease with the
increase of incubation time period .The results show the amount of pectinase
produced by P. lilacinum having similarity with the results of A. niger produced
more or less same amount of pectinase. The concentration of total sugars,
reducing sugars and total protein were decreased with the increase of
fermentation time period. pH was also changed with increase of fermentation
time it becomes basic after 240 hours.
112
Table-5.51: A. fumigatus was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was
adjusted at 6.5
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.3±0.2 0.955±0.007 1.898±0.004 10.165±0.002 8.878±0.005 4.71±0.03
48 4.0±0.4 0.991±0.004 1.9055±0.0002 9.893±0.006 8.134±0.003 5.12±0.04
72 3.85±0.03 1.154±0.005 2.0708±0.0004 8.819±0.003 7.513±0.002 6.47±0.05
96 3.35±0.04 1.219±0.006 2.183±0.004 7.66±0.02 6.554±0.004 5.31±0.07
120 3.1±0.01 1.271±0.004 2.2429±0.0006 6.786±0.003 6.209±0.006 3.97±0.05
144 3.35±0.03 1.323±0.004 2.2599±0.0005 5.329±0.004 4.983±0.002 3.58±0.04
168 3.45±0.02 1.365±0.003 2.3186±0.0004 4.965±0.002 3.8252±0.0004 2.75±0.06
192 3.5±0.5 1.422±0.004 1.952±0.003 4.279±0.0004 3.749±0.005 2.7±0.2
216 3.54±0.06 1.45±0.06 1.948±0.004 2.416±0.003 1.21420±0.0002 2.62±0.03
240 3.7±0.4 1.549±0.002 1.877±0.006 0.788±0.006 0.569±0.006 1.87±0.02
Table-5.52: A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 + 2 ºC
and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml Broth
Total Proteins (mg/ml)
Total Sugar (mg/ml)
Reducing Sugars (mg/ml)
Pectinase Activity (U/ml)
24 3.85±0.02 0.996±0.001 1.056±0.003 11.16±0.02 8.83±0.04 4.87±0.06
48 3.5±0.3 1.07±0.04 1.159±0.004 10.35±0.06 8.27±0.06 5.25±0.03
72 3.35±0.04 1.16±0.03 1.236±0.005 9.37±0.05 7.67±0.04 6.23±0.02
96 3.15±0.02 1.27±0.05 1.282±0.003 7.98±0.05 6.48±0.02 6.12±0.05
120 2.8±0.4 1.29±0.04 1.210±0.04 5.34±0.03 5.69±0.03 5.89±0.06
144 2.9±0.4 1.31±0.03 1.191±0.003 5.19±0.06 4.8±0.4 3.63±0.05
168 3.00±0.2 1.33±0.02 1.174±0.004 3.98±0.04 3.18±0.03 3.57±0.04
192 3.45±0.04 1.37±0.03 1.166±0.004 2.93±0.06 2.55±0.05 1.97±0.05
216 3.5±0.2 1.36±0.03 1.153±0.002 1.99±0.02 1.41±0.04 1.35±0.02
240 4.05±0.04 1.3±0.2 1.139±0.007 1.037±0.004 0.94±0.05 1.11±0.04
113
Table-5.53: A. niger was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 3.95±0.06 0.899±0.004 2.1±0.1 9.74±0.03 8.54±0.02 5.67±0.04
48 3.4±0.3 1.116±0.003 2.11±0.04 9.14±0.05 8.37±0.06 6.43±0.03
72 3.35±0.03 1.182±0.003 2.13±0.07 8.49±0.04 7.94±0.03 7.36±0.03
96 3.5±0.4 1.198±0.004 2.18±0.04 6.67±0.04 6.13±0.03 6.89±0.07
120 3.95±0.07 1.256±0.001 2.21±0.02 6.33±0.03 6.04±0.01 4.97±0.02
144 4.5±0.3 1.204±0.002 2.25±0.03 5.62±0.04 4.74±0.02 4.83±0.03
168 4.25±0.01 1.281±0.004 2.28±0.05 4.291±0.003 3.45±0.05 3.89±0.04
192 4.4±0.2 1.322±0.004 2.28±0.03 3. 25±0.02 2.87±0.02 3.86±0.02
216 4.47±0.06 1.416±0.003 2.27±0.02 2.49±0.06 1.65±0.03 2.85±0.04
240 4.85±0.05 1.508±0.004 2.63±0.03 0.98±0.04 0.67±0.05 2.27±0.03
Table-5.54: M. geophillus was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.4±0.3 1.542±0.003 1.067±0.002 13.98±0.03 11.179±0.002 4.58±0.03
48 5.9±0.5 1.442±0.001 1.179±0.006 9.536±0.004 7.329±0.004 5.97±0.04
72 6.1±0.5 1.367±0.003 1.197±0.001 5.212±0.003 4.887±0.004 6.27±0.06
96 6.45±0.03 1.411±0.003 1.229±0.002 4.791±0.004 3.939±0.003 5.67±0.02
120 6.15±0.04 1.487±0.004 1.263±0.003 3.89±0.03 2.98±0.02 4.14±0.01
144 6.5±0.2 1.527±0.002 1.286±0.004 3.584±0.001 2. 58±0.02 3.75±0.02
168 6.35±0.02 1.552±0.002 1.337±0.002 2.846±0.002 1.976±0.002 2.541±0.002
192 6.3±0.2 1.587±0.003 0.995±0.001 2.22±0.02 1.696±0.003 1.96±0.03
216 7.1±0.4 1.592±0.002 0.976±0.002 1.99±0.04 1.579±0.004 1.765±0.003
240 7.3±0.3 1.599±0.003 0.964±0.001 0.97±0.04 0.773±0.003 1.577±0.002
114
Table-5.55: P. lilacinum was grown on mineral medium supplemented with 2.5% CCP (commercial citrus pectin) at 30 + 2ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/100 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 3.95±0.01 1.542±0.002 1.0236±0.003 9.2298±0.004 8.073±0.002 4.412±0.004
48 3.6±0.3 1.627±0.002 1.0202±0.003 6.858±0.003 5.949±0.004 5.485±0.004
72 3.75±0.03 1.637±0.004 1.154±0.001 4.505±0.004 4.772±0.002 7.14±0.01
96 4.00±0.2 1.663±0.003 1.176±0.001 4.263±0.001 3.199±0.006 5.158±0.003
120 4.2±0.2 1.674±0.002 1.213±0.003 3.848±0.003 3.978±0.007 4.963±0.003
144 4.5±0.3 1.697±0.002 1.324±0.001 3.727±0.004 3.098±0.002 3.827±0.002
168 4.85±0.02 1.752±0.002 1.346±0.003 2.568±0.003 2.163±0.001 2.886±0.005
192 5.4±0.3 1.827±0.004 1.395±0.001 1.971±0.001 1.668±0.003 2.484±0.004
216 5.95±0.03 1.887±0.003 1.425±0.005 1.301±0.001 0.569±0.003 1.972±0.004
240 7.00±0.1 1.894±0.001 1.582±0.001 0.723±0.003 0.391±0.001 1.259±0.007
The results of pectinase synthesis by A. fumigatus, (A. niger + A. fumigatus),
A. niger, M. geophillus and P. lilacinum are presented in Tables - 5.56 to 5.60 when
M.O grown in a culture medium with 5% CCP (commercial citrus pectin) as a
carbon source.The maximum production of pectinase 8.95 U / ml ; 8.76 U / ml ;
10.19 U /ml ; 8.92 U / ml and 10.1 U / ml respectively obtained after incubation
at 72 hours and then decreased with the increase of time period . A. niger and P.
lilacinum produced more or less the same amount of Pectinase, and reduction in
activity by pectin may be due to the presence of catabolic repression formed by the
high galacturonic acid concentration by pectin degradation (Aguilar and Huitron,
(1987); Maldonado and Callieri, (1989). Catabolic repression can occur not only due to
pectin degradation, but also due to the natural sugars attached to the pectin molecule.
Siddiqui et al., (2013 ) reported pure pectin as a best carbon source while working with
Rhizomucor pusillus , pectin as a good carbon source also reported by (Maria et al., 2002)
for Mucor sp., for Mucor ramosissimus Marques et al., ( 2006) and for Mucor circinelloides
Thakur et al. (2010) . Arijit et al., (2013) observed that maximum pectinase activity was
achieved by using pectin as a carbon source.
115
Table-5.56: A. fumigatus was grown on mineral medium supplemented with 5% CCP (commercial citrus pectin) at 30 + 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.75±0.02 1.11±0.04 1.753±0.001 13.126±0.004 11.975±0.003 5.86±0.01
48 4.55±0.05 1.202±0.002 1.892±0.002 10.028±0.003 9.37±0.05 7.608±0.003
72 4.0±0.2 1.233±0.002 1.923±0.003 7.526±0.005 6.23±0.03 8.95±0.01
96 4.1±0.1 1.278±0.003 2.094±0.003 6.455±0.005 6.01±0.01 7.35±0.05
120 3.8±0.3 1.286±0.003 2.18±0.03 6.303±0.002 5.85±0.05 6.17±0.05
144 3.5±0.2 1.313±0.00 2.187±0.001 3.874±0.001 3.127±0.006 4.96±0.01
168 3.7±0.1 1.425±0.003 2.242±0.002 2.229±0.007 1.895±0.002 3.92±0.05
192 4.15±0.04 1.519±0.008 2.22±0.04 1.265±0.002 1.41±0.05 2.85±0.04
216 4.2±0.2 1.567±0.005 0.16±0.03 1.806±0.002 1.261±0.002 1.53±0.03
240 5.85±0.03 0.105±0.004 0.19±0.03 1.97±0.05 0.884±0.002 0.48±0.05
Table-5.57:A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 + 2 ºC and
pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.2±0.2 0.99±0.04 1.64±0.03 11.9±0.5 9.18±0.04 6.64±0.05
48 4.1±0.3 1.19±0.02 1.964±0.001 9.42±0.04 8.32±0.02 7.28±0.03
72 4.0±0.3 1.29±0.02 2.168±0.003 9.83±0.03 7.46±0.03 8.76±0.01
96 5.15±0.04 1.30±0.07 2.281±0.005 6.47±0.01 5.93±0.03 7.1±0.1
120 5.45±0.02 1.29±0.04 2.285±0.005 4.62±0.02 4.15±0.02 6.87±0.05
144 5.5±0.3 1.39±0.03 2.172±0.004 3.57±0.02 3.44±0.01 5.97±0.02
168 5.25±0.04 1.4±0.1 2.164±0.002 2.86±0.05 2.73±0.01 3.26±0.03
192 5.1±0.1 1.44±0.04 2.089±0.004 2.56±0.03 2.42±0.04 2.62±0.02
216 4.9±0.4 1.49±0.07 2.039±0.007 1.48±0.03 1.34±0.03 1.55±0.04
240 4.4±0.2 1.56±0.01 2.015±0.005 0.94±0.01 0.67±0.03 0.85±0.02
116
Table-5.58: A. niger was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.5±0.3 1.078±0.002 1.219±0.004 11.896±0.003 9.987±0.005 7.83±0.02
48 4.85±0.02 1.125±0.002 1.233±0.003 9.978±0.004 8.358±0.005 8.64±0.01
72 4.15±0.02 1.249±0.005 1.317±0.004 7.62±0.02 6.986±0.004 10.06±0.04
96 5.85±0.01 1.298±0.005 1.385±0.003 6.541±0.003 5.987±0.001 8.31±0.04
120 5.4±0.1 1.313±0.003 1.382±0.004 4.868±0.002 4.119±0.002 7.35±0.06
144 5.75±0.07 1.356±0.006 1.392±0.007 4.60±0.03 3.86±0.01 5.03±0.02
168 6.25±0.06 1.387±0.005 1.376±0.003 3.766±0.003 3.319±0.006 4.76±0.01
192 5.0±0.2 1.391±0.001 1.265±0.004 2.894±0.003 2.316±0.005 3.49±0.03
216 4.4±0.2 1.411±0.005 1.263±0.003 1.982±0.002 1.345±0.005 3.51±0.01
240 5.8±0.1 1.523±0.002 1.252±0.004 0.914±0.001 0.772±0.001 2.75±0.02
Table-5.59 : M. geophillus was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.9±0.2 1.022±0.003 1.19±0.06 12.03±0.04 10.80±0.04 6.98±0.03
48 4.7±0.2 1.19±0.04 1.72±0.04 10.90±0.02 8.72±0.02 7.1±0.1
72 6.4±0.1 1.213±0.003 1.79±0.04 6.17±0.04 5.97±0.04 8.92±0.01
96 6.3±0.1 1.134±0.004 1.790±0.007 5.8±0.05 4.57±0.05 7.14±0.03
120 5.5±0.3 1.285±0.002 1.783±0.003 4.117±0.010 3.949±0.001 6.28±0.06
144 3.2±0.1 1.305±0.003 1.83±0.05 3.116±0.004 3.461±0.004 4.96±0.03
168 5.85±0.05 1.335±0.004 1.8±0.3 3.985±0.005 3.127±0.005 4.6±0.5
192 6±0.2 1.413±0.005 1.721±0.004 2.863±0.02 2.23±0.02 3.0±0.3
216 6.1±0.1 1.449±0.008 1.714±0.001 1.982±0.03 1.56±0.03 2.2±0.1
240 5.9±0.3 1.510+0.004 1.68±0.03 0.869±0.002 0.397±0.002 1.8±0.2
117
Table-5.60: P. lilacinum was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.1±0.1 1.085±0.003 1.737±0.005 11.76±0.03 9.94±0.01 7.985±0.001
48 4.3±0.2 1.11±0.01 1.84±0.03 11.2±0.1 8.76±0.06 8.83±0.05
72 4.5±0.2 1.135±0.004 1.816±0.003 9.85±0.01 8.36±0.06 9.87±0.1
96 4.35±0.05 1.281±0.003 1.874±0.001 8.931±0.004 8.13±0.03 7.91±0.04
120 4.75±0.03 1.209±0.003 1.784±0.002 6.69±0.04 6.35±0.01 7.57±0.04
144 5.2±0.2 1.295±0.002 1.775±0.006 5.58±0.01 5.143±0.003 6.13±0.02
168 5.0±0.3 1.355±0.001 1.767±0.006 3.67±0.04 3.19±0.02 4.33±0.02
192 4.8±0.5 1.395±0.003 1.728±0.006 2.41±0.03 2.12±0.03 3.19±0.04
216 4.65±0.01 1.485±0.001 1.633±0.007 1.40±0.06 1.18±0.03 2.85±0.05
240 4.38±0.03 1.548±0.004 1.612±0.003 0.831±0.002 0.65±0.04 1.16±0.02
Table-5.61 shows the results of pectinase synthesis by different filamentous fungi
like A. fumigatus, (A. niger + A. fumigatus ), A. niger, M. geophillus and P.lilacinum
when grown on 2.5% and 5% of CCP (commercial citrus pectin) incorporated
with mineral medium as carbon source. The highest Pectinase activity and
growth were obtained in comparison to other organisms by A. niger 10.06 U/ml
and 1.249 g/50 ml and P. lilacinum 9.87 U/ml and 1.135 g/50 ml respectively.
The results are in agreement with Akhilesh et al., (2010) and Favela-Torres et al.,
(2006) indicate that pectin can induce the polygalacturonase activity.
118
Table-5.61:Effect on growth and pectinase production by different fungi when grown on mineral medium supplemeted with 2.5 and 5% CCP (commercial
citrus pectin) at 30 + 2 ºC and pH was adjusted at 6.5. Filamentous Fungi
Biomass g/50ml Broth
Pectinase Activity U/ml
Biomass g/ 50 ml Broth
Pectinase Activity U/ml
2.5% Synthetic pectin 5% Synthetic pectin
A. fumigatus 1.154±0.003 6.47±0.004 1.233±0.005 8.9±0.2
A. niger+ A. fumigatus
1.16±0.03 6.12±0.001 1.198±0.005 8.76±0.03
A. niger 1.182±0.004 7.36±0.002 1.249±0.006 10.06±0.01
M. geophillus 1.367±0.001 6.27±0.002 1.213±0.002 8.92±0.01
P. lilacinum 1.637±0.006 7.142±0.0001 1.135±0.005 9.87±0.1
v- Effect of sugars as carbon sources:
In concern with the use of molasses which is an agro-industrial
byproduct in the present study suggested high pectinase production by
Aspergillus niger and Penicillium lilacinum with molasses as sole carbon source. It
is economic to use molasses for the production of pectinase as compared to pure
pectin. In Pakistan molasses is a byproduct of sugar industry and it is used to
produce ethanol; however it can be successfully used in enzyme production. Its
careless dumping in nature can causes environmental pollution, hence it can be
eco-friendly used as a good substrate for enzyme production. After selection of
molasses as a carbon source different sugars were added to molasses to
investigate their effect as a carbon source with selected microorganisms i-e A.
niger and P. lilacinum
119
Fig # 5.10 Comparison of pectinase production by different organisms grown on
5 % Molasses as a carbon source.
Tables -5.63 and 5.64 show the results of pectinase synthesis by A. niger when
grown on mineral medium containing (2.5 % and 5%) fructose and 5% molasses
as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. The maximum
production of pectinase 12.00 U/ml and 13.89 U/ ml respectively, were obtained
at 72 hours. The concentration of total sugars, reducing sugars and total protein
was decreased with the increase of the fermentation time period. Our results are
supported by previous work as reported that polygalacturonase production in
Geotrichum candidum (Shastri et at., 1988).
Tables-5.64 and 5.65 represents the production of pectinase by P. lilacinum
grown on mineral medium containing (2.5% and 5%) fructose and 5% molasses
incubated at 30 ± 2 ºC and the pH was adjusted at 6.5. The higher amount of
pectinase was produced by P. lilacinum 10.73 U/ml and 12.11 U/ml respectively.
pectinase production by A. niger, A. alliaceus, Geotrichum lactis, Neurospora crassa,
induced by fructose ( McKay, 1988; Pardo et al., 1991).
0
2
4
6
8
10
12
Pect
ina
se A
ctiv
ity
U/
ml
120
Table-5.62: A. niger was grown on a mineral medium supplemented with 2.5%
fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar (mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
U/ml
24 5.3±0.2 0.879±0.008 1.591±0.006 14.154±0.003 13.078±0.003 5.53±0.01
48 5±0.2 0.879±0.001 1.599±0.003 12.463±0.003 11.197±0.004 6.96±0.01
72 4.85±0.05 0.897±0.005 1.607±0.003 10.128±0.003 9.625±0.005 12.00±0.2
96 4.35±0.02 0.996±0.001 1.686±0.003 8.783±0.002 5.513±0.002 7.17±0.04
120 4.1±0.1 1.137±0.004 1.612±0.004 5.0375±0.0006 3.202±0.001 5.96±0.03
144 4.35±0.06 1.269±0.004 1.599±0.005 3.098±0.005 2.918±0.005 4.53±0.01
168 4.45±0.03 1.365±0.007 1.573±0.003 1.987±0.001 1.825±0.002 3.48±0.04
192 4.5±0.2 1.452±0.005 1.567±0.002 1.882±0.004 1.862±0.001 3.06±0.04
216 4.5±0.4 1.464±0.001 1.556±0.002 1.339±0.002 1.214±0.003 2.95±0.02
240 4.7±0.2 1.517±0.004 1.544±0.001 1.196±0.005 1.059±0.002 1.62±0.01
Table-5.63: A. niger was grown on a mineral medium supplemented with 5%
fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity U/ml
24 5.2±0.1 0.875±0.002 1.768±0.004 17.997±0.001 15.169±0.004 6.05±0.04
48 4.45±0.02 0.937±0.003 1.688±0.003 12.978±0.006 9.919±0.002 6.59±0.06
72 4.7±0.1 1.192±0.005 1.726±0.005 9.988±0.005 7.251±0.001 13.89±0.04
96 4.7±0.3 1.115±0.009 1.687±0.004 7.842±0.001 6.159±0.002 5.87±0.06
120 5.45±0.05 1.138±0.003 1.677±0.001 6.580±0.004 5.079±0.002 4.67±0.04
144 5.75±0.07 2.285±0.002 1.638±0.002 4.782±0.003 3.487±0.004 4.13±0.02
168 6.35±0.02 1.318±0.003 1.616±0.003 4.017±0.004 3.102±0.001 3.72±0.02
192 6.4±0.2 1.367±0.004 1.524±0.004 3.603±0.001 2.276±0.005 2. 05±0.03
216 6.65±0.03 1.466±0.001 1.513±0.002 2.17±0.04 1.395±0.006 1.28±0.03
240 6.9±0.1 1.592±0.002 1.484±0.001 1.037±0.006 0. 952±0.005 1.16±0.03
121
Table-5.64: P. lilacinum was grown on a mineral medium supplemented with 2.5% fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ ml)
Pectinase
Activity (U/ ml)
24 5.15±0.02 1.025±0.004 1.47±0.0005 16.899±0.006 14.165±0.0002 6.14±0.03
48 5.0±0.1 1.317±0.004 1.598±0.001 13.395±0.005 11.45±0.001 8.52±0.02
72 4.85±0.02 1.365±0.002 1.706±0.003 9.501±0.001 7.083±0.005 10.73±0.02
96 4.2±0.1 1.421±0.004 1.741±0.003 8.894±0.003 6.385±0.0003 7.03±0.02
120 4.15±0.01 1.592±0.006 1.755±0.003 7.825±0.002 5.67±0.05 6.32±0.04
144 4.3±0.4 1.87±0.04 1.76±0.0003 5.778±0.002 4.326±0.001 5.87±0.04
168 4.4±0.1 2.196±0.004 1.799±0.006 3.351±0.003 2.81±0.003 5.22±0.05
192 4.45±0.07 2.24±0.01 1.816±0.003 2.259±0.004 1.83±0.003 3.85±0.01
216 4.6±0.3 2.48±0.02 1.803±0.002 1.627±0.005 1.50±0.001 2.29±0.04
240 5.8±0.1 2.512±0.001 1.76±0.04 1.076±0.002 0.917±0.001 2.02±0.01
Table-5.65: P. lilacinum was grown on a mineral medium supplemented with 5%
fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.7±0.3 1.015±0.004 1.566±0.003 17.458±0.003 1.623±0.002 4.94±0.01
48 4.8±0.3 1.225±0.002 1.629±0.004 15.842±0.002 12.766±0.003 5.94±0.07
72 5.75±0.02 1.339±0.002 1.723±0.002 13.541±0.004 11.528±0.003 12.11±0.06
96 5.2±0.1 1.371±0.003 1.784±0.002 9.875±0.002 8.176±0.001 8.58±0.02
120 5.8±0.3 1.386±0.003 1.684±0.003 7.634±0.003 6.943±0.002 7.85±0.04
144 6.05±0.03 1.391±0.004 1.678±0.002 5.917±0.002 3.395±0.002 5.74±0.05
168 6.15±0.04 1.403±0.002 1.615±0.002 3.257±0.002 3.146±0.002 4.87±0.04
192 6.3±0.2 1.434±0.003 1.607±0.002 3.171±0.003 2.68±0.03 3.04±0.03
216 5.85±0.06 1.497±0.004 1.598±0.005 1.998±0.007 1.518±0.005 3.00±0.2
240 6.15±0.02 1.529±0.004 1.591±0.001 1.319±0.002 0.792±0.002 1.62±0.05
122
Tables–5.66 to 5.69 show the results of pectinase synthesis by A. niger and P.
lilacinum were grown on mineral medium containing 2.5% and 5% maltose and
5% molasses as carbon source at 30 ± 2 ºC and the pH was adjusted at 6.5.The
maximum production of pectinase achieved 12.00 U/ml and 12.75U/ml by A.
niger and 11.04 U/ml and 11.20 U/ml by P. lilacinum respectively. Pectinase
production increased with fermentation duration up to 72 hours beyond that, the
production of the enzymes decreased gradually may be due to the depletion of
nutrients. Singh and Mandal (2012) reported that fermentation time period
depends on the growth rate of the microorganism and its pattern of enzyme
synthesis.
Table-5.66: A.niger was grown on mineral medium supplemented with 2.5% maltose and 5% molasses as carbon source at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.7±0.2 1.129±0.003 1.647±0.004 17.514±0.003 14.179±0.003 6.30±0.07
48 4.6±0.4 1.285±0.005 1.701±0.001 15.298±0.003 13.465±0.002 7.15±0.04
72 4.5±0.2 1.31±0.04 1.739±0.002 12.743±0.002 9.158±0.004 12.00±0.5
96 4.35±0.02 1.425±0.001 1.743±0.002 10.151±0.003 8.916±0.003 8.31±0.02
120 4.4±0.3 1.525±0.004 1.751±0.003 7.033±0.002 5.958±0.003 5.32±0.06
144 4.5±0.2 1.58±0.04 1.774±0.001 6.913±0.002 5.165±0.004 4.85±0.02
168 4.8±0.3 1.625±0.002 1.771±0.003 5.952±0.004 4.179±0.006 4.40±0.03
192 5.05±0.03 1.63±0.05 1.759±0.002 4.138±0.001 3.509±0.004 3.44±0.01
216 5.25±0.04 1.644±0.003 1.717±0.003 2.496±0.003 1.692±0.001 2.31±0.04
240 5.5±0.1 1.753±0.003 1.696±0.003 1.312±0.001 0.866±0.003 1 .13±0.02
123
Table-5.67: A.niger was grown on mineral medium supplemented with 5% maltose and 5% molasses as carbon source at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.4±0.3 1.098±0.005 1.622±0.005 19.482±0.006 14.795±0.001 7.32±0.01
48 5.05±0.02 1.206±0.0004 1.695±0.002 16.355±0.004 13.215±0.007 8.20±0.03
72 4.85±0.01 1.409±0.004 1.791±0.006 12.734±0.001 10.198±0.003 12.75±0.03
96 4.4±0.3 1.568±0.0003 1.786±0.003 11.706±0.004 9.165±0.001 10.21±0.03
120 3.85±0.06 1.568±0.0003 1.769±0.002 8.761±0.0004 6.169±0.007 7.20±0.07
144 3.55±0.02 1.626±0.0007 1.765±0.001 6.656±0.003 5.417±0.004 7.02±0.01
168 3.41±0.04 1.631±0.004 1.75±0.04 4.927±0.0003 3.242±0.002 6.90±0.04
192 3.5±0.4 1.656±0.003 1.851±0.003 3. 615±0.002 2.269±0.004 4.18±0.03
216 3.6±0.2 1.639±0.003 1.825±0.002 1.869±0.004 1.197±0.004 2.92±0.01
240 3.9±0.4 1.617±0.004 1.806±0.001 1.476±0.002 0.875±0.001 2.01±0.01
Table-5.68: P. lilacinum was grown on mineral medium supplemented with 2.5%
maltose and 5% molasses at 30 ± 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml Broth
Total Proteins (mg/ml)
Total Sugar (mg/ml)
Reducing Sugars (mg/ml)
Pectinase Activity (U/ml)
24 4.90±0.04 1.105±0.004 1.10±0.03 17.618±0.003 13.439±0.003 5.44±0.03
48 4.88±0.03 1.162±0.002 1.11±0.07 15.429±0.006 13.534±0.003 6.85±0.07
72 4.82±0.04 1.205±0.001 1.125±0.004 12.858±0.002 9.180±0.0003 11.04±0.02
96 4.74±0.01 1.273±0.001 1.143±0.005 10.995±0.002 7.034±0.0005 8.07±0.02
120 4.45±0.05 1.336±0.005 1.164±0.001 8.935±0.004 5.454±0.0002 5.427±0.006
144 4.4±0.2 1.355±0.003 1.174±0.006 7.448±0.004 5.240±0.0002 5.17±0.04
168 4.55±0.07 1.425±0.002 1.181±0.005 5.216±0.003 3.816±0.003 5.91±0.04
192 4.85±0.02 1.483±0.005 1.185±0.002 4.737±0.002 2.448±0.001 4.47±0.04
216 5.05±0.03 1.498±0.005 1.182±0.006 1.447±0.004 1.065±0.003 2.83±0.04
240 5.20±0.03 1.561±0.005 1.18±0.03 0.869±0.006 0.703±0.002 1.13±0.02
124
Table-5.69: P. lilacinum was grown on mineral medium supplemented with 5% maltose and 5% molasses as carbon source at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final
pH
Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.6±0.1 1.11±0.05 1.30±0.06 23.38±0.06 22.83±0.01 6.02±0.01
48 4.5±0.4 1.12±0.01 1.28±0.07 21.05±0.03 18.02±0.01 7.47±0.04
72 4.2±0.1 1.125±0.002 1.29±0.02 17.70±0.04 16.76±0.03 11.20±0.07
96 3.85±0.03 1.13±0.08 1.29±0.04 13.61±0.04 12.72±0.01 8.12±0.03
120 3.65±0.01 1.18±0.02 1.29±0.07 11.47±0.06 9.93±0.05 7.32±0.01
144 3.25±0.04 1.36±0.05 1.30±0.01 9.30±0.02 8.86±0.04 6.51±0.05
168 4.15±0.02 1.44±0.04 1.30±0.04 6.54±0.03 5.457±0.005 5.11±0.06
192 4.45±0.06 1.47±0.04 1.31±0.06 5.17±0.04 4.70±0.03 3.68±0.05
216 4.5±0.2 1.53±0.02 1.35±0.02 3.86±0.04 3.44±0.02 2.64±0.02
240 4.15±0.04 1.55±0.01 1.40±0.03 2.47±0.02 2.12±0.01 1.27±0.03
Tables-5.70 to 5.73 show the results of pectinase synthesis by A. niger and
P. lilacinum when grown on mineral medium containing 2.5% and 5% sucrose
and 5% molasses as carbon source at 30 ± 2 ºC and the pH was adjusted at 6.5.
The maximum production of pectinase was achieved 13.66 U/ml and 16.16 U/ml
by A.niger but 12.29 U/ml and 15.54 U/ml by P.lilacinum respectively after
incubation of 72 hours. By A. niger and then its concentration was decreased
with the increase of the time period. The present study is in full agreement with
Tariq and Reyaz ( 2012), Reda et al., (2008) and Solis-Pereyra et al., (1993), who
reported that the presence of sucrose increases pectinase production. Similar
results were presented by Hoa and Hung (2013).
125
Table-5.70: A.niger was grown on mineral medium supplemented with 2.5 % sucrose and 5 % molasses as carbon source at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.40±0.04 0.93±0.04 1.56±0.05 17.24±0.01 10.47±0.04 7.82±0.04
48 4.95±0.04 1.05±0.03 1.62±0.04 15.80±0.04 10.41±0.04 8.04±0.02
72 4.45±0.01 1.18±0.05 1.62±0.02 12.32±0.02 9.25±0.04 13.66±0.03
96 4.35±0.01 1.29±0.03 1.62±0.07 9.54±0.03 7.27±0.04 7.39±0.04
120 3.75±0.06 1.24±0.01 1.63±0.02 8.18±0.03 5.93±0.06 7.30±0.08
144 3.85±0.03 1.35±0.04 1.62±0.04 5.36±0.05 3.99±0.04 6.17±0.04
168 3.65±0.01 1.36±0.02 1.58±0.01 4.79±0.03 3.12±0.02 5.95±0.01
192 3.45±0.02 1.48±0.06 1.55±0.07 3.84±0.06 2.16±0.03 3.40±0.04
216 3.15±0.07 1.55±0.02 1.48±0.04 1.98±0.03 1.13±0.02 2.13±0.01
240 3.10±0.06 1.58±0.08 1.48±0.06 1.02±0.01 0.87±0.01 1.23±0.02
Table-5.71: A.niger was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 3.75±0.04 0.84±0.01 1.46±0.03 20.73±0.01 12.33±0.05 6.54±0.03
48 4.05±0.02 1.14±0.03 1.51±0.04 18.23±0.03 11.97±0.06 8.22±0.05
72 3.5±0.3 1.25±0.07 1.58±0.03 15.15±0.02 11.49±0.01 16.16±0.03
96 3.25±0.08 1.30±0.04 1.64±0.01 12.84±0.02 8.07±0.02 10.56±0.04
120 2.85±0.07 1.32±0.06 1.62±0.05 9.50±0.03 6.19±0.04 7.04±0.03
144 4.15±0.02 1.33±0.1 1.57±0.02 7.25±0.02 4.70±0.07 6.29±0.01
168 3.85±0.02 1.48±0.03 1.53±0.01 6.85±0.04 4.23±0.01 5.13±0.08
192 3.75±0.01 1.55±0.02 1.53±0.06 4.53±0.04 3.09±0.04 3.93±0.06
216 3.7±0.2 1.59±0.02 1.52±0.01 3.26±0.04 2.81±0.05 1.27±0.04
240 3.95±0.04 1.61±0.01 1.51±0.07 1.28±0.01 0.73±0.02 1.18±0.03
126
Table-5.72: P. lilacinum was grown on mineral medium supplemented with 2.5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Protein
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.8±0.3 1.00±0.3 1.20±0.05 17.91±0.04 11.30±0.05 7.61±0.04
48 5.4±0.2 1.18±0.05 1.58±0.06 16.12±0.02 10.99±0.08 8.04±0.02
72 4.85±0.04 1.23±0.01 1.68±0.03 14.94±0.03 9.36±0.05 12.20±0.07
96 4.75±0.07 1.32±0.05 1.67±0.01 12.28±0.03 8.96±0.03 7.04±0.03
120 4.15±0.02 1.46±0.03 1.57±0.04 9.09±0.03 6.66±0.04 6.15±0.06
144 4.45±0.01 1.49±0.04 1.55±0.01 7.48±0.05 4.07±0.02 5.64±0.02
168 4.75±0.06 1.51±0.02 1.55±0.06 5.23±0.06 5.15±0.02 5.43±0.02
192 5.05±0.02 1.52±0.01 1.53±0.02 4.75±0.04 3.56±0.02 2.93±0.05
216 5.25±0.04 1.53±0.06 1.52±0.02 2.92±0.06 1.53±0.05 2.35±0.03
240 5.35±0.02 1.55±0.02 1.44±0.02 1.47±0.05 1.02±0.01 1.74±0.01
Table-5.73: P. lilacinum grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml Broth
Total Protein (mg/ml)
Total Sugar (mg/ml)
Reducing Sugars (mg/ml)
Pectinase Activity (U/ml)
24 4.8±0.2 0.94±0.07 1.65±0.03 28.87±0.05 13.67±0.04 6.97±0.05
48 4.15±0.02 1.03±0.02 1.67±0.02 22.14±0.03 21.87±0.02 8.90±0.07
72 3.85±0.07 1.30±0.05 1.68±0.04 16.15±0.06 15.08±0.04 15.54±0.04
96 3.65±0.01 1.32±0.01 1.68±0.07 12.62±0.05 11.53±0.05 9.80±0.02
120 3.5±0.1 1.34±0.08 1.69±0.01 10.31±0.02 9.55±0.02 7.63±0.01
144 3.6±0.3 1.38±0.03 1.63±0.03 8.88±0.06 8.59±0.03 6.56±0.03
168 3.75±0.01 1.54±0.06 1.61±0.03 6.42±0.03 6.41±0.05 5.63±0.06
192 3.95±0.02 1.56±0.04 1.58±0.06 3.89±0.03 2.97±0.04 2.06±0.04
216 4.3±0.1 1.57±0.06 1.57±0.04 3.15±0.02 2.62±0.01 1.46±0.04
240 4.15±0.07 1.58±0.02 1.53±0.05 1.59±0.03 1.07±0.02 1.19±0.04
The pH values fluctuate during fermentation and remain acidic. Pectinase synthesis
by A.niger when grown on mineral medium containing 2.5% and 5% galactose and
5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. Tables -5.74
and 5.75 clearly show that when A.niger was grown on above described cultural
conditions at 72 h incubation showed the maximum pectinase activity 12.22 and
127
13.98 U/ml respectively. Both tables -5.76 and 5.77 show more or less same trend for
pectinase production by P.lilacinum in the presence of the same substrate. Galactose
induced pectinase production in Bacteroides ovatus rather than pectin as reported by
Macfarlane et al., (1990). The observation indicates the constitutive nature of the
enzymes and also the stimulating capacity of carbon source for the production of
pectinase enzymes, and also varies from strain to strain (Ramachandran and Kurup,
2013).
Table-5.74: A. niger was grown on mineral medium supplemented with 2.5% galactose and 5% molasses as carbon source at 30 ± 2 ºC and
pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml Broth
Total Proteins (mg/ml)
Total Sugar (mg/ml)
Reducing Sugars (mg/ml)
Pectinase Activity (U/ml)
24 4.35±0.06 0.98±0.05 1.61±0.03 16.13±0.02 13.26±0.05 6.19±0.06
48 4.2±0.01 0.99±0.04 1.49±0.03 15.77±0.04 12.75±0.02 6.95±0.03
72 3.65±0.01 1.11±0.05 1.47±0.04 13.52±0.04 10.90±0.05 12.22±0.07
96 3.85±0.03 1.12±0.03 1.44±0.03 8.19±0.02 6.41±0.04 9.19±0.05
120 3.65±0.02 1.13±0.06 1.46±0.02 5.96±0.04 4.33±0.02 8.27±0.04
144 3.45±0.02 1.14±0.03 1.44±0.01 4.84±0.05 3.24±0.01 6.24±0.03
168 3.15±0.04 1.18±0.03 1.43±0.05 4.10±0.05 3.04±0.01 4.08±0.06
192 2.8±0.2 1.18±0.03 1.42±0.04 3.33±0.01 2.38±0.04 2.82±0.04
216 3.2±0.2 1.20±0.07 1.40±0.05 2.86±0.01 2.02±0.01 2.66±0.01
240 3.6±0.3 1.23±0.02 1.36±0.03 1.38±0.07 0.65±0.02 1.84±0.04
Table-5.75: A niger was grown on mineral medium supplemented with 5% galactose
and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.00±0.3 1.08±0.05 1.57±0.02 17.95±0.02 14.69±0.04 4.167±0.004
48 5.25±0.02 1.12±0.01 1.72±0.02 14.95±0.04 11.196±0.003 8.69±0.02
72 5.05±0.01 1.14±0.05 1.31±0.05 11.78±0.03 8.95±0.04 13.98±0.05
96 4.9±0.4 1.21±0.06 1.36±0.04 10.93±0.04 7.94±0.04 9.92±0.03
120 2.95±0.02 1.26±0.04 1.43±0.04 9.24±0.04 6.72±0.05 8.93±0.06
144 2.65±0.07 1.27±0.04 1.44±0.02 7.73±0.01 5.20±0.05 6.52±0.02
168 3.4±0.1 1.36±0.01 1.45±0.05 5.01±0.01 3.93±0.02 5.07±0.01
192 3.65±0.03 1.39±0.08 1.51±0.03 4.28±0.05 2.92±0.06 2.39±0.08
216 6.1±0.1 1.47±0.04 1.10±0.06 2.44±0.03 2.02±0.01 2.34±0.01
240 6.2±0.1 1.47±0.01 1.43±0.06 1.18±0.03 0.83±0.05 1.13±0.02
128
Table-5.76: P. lilacinum was grown on mineral medium supplemented with 2.5% galactose and 5% molasses as carbon source at 30 ± 2 ºC and
pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.00±0.3 0.976±0.003 1.21±0.08 17.19±0.02 13.68±0.03 5.51±0.03
48 4.45±0.01 0.99±0.08 1.27±0.02 16.94±0.03 13.48±0.04 8.64±0.03
72 4.15±0.02 1.22±0.05 1.39±0.02 15.41±0.04 12.27±0.06 11.14±0.01
96 3.9±0.2 1.22±0.03 1.40±0.06 12.46±0.03 10.20±0.05 7.16±0.05
120 3.65±0.04 1.24±0.02 1.40±0.03 9.80±0.02 8.68±0.06 7.11±0.04
144 3.75±0.02 1.25±0.04 1.42±0.02 8.30±0.06 6.27±0.05 6.07±0.04
168 3.3±0.3 1.26±0.02 1.43±0.01 5.46±0.04 4.13±0.02 5.86±0.04
192 2.95±0.06 1.42±0.02 1.44±0.05 4.07±0.04 3.88±0.05 2.69±0.06
216 2.65±0.04 1.47±0.04 1.44±0.01 1.54±0.03 1.27±0.02 1.87±0.04
240 2.85±0.02 1.51±0.02 1.45±0.04 1.08±0.05 0.96±0.03 1.25±0.01
Table-5.77: P. lilacinum was grown on mineral medium supplemented with 5% galactose and 5% molasses as carbon source at 30 ± 2 ºC and
pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 4.70±0.06 0.89±0.07 1.47±0.04 18.38±0.01 15.14±0.03 6.85±0.03
48 4.75±0.02 1.20±0.07 1.49±0.04 16.15±0.01 13.29±0.04 9.66±0.03
72 3.15±0.02 1.22±0.01 1.41±0.04 13.28±0.06 11.06±0.03 12.13±0.02
96 2.95±0.05 1.38±0.03 1.46±0.02 12.84±0.04 10.45±0.04 7.25±0.01
120 2.70±0.03 1.43±0.03 1.45±0.03 10.80±0.09 7.57±0.01 5.52±0.04
144 2.8±0.2 1.49±0.04 1.44±0.03 9.94±0.01 6.88±0.03 5.51±0.08
168 4.30±0.01 1.51±0.08 1.32±0.05 8.34±0.04 5.88±0.07 4.16±0.03
192 4.15±0.02 1.57±0.02 1.30±0.07 5.70±0.07 4.57±0.04 3.24±0.03
216 5.10±0.07 1.60±0.02 1.28±0.03 3.36±0.04 2.19±0.02 2.47±0.01
240 5.25±0.02 1.63±0.02 1.24±0.02 1.47±0.06 1.11±0.05 2.31±0.04
129
Tables-5.78 -5.79 showed the result of A. niger grown on mineral medium
containing 2.5 % and 5% starch, 5% molasses at 30 ± 2 ºC and pH was adjusted at
6.5. Like other carbon sources when starch was used as a carbon source in above
defined media A. niger produces greater 12.85 U/ml and 15.64 U/ml quantity of
pectinase respectively at 72 h incubation. Total and reducing sugars were
decreased with the increase of the time period. The pH values continuously
changed during the fermentation period
Tables-5.80 and 5.81 shows the P. lilacinum growth pattern and enzyme
production on mineral medium containing 2.5% and 5% starch , 5% molasses at
30 ± 2 ºC and pH was adjusted at 6.5. It shows a maximum production of
pectinase 12.21 U/ml and 15.17 U/ml respectively, at 72 hours and then its
concentration was decreased with the increase of the time period. The
concentration of total and reducing sugars was decreased with the increase of
fermentation time period. Total protein concentration increased during
fermentation. The present study was supported by the work of Singh and
Mandal (2012) who reported that starch was identified as best carbon source for
the production of Pectinolytic enzymes by a mixed culture of Aspergillus species
like A. fumigatus and A. sydowii. It is also reported by Singh and Mandal (2012)
that with the increase in concentration of carbon source the decrease in enzyme
activity starts due to substrate inhibition and catabolic repression, probably due
to the presence of high concentration of galacturonic acid. These investigations
are also supported by Aguilar and Huitron, (1987) and Maldonado et al., (1989).
130
Table-5.78: A. niger was grown on mineral medium supplemented with 2.5% starch and 5% molasses as carbon source at 30 ± 2 ºC and
pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.00±0.3 0.81±0.04 1.61±0.03 18.97±0.05 10.90±0.02 5.23±0.01
48 5.25±0.02 0.83±0.02 1.62±0.02 14.30±0.04 10.13±0.03 8.07±0.02
72 5.90±0.02 0.89±0.06 1.78±0.07 11.35±0.04 8.12±0.05 12.85±0.04
96 5.00±0.1 0.89±0.04 1.79±0.03 9.93±0.03 6.51±0.05 9.65±0.02
120 4.05±0.02 1.17±0.06 1.87±0.05 6.74±0.01 5.32±0.04 7.38±0.06
144 3.90±0.04 1.22±0.03 1.89±0.04 5.64±0.06 3.92±0.02 6.96±0.03
168 3.65±0.02 1.25±0.03 1.65±0.02 3.99±0.06 2.22±0.01 5.41±0.04
192 3.85±0.04 1.27±0.04 1.62±0.03 2.88±0.06 1.76±0.04 4.14±0.03
216 4.25±0.03 1.29±0.02 1.55±0.01 2.34±0.03 1.63±0.02 2.95±0.01
240 4.60±0.01 1.32±0.04 1.54±0.03 2.00±0.3 1.08±0.03 1.71±0.03
Table-5.79: A .niger was grown on mineral medium supplemented with 5% starch and 5% molasses as carbon source at 30 ± 2 ºC and
pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
U/ml
24 5.2±0.1 1.04±0.03 1.76±0.04 21.13±0.05 12.98±0.08 6.89±0.04
48 4.45±0.02 1.19±0.06 1.85±0.04 19.15±0.04 12.16±0.03 9.89±0.07
72 4.7±0.2 1.22±0.04 1.92±0.05 17.69±0.04 11.65±0.03 15.64±0.03
96 4.7±0.3 1.25±0.03 1.95±0.02 14.75±0.04 9.161±0.003 10.24±0.01
120 5.45±0.05 1.28±0.02 1.98±0.06 11.98±0.05 9.246±0.003 8.43±0.04
144 5.75±0.02 1.31±0.01 1.97±0.02 7.98±0.01 5.88±0.04 7.64±0.02
168 6.35±0.03 1.33±0.01 1.90±0.04 4.60±0.04 3.30±0.05 5.80±0.04
192 6.4±0.4 1.37±0.06 1.80±0.03 2.86±0.04 2.13±0.02 4.65±0.02
216 6.55±0.01 1.39±0.04 1.71±0.03 2.29±0.02 1.98±0.01 2.67±0.06
240 8.50±0.02 1.40±0.07 1.69±0.04 1.95±0.01 0.82±0.03 2.54±0.01
131
Table-5.80: P. lilacinum was grown on mineral medium supplemented with 2.5% starch and 5% molasses as carbon source at 30 ± 2 ºC and
pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.95±0.01 0.89±0.02 1.39±0.08 21.9±0.2 18.99±0.04 6.02±0.01
48 5.15±0.02 0.72±0.04 1.48±0.04 16.60±0.02 14.91±0.04 8.14±0.03
72 4.85±0.03 0.97±0.04 1.54±0.02 12.53±0.02 11.98±0.05 12.21±0.05
96 4.3±0.2 0.99±0.04 1.55±0.01 9.89±0.04 8.88±0.06 7.23±0.01
120 4.45±0.04 1.15±0.02 1.58±0.08 6.823±0.002 5.07±0.02 6.09±0.01
144 4.6±0.1 1.30±0.05 1.77±0.04 3.770±0.004 3.43±0.03 5. 93±0.02
168 4.8±0.1 1.38±0.06 1.83±0.04 3.451±0.003 3.28±0.03 5.82±0.04
192 4.65±0.02 1.47±0.04 1.89±0.06 3.157±0.004 2.64±0.03 4.86±0.04
216 4.6±0.4 1.49±0.04 1.89±0.04 2.626±0.004 2.50±0.05 2.98±0.03
240 5.1±0.1 1.51±0.03 1.70±0.05 1.73±0.02 1.62±0.01 1.47±0.03
Table-5.81: P. lilacinum was grown on mineral medium supplemented with 5 %
starch, 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 4.40±0.03 0.907±0.003 1.52±0.05 21.58±0.03 15.19±0.02 6.32±0.05
48 4.95±0.02 0.922±0.003 1.60±0.05 17.26±0.03 14.59±0.06 7.70±0.05
72 5.65±0.02 0.935±0.004 1.66±0.03 16.54±0.03 14.33±0.02 15.17±0.04
96 5.40±0.01 0.97±0.04 1.69±0.02 12.77±0.04 9.17±0.06 9.65±0.03
120 5.65±0.03 1.038±0.006 1.72±0.02 10.57±0.02 7.43±0.02 8.72±0.03
144 6.05±0.01 1.255±0.002 1.79±0.05 9.77±0.02 6.93±0.05 6.27±0.04
168 6.15±0.04 1.278±0.006 1.83±0.01 5.72±0.04 3.62±0.02 5.80±0.04
192 6.30±0.03 1.295±0.001 1.82±0.03 5.18±0.05 3.30±0.04 4.74±0.01
216 5.85±0.02 1.305±0.002 1.80±0.03 2.68±0.03 2.13±0.03 2.25±0.03
240 6.15±0.02 1.355±0.003 1.82±0.04 1.68±0.01 1.06±0.02 1.76±0.05
Table-5.82 and 83 show the results of two different concentrations (2.5% and 5%)
of additional carbon sources incorporated in mineral medium to screen out the
best productivity and cost effective secondary carbon source. The secondary
132
carbon sources were used like fructose, maltose, sucrose, galactose and starch
and both selected organisms A. niger and P. lilacinum were grown on these
carbon sources. The results compiled in the Table-5.82 shown that most of the
carbon source when added in optimized medium, all of those have produced a
good amount of pectinase, but sucrose proved as a best inducer with highest
activity and growth 16.16 U/ml, while second most promising substrate was
starch with 15.64 U/ml. This induced effect of sucrose has been reported by
Hours et al., (1988) and different isoenzymes production was affected by the type
and concentration of the substrate present in the culture medium (Leone and Van
Den Heuvel, 1987). The present study is also in agreement with Phutela et al.,
(2005), Crotti et al.,(1998), Baracat-Pereira et al., (1994) and Minussi et al., (1996).
Table-5.82: Effect on growth and pectinase production by Aspergillus niger when grown on mineral medium supplementedwith 5% molasses and sugars (2.5%
and 5%) as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.
Carbon sources Biomass Pectinase Activity
% g/50 ml Broth U/ml
Fructose 2.5 0.897 ±0.004 12.00±0.3 5 1.192 ±0.004 13.89 ±0.03
Maltose 2.5 1.339 ±0.006 12.00±0.2 5 1.31 ±0.05 12.75 ±0.03 Sucrose 2.5 1.18 ±0.03 13.66±0.02
5 1.25 ±0.04 16.16±0.03 Galactose 2.5 1.11 ±0.03 12.22±0.02
5 1.14 ±0.02 13.98±0.03 Starch 2.5 0.89 ±0.03 12.85±0.02
5 1.22 ±0.01 15.64±0.03
133
Table-5.83: Effect on growth and pectinase production by Penecillium lilacinum grown on mineral medium supplemented with 5% molasses and sugars (2.5%
and 5%) as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.
Carbon sources Biomass Pectinase Activity
% g/50 ml Broth U/ml
Frutose 2.5 1.365± 0.006 10.73±0.04 5 1.339 ±0.003 12.0±0.1
Maltose 2.5 1.205±0.002 11.04±0.02 5 1.125± 0.002 11.2±0.1
Sucrose 2.5 1.23±0.03 12.178±0.005 5 1.30±0.03 15.54 ±0.03 Galactose 2.5 1.22±0.01 11.14±0.01
5 1.22±0.04 12.13±0.03 Starch 2.5 0.97±0.01 12.21±0.04
5 0.935±0.002 15.17 ±0.03
vi- Effect of nitrogen source:
Nitrogen source is an important nutrient in fermentation medium and
has a significant role in the growth, development and production of metabolites
by microorganisms. Most of the industrially useful microbes are grown on
organic and inorganic sources, (Hunter, 1972).
Nutritional Factors such as carbon and nitrogen have always been of great
attention to the investigators to design cost effective media in the enzyme
production. About 30–40% of the manufacturing cost of industrial enzymes are
predictable as the cost of growth medium. Therefore, it is of enormous worth to
optimize the conditions for low cost enzyme production (Palaniyappan et al.,
2009). Nonetheless, a study on the impact of carbon and nitrogen sources had
shown that not all carbon and nitrogen supplements would proceed as boosters
for the concurrent production of enzymes in a single fermentation system (Negi
and Banerjee, 2010).
Organic nitrogen may be used as amino acids, proteins and peptone and urea
where as inorganic nitrogen source used like ammonium sulphate, potassium
nitrate, sodium nitrate etc. In this study ammonium sulphate was used as
control in culture media to grow fungi and best results were achieved with
134
inorganic nitrogen source which is in contrast with observations of Narasimha et
al., (2006) who reported that organic nitrogen sources maintained the superior
growth of fungi more than the nitrogen from inorganic sources. Vahidi et al.,
(2004) also reported same statement that good growth was achieved by using
complex nitrogen sources like yeast extract, peptone as compared to inorganic
nitrogen sources. Addition of yeast extract in media increase pectic lyase
synthesis (Phutela et al., 2005). Fadel ( 2000 ) has reported that irrespective of
substrate used the amount of enzyme production is influenced due to nitrogen
source as well as other supplements used in the media. The source of nitrogen in
a culture medium plays a substantial role for the growth of fungi and production
of enzyme (Juwon and Emmanuel, 2012).
Tables 5.84 to 5.87 showed the results of A. niger and P. lilacinum grown
on optimized mineral medium containing 0.2% and 0.4% corn steep liquor, the
maximum production of pectinase was achieved 23.29 U/ml and 23.23 U/ml
respectively.
Table-5.84: A niger was grown on mineral medium supplemented with 5%
sucrose and 5% molasses as carbon source and 0.2% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml Broth
Total Proteins (mg/ml)
Total Sugar (mg/ml)
Reducing Sugars (mg/ml)
Pectinase Activity (U/ml)
24 6.10±0.03 1.05±0.02 1.45±0.02 23.92±0.03 17.57±0.05 7.2±0.1
48 5.85±0.02 1.17±0.04 1.58±0.03 20.63±0.05 15.20±0.04 8.97±0.04
72 6.20±0.03 1.27±0.04 1.58±0.07 18.15±0.04 14.28±0.05 21.19±0.06
96 6.10±0.01 1.31±0.01 1.67±0.04 15.86±0.04 11.53±0.03 11.86±0.04
120 5.75±0.02 1.36±0.01 1.69±0.02 13.73±0.02 9.25±0.03 9.87±0.02
144 5.25±0.03 1.38±0.07 1.70±0.01 9.88±0.03 6.59±0.06 6.90±0.01
168 5.55±0.02 1.39±0.02 1.74±0.01 5.41±0.03 3.31±0.03 4.68±0.03
192 5.70±0.01 1.41±0.02 1.79±0.03 3.13±0.02 2.33±0.03 3.66±0.05
216 5.15±0.01 1.41±0.01 1.78±0.03 2.29±0.04 1.62±0.05 2.45±0.02
240 5.35±0.02 1.43±0.05 1.70±0.03 1.70±0.04 0.67±0.05 1.87±0.01
135
Table-5.85: A. niger was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source and 0.4% corn steep
liquor at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.15±0.01 0.98±0.05 1.62±0.04 22.73±0.02 16.23±0.05 8.73±0.03
48 6.05±0.04 1.17±0.04 1.71±0.02 19.13±0.02 14.75±0.02 9.81±0.03
72 5.55±0.03 1.20±0.03 1.75±0.02 14.94±0.02 11.49±0.04 23.39±0.02
96 5.25±0.02 1.21±0.06 1.77±0.02 13.93±0.04 11.07±0.04 13.56±0.04
120 4.85±0.02 1.28±0.06 1.79±0.07 9.50±0.07 6.10±0.02 8.04±0.01
144 5.15±0.03 1.30±0.02 1.82±0.02 8.58±0.03 6.98±0.05 6.29±0.04
168 5.35±0.03 1.33±0.02 1.84±0.03 6.35±0.03 4.23±0.01 5.13±0.02
192 4.90±0.03 1.35±0.02 1.88±0.03 4.21±0.04 3.89±0.06 4.94±0.01
216 4.70±0.02 1.44±0.04 1.79±0.06 1.98±0.05 1.21±0.06 2.27±0.03
240 3.95±0.02 1.47±0.02 1.79±0.02 1.31±0.03 0.98±0.05 1.18±0.03
Table 5.86: P. lilacinum was grown on mineral medium supplemented with 5%
sucrose and 5% molasses as carbon source and 0.2% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugar (mg/ml)
Pectinase
Activity (U/ml)
24 5.75±0.02 1.21±0.04 1.52±0.04 22.73±0.05 15.34±0.03 6.935±0.004
48 5.65±0.03 1.24±0.01 1.57±0.04 20.23±0.03 15.25±0.03 9.217±0.004
72 5.5±0.1 1.30±0.04 1.65±0.03 19.95±0.03 13.99±0.06 21.172±0.004
96 5.25±0.03 1.36±0.04 1.66±0.03 14.83±0.05 11.67±0.02 13.264±0.002
120 4.85±0.02 1.38±0.05 1.72±0.02 9.99±0.04 6.90±0.07 8.039±0.002
144 4.15±0.01 1.39±0.02 1.74±0.03 8.35±0.02 6.29±0.07 7.294±0.004
168 3.95±0.03 1.42±0.03 1.78±0.03 6.25±0.02 4.23±0.06 4.126±0.004
192 4.75±0.02 1.45±0.02 1.83±0.05 4.21±0.04 3.19±0.06 3.428±0.003
216 4.40±0.02 1.56±0.04 1.89±0.06 3.33±0.01 1.81±0.03 2.27±0.01
240 3.90±0.01 1.67±0.06 1.89±0.04 2.07±0.04 1.12±0.03 1.18±0.04
136
Table-5.87: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% corn steep liquor at 30 ±2 ºC and
pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugar (mg/ml)
Pectinase
Activity (U/ml)
24 5.55±0.01 0.94±0.03 1.55±0.02 23.69±0.07 16.167±0.005 7.97±0.02
48 5.75±0.02 1.23±0.03 1.57±0.05 20.74±0.03 15.27±0.02 9.91±0.02
72 5.85±0.02 1.30±0.04 1.58±0.03 17.95±0.02 12.68±0.05 23.23±0.04
96 5.65±0.03 1.37±0.04 1.58±0.01 14.55±0.04 10.23±0.05 12.83±0.02
120 4.5±0.1 1.38±0.03 1.63±0.04 12.13±0.03 8.95±0.02 7.27±0.05
144 4.6±0.4 1.41±0.04 1.66±0.03 11.88±0.06 7.99±0.01 6.17±0.04
168 4.75±0.02 1.54±0.02 1.68±0.03 9.41±0.03 6.92±0.02 5.84±0.04
192 4.95±0.03 1.56±0.01 1.72±0.03 6.13±0.08 4.37±0.04 3.91±0.06
216 4.3±0.3 1.59±0.01 1.74±0.01 3.97±0.05 2.62±0.01 2.89±0.06
240 4.1±0.2 1.62±0.03 1.75±0.05 1.82±0.02 1.07±0.02 1.86±0.04
Urea was used as nitrogen source more or less same the result 22.16 U/ml
and 23.94 U/ml respectively obtained when A.niger and P. lilacinum grown on
0.4% urea at 72 hours as shown in Tables 5.88 to 5.91. This type of shorter
incubation time period can be advantageous for industrial production. Said et al.,
(1991) have reported maximum pectinase production by Penicillium frequentans
after 48 h in media containing urea and trace elements Rashmi et al., (2008) and
Neeta et el., (2011) reported that urea was a good nitrogen source enhancing
pectinase production after peptone. Hoa and Hung (2013) reported urea as a best
nitrogen source for the production of pectinase by A. oryzae .
137
Table-5.88: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses as carbon source and 0.2% urea at 30 ± 2 ºC and
pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.05±0.02 1.11±0.05 1.57±0.02 20.38±0.04 14.51±0.03 6.70±0.02
48 5.55±0.03 1.13±0.04 1.62±0.02 17.87±0.04 13.25±0.02 8.94±0.04
72 5.1±0.1 1.16±0.01 1.63±0.06 14.56±0.01 11.36±0.04 21.29±0.02
96 5.05±0.01 1.18±0.05 1.67±0.04 13.96±0.03 11.14±0.03 11.54±0.03
120 5.25±0.02 1.21±0.03 1.68±0.06 9.52±0.02 7.21±0.02 7.99±0.04
144 5.5±0.3 1.23±0.01 1.69±0.04 7.46±0.04 5.22±0.01 6.27±0.04
168 5.55±0.03 1.24±0.03 1.71±0.02 6.30±0.03 4.18±0.03 5.44±0.03
192 5.75±0.01 1.28±0.03 1.74±0.03 4.45±0.04 3.18±0.01 3.18±0.03
216 5.87±0.04 1.44±0.05 1.77±0.04 3.23±0.03 2.49±0.06 1.39±0.02
240 5.95±0.01 1.56±0.04 1.81±0.03 2.67±0.04 1.14±0.02 1.34±0.01
Table-5.89: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses as carbon source and 0.4% urea at 30 ± 2 ºC and
pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar (mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 5.8±0.3 1.071±0.003 1.56±0.05 22.65±0.02 16.27±0.04 7.99±0.06
48 6.05±0.02 1.132±0.002 1.57±0.04 17.92±0.05 13.83±0.03 9.90±0.03
72 5.85±0.03 1.193±0.003 1.59±0.02 16.69±0.04 12.48±0.03 23.94±0.03
96 5.65±0.01 1.219±0.002 1.62±0.03 15.89±0.03 11.54±0.01 11.90±0.02
120 5.5±0.2 1.242±0.004 1.73±0.02 12.87±0.05 9.15±0.04 8.86±0.02
144 5.1±0.2 1.2825±0.0002 1.75±0.01 8.69±0.06 6.60±0.02 7.40±0.03
168 4.55±0.02 1.2905±0.0004 1.79±0.02 5.67±0.01 3.27±0.05 5.73±0.03
192 4.45±0.03 1.3605±0.0002 1.79±0.06 4.13±0.02 3.96±0.03 4.18±0.03
216 4.3±0.2 1.37±0.05 1.83±0.03 2.69±0.04 1.43±0.02 3.71±0.03
240 4.15±0.01 1.41±0.04 1.84±0.02 1.15±0.02 0.88±0.04 1.23±0.03
138
Table-5.90: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses as carbon source and 0.2% urea at 30 ± 2 ºC and
pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
activity (U/ml)
24 6.10±0.02 0.92±0.03 1.58±0.04 22.34±0.02 15.97±0.02 7.82±0.02
48 5.95±0.02 1.49±0.02 1.60±0.02 19.39±0.02 15.41±0.04 11.18±0.03
72 5.45±0.04 1.21±0.03 1.62±0.02 16.32±0.02 11.12±0.04 20.14±0.03
96 5.35±0.03 1.23±0.02 1.65±0.02 15.77±0.02 10.89±0.07 12.54±0.03
120 5.75±0.02 1.23±0.03 1.67±0.02 13.18±0.03 9.92±0.03 8.15±0.07
144 5.25±0.05 1.24±0.01 1.68±0.01 10.56±0.05 7.20±0.05 7.97±0.04
168 4.85±0.03 1.26±0.04 1.70±0.06 7.28±0.06 5.13±0.02 5.95±0.03
192 4.45±0.01 1.32±0.03 1.75±0.02 4.24±0.04 3.96±0.01 2.40±0.03
216 4.20±0.01 1.42±0.04 1.79±0.02 2.98±0.05 1.69±0.06 2.13±0.03
240 4.10±0.01 1.48±0.04 1.83±0.03 1.12±0.02 0.69±0.01 1.24±0.02
Table-5.91: P. lilacinum was grown on mineral medium supplemented with 5%
sucrose, 5% molasses as carbon source and 0.4% urea at 30 ± 2 ºC and pH was adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml Broth
Total Proteins (mg/ml)
Total Sugar (mg/ml)
Reducing Sugars (mg/ml)
Pectinase Activity (U/ml)
24 6.10±0.02 1.10±0.03 1.43±0.06 22.94±0.02 14.61±0.05 6.96±0.03
48 5.8±0.1 1.12±0.07 1.46±0.03 18.72±0.06 13.91±0.04 10.84±0.02
72 5.55±0.03 1.14±0.03 1.52±0.02 14.94±0.03 9.85±0.04 22.16±0.01
96 5.75±0.02 1.22±0.04 1.56±0.04 11.39±0.02 8.79±0.01 13.64±0.03
120 5.15±0.01 1.32±0.02 1.58±0.03 9.69±0.06 6.82±0.07 9.75±0.07
144 4.95±0.02 1.35±0.02 1.65±0.03 9.36±0.05 7.04±0.02 7.14±0.05
168 4.75±0.02 1.40±0.01 1.68±0.03 6.74±0.02 4.15±0.01 4.24±0.02
192 4.05±0.03 1.46±0.05 1.75±0.02 3.04±0.03 2.86±0.03 3.77±0.04
216 4.25±0.01 1.54±0.02 1.78±0.07 2.98±0.05 1.54±0.03 1.65±0.01
240 4.65±0.04 1.61±0.05 1.83±0.03 1.42±0.05 1.13±0.03 1.29±0.06
Tables 5.92 to 5.95 indicates that A. niger and P. lilacinum was grown on optimized
culture medium with addition to 0.2% and 0.4% NaNO3 and the maximum pectinase
was obtained 22.88 U/ml and 22.90 U/ml respectively. The present results are in
agreement with the findings of Arijit et al., (2013). Neeta et al., ( 2011) reported that
next to peptone NaNO3 exhibited higher pectinase production from A. niger.
139
Table-5.92: A. nigar was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% NaNO3 at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.30±0.03 1.05±0.03 1.39±0.06 23.17±0.04 15.86±0.01 7.23±0.04
48 5.90±0.04 1.21±0.02 1.42±0.02 21.84±0.02 15.74±0.03 9.14±0.01
72 6.65±0.01 1.24±0.01 1.49±0.06 17.98±0.03 14.61±0.01 21 .11±0.01
96 5.2±0.1 1.26±0.05 1.50±0.04 12.90±0.03 9.13±0.02 9.73±0.05
120 4.75±0.02 1.28±0.01 1.56±0.04 11.72±0.02 7.24±0.06 8.94±0.07
144 4.25±0.04 1.32±0.02 1.69±0.04 9.92±0.03 6.39±0.03 6.80±0.04
168 4.55±0.03 1.34±0.03 1.70±0.02 8.68±0.05 6.17±0.04 5.39±0.02
192 4.7±0.2 1.42±0.05 1.72±0.02 4.42±0.02 3.98±0.06 2.13±0.02
216 4.15±0.02 1.43±0.02 1.78±0.05 3.80±0.07 3.13±0.06 1.27±0.05
240 4.2±0.2 1.46±0.01 1.79±0.04 1.67±0.05 1.11±0.02 1.13±0.03
Table-5.93: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% NaNO3 at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 6.30±0.03 0.99±0.04 1.59±0.01 31.13±0.02 27.33±0.01 7.61±0.05
48 5.65±0.02 1.05±0.03 1.63±0.03 25.97±0.04 24.12±0.01 9.98±0.05
72 5.55±0.03 1.09±0.04 1.67±0.04 23.94±0.02 22.30±0.07 22.90±0.07
96 5.20±0.01 1.14±0.03 1.69±0.03 16.73±0.03 15.81±0.03 11.61±0.04
120 5.10±0.02 1.18±0.03 1.69±0.02 11.32±0.04 11.00±0.3 8.61±0.06
144 4.90±0.04 1.29±0.04 1.73±0.02 9.85±0.03 8.92±0.06 7.69±0.02
168 4.85±0.02 1.32±0.02 1.76±0.01 7.98±0.05 7.22±0.05 5.77±0.01
192 4.75±0.01 1.36±0.03 1.79±0.06 5.32±0.04 5.17±0.04 4.92±0.02
216 4.50±0.02 1.43±0.03 1.80±0.05 3.81±0.01 3.52±0.03 2.10±0.04
240 3.95±0.02 1.49±0.05 1.80±0.01 1.68±0.03 1.56±0.05 1.71±0.08
140
Table-5.94 : P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% NaNO3 at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugarss (mg/ml)
Pectinase
Activity (U/ml)
24 6.25±0.02 1.16±0.03 1.62±0.05 18.13±0.03 13.13±0.06 6.12±0.05
48 6.05±0.01 1.14±0.02 1.67±0.05 17.12±0.04 13.21±0.01 9.67±0.04
72 6.10±0.01 1.21±0.01 1.75±0.02 14.62±0.04 11.20±0.06 20.39±0.02
96 5.75±0.02 1.25±0.04 1.79±0.04 11.12±0.01 8.81±0.03 11.40±0.06
120 5.35±0.03 1.29±0.04 1.80±0.03 11.00±0.5 7.89±0.04 9.61±0.01
144 5.15±0.03 1.30±0.03 1.88±0.05 7.15±0.06 5.61±0.05 7.82±0.05
168 4.85±0.01 1.32±0.05 1.84±0.03 5.26±0.03 4.12±0.02 6.34±0.03
192 4.75±0.02 1.36±0.04 1.87±0.01 4.72±0.02 2.62±0.04 5.72±0.04
216 4.3±0.2 1.37±0.02 1.89±0.03 2.91±0.08 1.72±0.05 3.72±0.01
240 3.95±0.03 1.39±0.03 1.90±0.01 1.12±0.01 0.98±0.06 2.91±0.06
Table- 5.95 : P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% NaNO3 at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 6.40±0.03 1.10±0.03 1.65±0.02 21.79±0.06 15.79±0.06 6.19±0.03
48 6.10±0.02 1.12±0.01 1.67±0.05 19.00±0.3 14.81±0.01 8.16±0.03
72 5.85±0.02 1.17±0.04 1.71±0.04 15.45±0.04 12.22±0.03 22.50±0.06
96 5.65±0.03 1.22±0.02 1.76±0.03 11.87±0.05 8.10±0.06 12.85±0.03
120 5.10±0.01 1.29±0.04 1.80±0.04 10.74±0.03 7.21±0.03 8.88±0.04
144 4.60±0.07 1.31±0.04 1.83±0.03 8.91±0.03 6.25±0.03 7.98±0.05
168 4.25±0.01 1.34±0.03 1.84±0.02 6.19±0.04 4.97±0.02 5.12±0.02
192 3.95±0.02 1.36±0.03 1.87±0.02 4.92±0.02 4.13±0.02 2.81±0.04
216 3.80±0.02 1.49±0.07 1.88±0.01 2.98±0.06 1.70±0.07 2.16±0.05
240 3.70±0.03 1.52±0.02 1.89±0.09 1.27±0.05 0.53±0.02 1.94±0.02
Tables 5.96 to 5.99 shows the growth pattern and pectinase production when A. niger
and P. lilacinum was grown on 5% sucrose, 5% molasses ,0.2%and 0.4% KNO3 as
nitrogen source at 30 ± 2 ºC and pH was adjusted at 6.5. The maximum production
141
of pectinase 20.67 U/ml and 19.14 U/ml respectively was observed at 72 hours and
then its concentration was decreased with the increase of time period.
Table-5.96: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses & 0.2% KNO3 at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 6.15±0.02 0.91±0.04 1.52±0.02 22.95±0.02 16.86±0.04 7.16±0.05
48 5.90±0.01 1.06±0.03 1.53±0.05 20.93±0.05 15.71±0.03 11.13±0.05
72 5.80±0.02 1.11±0.05 1.56±0.05 16.16±0.03 11.63±0.03 19.22±0.07
96 5.25±0.02 1.19±0.06 1.59±0.02 13.87±0.05 11.24±0.01 10.48±0.04
120 5.85±0.03 1.22±0.01 1.62±0.04 12.47±0.04 9.79±0.05 8.42±0.02
144 5.15±0.02 1.24±0.04 1.64±0.02 9.99±0.06 7.16±0.04 7.37±0.05
168 4.85±0.01 1.27±0.05 1.69±0.06 6.90±0.02 5.28±0.06 4.16±0.03
192 475±0.01 1.28±0.03 1.70±0.03 3.82±0.02 2.29±0.08 2.14±0.03
216 4.40±0.02 1.34±0.01 1.72±0.03 1.92±0.05 1.22±0.06 1.36±0.07
240 4.10±0.01 1.37±0.03 1.79±0.06 1.18±0.05 0.76±0.03 1.02±0.02
Table-5.97:A. niger was grown on mineral medium supplemented with 5%
sucrose, 5% molasses and 0.4% KNO3 at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.30±0.01 0.98±0.04 1.46±0.05 22.94±0.04 15.17±0.04 5.73±0.04
48 6.25±0.02 1.01±0.01 1.48±0.03 18.99±0.06 14.45±0.02 11.24±0.03
72 6.10±0.01 1.17±0.04 1.57±0.04 17.43±0.02 12.91±0.05 20.67±0.05
96 5.70±0.02 1.20±0.01 1.59±0.03 14.83±0.02 11.64±0.03 9.17±0.04
120 5.6±0.3 1.21±0.04 1.62±0.03 10.19±0.05 7.69±0.06 8.92±0.02
144 5.30±0.03 1.26±0.04 1.68±0.06 7.49±0.05 5.19±0.05 7.97±0.04
168 5.15±0.01 1.24±0.03 1.70±0.02 6.46±0.04 4.32±0.02 6.61±0.05
192 4.95±0.02 1.32±0.05 1.72±0.02 4.12±0.03 3.92±0.05 4.17±0.06
216 4.30±0.02 1.38±0.05 1.78±0.01 2.39±0.06 1.13±0.02 2.98±0.04
240 4.10±0.01 1.42±0.02 1.79±0.06 1.22±0.02 0.57±0.05 1.42±0.03
142
Table-5.98: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% KNO3 at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Protein (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.5±0.2 0.92±0.05 1.28±0.06 21.94±0.03 14.97±0.04 6.82±0.06
48 6.2±0.1 1.49±0.05 1.40±0.03 20.39±0.02 13.41±0.05 9.04±0.01
72 5.9±0.2 1.21±0.01 1.32±0.02 16.32±0.04 12.12±0.01 18.18±0.08
96 5.35±0.03 1.23±0.04 1.35±0.04 15.77±0.04 11.89±0.04 12.40±0.03
120 5.15±0.02 1.23±0.06 1.37±0.02 13.18±0.03 11.92±0.02 9.15±0.04
144 4.85±0.01 1.24±0.02 1.38±0.06 9.56±0.04 7.20±0.04 6.97±0.04
168 4.65±0.01 1.26±0.01 1.39±0.02 6.28±0.07 3.13±0.04 5.95±0.03
192 4.3±0.2 1.32±0.02 1.45±0.01 4.24±0.04 3.96±0.03 3.40±0.05
216 4.2±0.2 1.42±0.06 1.48±0.05 2.98±0.05 1.69±0.01 2.13±0.02
240 4.1±0.1 1.48±0.04 1.58±0.06 1.51±0.03 1.05±0.02 1.24±0.04
Table-5.99: P. lilacinum was grown on mineral medium supplemented with 5%
sucrose, 5% molasses & 0.4% KNO3 at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.4±0.3 1.00±0.3 1.53±0.02 21.16±0.03 14.19±0.06 6.86±0.04
48 6.1±0.1 1.94±0.04 1.65±0.02 18.54±0.03 13.13±0.06 8.11±0.05
72 5.85±0.02 1.97±0.02 1.76±0.04 15.65±0.05 11.75±0.02 19.14±0.03
96 5.65±0.03 1.12±0.04 1.77±0.07 13.39±0.02 10.12±0.02 11.16±0.01
120 5.15±0.01 1.21±0.04 1.79±0.06 11.89±0.07 8.13±0.04 9.33±0.03
144 4.45±0.02 1.22±0.07 1.84±0.03 8.97±0.02 5.13±0.08 5.98±0.02
168 4.75±0.02 1.28±0.05 1.89±0.07 7.99±0.06 6.15±0.02 4.89±0.07
192 4.05±0.01 1.29±0.01 1.90±0.03 4.82±0.04 3.89±0.02 3.07±0.02
216 4.25±0.03 1.31±0.05 1.88±0.02 2.99±0.01 2.61±0.01 2.13±0.02
240 3.95±0.02 1.34±0.03 1.82±0.06 1.57±0.04 1.03±0.02 1.11±0.05
143
Tables 5.100 to 5.103 shows the results of NH4NO3, when added as nitrogen source in optimized medium for the synthesis of pectinase by A. niger and P.
lilacinum and maximum production of pectinase was achieved 22.40 U/ml and 21.27 U/ml respectively
Table-5.100: A. niger was grown on mineral medium supplemented with 5%
sucrose, 5% molasses and 0.2% NH4NO3 at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.1±0.1 1.09±0.03 1.52±0.04 20.933±0.003 13.74±0.05 6.40±0.04
48 5.4±0.1 1.11±0.03 1.54±0.01 16.98±0.01 11.94±0.02 7.64±0.02
72 5.25±0.01 1.14±0.03 1.59±0.03 14.23±0.03 11.39±0.06 20.35±0.04
96 5.15±0.02 1.16±0.02 1.62±0.02 18.99±0.06 18.21±0.03 11.17±0.04
120 5.05±0.03 1.17±0.05 1.65±0.02 13.22±0.04 12.42±0.05 8.63±0.05
144 4.45±0.02 1.24±0.02 1.67±0.05 12.15±0.04 11.20±0.07 6.91±0.04
168 4.35±0.01 1.26±0.02 1.71±0.03 8.21±0.06 7.65±0.03 5.17±0.04
192 4.65±0.03 1.29±0.06 1.73±0.06 6.22±0.05 5.81±0.01 4.15±0.04
216 4.15±0.01 1.32±0.03 1.79±0.01 4.10±0.04 3.32±0.01 3.32±0.05
240 4.35±0.02 1.34±0.01 1.80±0.07 2.24±0.02 2.10±0.04 1.33±0.03
.
Table-5.101: A. niger was grown on mineral medium supplemented with 5%
sucrose, 5% molasses and 0.4% NH4NO3 at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.9±0.2 1.05±0.02 1.49±0.04 21.17±0.06 15.86±0.04 5.23±0.04
48 6.0±0.2 1.21±0.02 1.52±0.02 18.84±0.04 14.74±0.03 8.14±0.01
72 5.4±0.2 1.24±0.03 1.57±0.04 17.18±0.03 14.00±0.2 22.40±0.04
96 5.1±0.1 1.26±0.02 1.59±0.03 14.90±0.02 11.13±0.04 13.73±0.05
120 4.75±0.02 1.28±0.05 1.61±0.03 10.72±0.04 8.24±0.03 8.94±0.01
144 4.25±0.01 1.32±0.04 1.67±0.05 9.92±0.05 7.69±0.01 6.80±0.07
168 4.55±0.02 1.34±0.04 1.69±0.02 5.68±0.05 3.17±0.03 4.39±0.02
192 4.7±0.2 1.42±0.05 1.71±0.01 3.4 ±0.0 4 2.98±0.05 3.13±0.02
216 4.15±0.02 1.43±0.02 1.75±0.03 2.27±0.04 1.83±0.03 2.27±0.01
240 4.2±0.1 1.46±0.02 1.79±0.06 1.07±0.03 0.46±0.03 1.83±0.03
144
Table-5.102: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% NH4NO3 at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.25±0.01 0.99±0.06 1.92±0.02 22.97±0.04 16.01±0.01 5.13±0.05
48 6.15±0.01 1.23±0.02 1.93±0.02 20.82±0.04 15.12±0.06 8.21±0.02
72 6.3±0.1 1.33±0.03 1.95±0.01 16.20±0.03 11.80±0.04 20 .11±0.05
96 6.15±0.02 1.48±0.04 1.97±0.04 15.14±0.04 10.87±0.05 9.44±0.04
120 5.85±0.03 1.59±0.03 1.99±0.03 12.61±0.05 9.87±0.02 9.19±0.05
144 5.25±0.02 1.61±0.04 1.94±0.07 9.38±0.05 7.82±0.05 8.46±0.03
168 4.65±0.01 1.62±0.02 1.94±0.04 6.62±0.05 4.27±0.04 5.39±0.04
192 4.25±0.01 1.75±0.02 1.83±0.02 5.36±0.04 3.89±0.02 3.619±0.008
216 4.4±0.2 1.76±0.01 1.77±0.05 2.97±0.02 2.63±0.02 2.16±0.01
240 4.95±0.03 1.77±0.04 1.57±0.01 1.03±0.03 0.67±0.02 1.78±0.05
Table-5.103: P.lilacinam was grown on mineral medium supplemented with 5%
sucrose, 5% molasses and 0.4% NH4NO3 at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total
Sugar (mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.9±0.2 1.07±0.05 1.761±0.004 21.84±0.04 14.12±0.04 6.22±0.02
48 5.7±0.3 1.14±0.03 1.81±0.04 17.55±0.02 13.13±0.06 8.19±0.06
72 5.05±0.02 1.15±0.05 1.85±0.03 14.77±0.05 10.03±0.03 21.27±0.04
96 5.65±0.01 1.20±0.05 1.88±0.02 11.62±0.02 7.18±0.03 13.16±0.05
120 5.5±0.1 1.24±0.01 1.90±0.07 9.13±0.05 6.95±0.03 9.97±0.01
144 5.3±0.1 1.27±0.05 1.71±0.05 8.78±0.05 5.87±0.04 7.87±0.04
168 5.15±0.03 1.31±0.03 1.77±0.05 5.02±0.02 4.92±0.02 5.11±0.05
192 4.85±0.02 1.37±0.04 1.79±0.04 3.91±0.05 2.02±0.01 4.18±0.01
216 4.4±0.1 1.42±0.03 1.79±0.08 1.97±0.04 1.37±0.02 3.19±0.05
240 4.2±0.2 1.44±0.02 1.80±0.01 0.89±0.02 0.50±0.04 1.84±0.04
Previous results in this study show that A. niger produces greater amount
of pectinase than P. lilacinum as shown in tables 5.104 to 5.107. A niger and
P .lilacinum with the adition of (0.2 and 0.4%) peptone as a nitrogen source has
given significant production of pectinase 23.98 U/ml and 22.97 U/ml
145
respectively. The results are in agreement with Neeta et al., (2011) who achieved
maximum production of pectinase from A. niger in Smf system using peptone as
a nitrogen source. Peptone consists of various amino acids that liberate nitrogen
which enhance the growth of fungi (Martin et al., 2004; Margesin et al., 2005).
Table-5.104: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% peptone at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 7.1±0.1 0.98±0.05 1.55±0.04 20.22±0.08 13.97±0.04 7.80±0.03
48 6.85±0.02 1.07±0.03 1.58±0.03 18.65±0.03 12.89±0.07 8.27±0.02
72 6.3±0.2 1.07±0.04 1.58±0.09 15.76±0.03 11.08±0.03 21.80±0.01
96 6.2±0.2 1.12±0.06 1.59±0.02 12.89±0.03 8.33±0.03 13.73±0.03
120 5.70±0.02 1.16±0.02 1.59±0.06 9.98±0.03 6.60±0.04 9.12±0.01
144 5.25±0.02 1.22±0.02 1.60±0.02 8.88±0.06 5.19±0.05 7.82±0.02
168 5.15±0.03 1.29±0.04 1.59±0.03 5.45±0.05 4.12±0.02 4.78±0.02
192 4.7±0.2 1.41±0.04 1.58±0.06 3.24±0.03 2.99±0.04 3.95±0.03
216 4.15±0.01 1.41±0.01 1.42±0.02 1.69±0.04 1.44±0.02 2.45±0.04
240 4.35±0.03 1.43±0.07 1.42±0.01 1.19±0.01 1.07±0.01 2.17±0.04
Table-5.105: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% peptone at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time Hours
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 7.15±0.02 0.92±0.08 1.43±0.03 21.14±0.03 14.33±0.04 7.76±0.05
48 6.95±0.02 0.917±0.004 1.51±0.05 19.78±0.03 14.13±0.03 11.83±0.05
72 6.55±0.01 0.98±0.03 1.42±0.02 17.92±0.05 13.63±0.05 23.98±0.05
96 6.15±0.01 0.99±0.05 1.47±0.04 14.81±0.05 11.67±0.05 15.66±0.01
120 5.85±0.03 1.13±0.02 1.68±0.03 10.19±0.06 7.90±0.05 9.64±0.03
144 5.15±0.02 1.29±0.04 1.67±0.06 6.72±0.02 5.14±0.01 6.39±0.02
168 5.0±0.2 1.33±0.03 1.68±0.04 5.34±0.03 4.22±0.02 4.74±0.04
192 4.75±0.02 1.34±0.02 1.74±0.04 3.79±0.03 2.69±0.04 3.94±0.06
216 4.7±0.2 1.34±0.03 1.76±0.03 1.98±0.03 1.37±0.04 1.97±0.05
240 4.95±0.02 1.49±0.05 1.71±0.03 1.01±0.01 0.83±0.01 1.78±0.05
146
Table-5.106: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% peptone at 30 ± 2 ºC and pH was
adjusted at 6.5
Time
hours
Final
pH
Biomass
g/50 ml Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 6.95±0.01 1.09±0.04 1.12±0.04 21.93±0.03 15.17±0.05 6.91±0.04
48 6.15±0.02 1.12±0.04 1.27±0.06 17.32±0.02 12.95±0.02 9.40±0.03
72 5.5±0.1 1.10±0.06 1.32±0.06 14.25±0.02 10.99±0.04 20.57±0.02
96 5.25±0.01 1.12±0.02 1.37±0.04 12.81±0.05 9.11±0.04 11.99±0.03
120 4.85±0.01 1.15±0.04 1.47±0.02 11.98±0.05 8.82±0.05 9.62±0.02
144 4.15±0.02 1.23±0.06 1.47±0.04 9.75±0.03 7.20±0.08 8.40±0.03
168 4.85±0.03 1.24±0.03 1.48±0.02 5.69±0.01 4.22±0.02 5.71±0.06
192 4.75±0.03 1.26±0.01 1.49±0.04 3.41±0.04 3.12±0.03 3.94±0.04
216 4.7±0.2 1.27±0.05 1.49±0.08 2.13±0.07 1.96±0.03 2.36±0.03
240 3.95±0.02 1.28±0.02 1.49±0.02 1.17±0.04 0.53±0.03 1.38±0.06
Table-5.107: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% peptone at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time hours
Final pH Biomass g/50 ml
Broth
Total Proteins (mg/ml)
Total Sugar (mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
24 6.9±0.2 0.93±0.03 1.37±0.05 20.62±0.05 14.04±0.03 7.20±0.07
48 6.65±0.01 1.03±0.03 1.35±0.01 18.15±0.02 13.94±0.07 8.12±0.02
72 6.55±0.01 1.15±0.04 1.37±0.06 16.05±0.03 12.88±0.03 22.97±0.04
96 6.15±0.02 1.17±0.03 1.40±0.01 13.56±0.05 11.84±0.03 12.86±0.01
120 5.8±0.3 1.18±0.03 1.46±0.01 10.13±0.07 8.95±0.05 7.13±0.03
144 5.6±0.2 1.21±0.01 1.47±0.07 7.88±0.05 6.64±0.04 6.12±0.06
168 5.1±0.1 1.24±0.07 1.50±0.07 4.79±0.07 4.34±0.01 4.82±0.05
192 4.9±0.3 1.26±0.03 1.53±0.04 2.23±0.03 1.94±0.02 2.85±0.03
216 4.7±0.2 1.39±0.03 1.54±0.04 1.36±0.05 1.13±0.07 2.37±0.06
240 5.3±0.2 1.42±0.03 1.52±0.05 0.54±0.04 0.29±0.06 1.11±0.05
Tables 5.108 to 5.111 A. niger and P. lilacinum was grown on 5% sucrose, 5%
molasses and incorporation 0.2% or 0.4% (NH4)2SO4 at 30 ± 2 ºC when pH was
adjusted at 6.5. The maximum production of pectinase 25.14 U/ml and
147
23.13 U/ml respectively was obtained at 72 hours. Loera et al., (1999) and
Scopes (1985) have reported 73 hours to be optimum incubation for maximal
polygalactouranase activity by a diploid construct from two Aspergillus niger
overproducing mutants. Ammonium sulphate appeared to be the most optimal
nitrogen source for pectinase production, which also caused a stabilizing effect
on enzyme. In the literature, ammonium sulphate and potassium phosphate
have been reported to have no significant influence on the production of
pectinase at lower concentrations Hours et al., (1988) but in present study
ammonium sulphate had shown a greater influence on the production of
pectinase after 72 hours with both filamentous fungi used. The present study is
in agreement with Phutela et al., (2005) contrast with , Arijit et al., (2013) reported
that (NH4)2 SO4 showed minimum production of pectinase enzyme by
Streptomyces sp. while Banu et el ., (2010) obtained highest pectinase production
by using ammonium per sulphate as a nitrogen source. Joshi et al., (2006)
reported that ammonium sulphate acted as best nitrogen source for the
production of Pectin methylestrase. Patil and Dayanand (2006 a) also reported
that both ammonium phosphate and ammonium sulphate did influence
production of pectinase positively in both submerged and solid-state conditions.
According to the observation of Phutela et al., (2005) fungi did not produce
pectinase when culture media was devoid of ammonium sulphate. Tariq and
Reyaz (2012) and Reda et al., (2008 ) reported that ammonium sulphate provides
additional nitrogen to the fermentation systems and in result more pectinase was
produced.
148
Table-5.108: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% (NH4)2SO4 at 30 ± 2 ºC and pH was
adjusted at 6.5
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.6±0.3 1.11±0.04 1.48±0.06 21.44±0.02 14.41±0.04 8.82±0.05
48 5.9±0.1 1.18±0.03 1.53±0.03 19.64±0.01 13.45±0.04 11.74±0.04
72 5.4±0.2 1.32±0.03 1.56±0.03 14.84±0.04 10.79±0.04 23.19±0.04
96 5.35±0.01 1.36±0.03 1.58±0.03 11.37±0.05 8.81±0.01 12.40±0.01
120 4.7±0.1 1.44±0.04 1.60±0.09 9.18±0.03 6.93±0.06 8.79±0.06
144 4.8±0.2 1.25±0.04 1.61±0.01 7.36±0.04 5.95±0.09 7.72±0.02
168 4.6±0.1 1.27±0.02 1.66±0.04 5.79±0.03 4.33±0.03 5.94±0.02
192 4.45±0.02 1.33±0.05 1.69±0.04 2.04±0.03 1.96±0.03 3.40±0.04
216 4.25±0.03 1.36±0.04 1.72±0.04 1.98±0.06 1.68±0.06 2.33±0.03
240 4.1±0.1 1.49±0.05 1.78±0.05 0.54±0.04 0.50±0.06 1.14±0.05
Table-5.109 : A.niger was grown on mineral medium supplemented with 5%
sucrose, 5% molasses and 0.4% (NH4)2SO4 at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.85±0.02 0.93±0.05 1.53±0.03 21.92±0.06 13.73±0.05 7.73±0.03
48 5.55±0.01 1.12±0.03 1.56±0.03 17.93±0.03 13.62±0.02 12.92±0.02
72 5.5±0.1 1.19±0.06 1.58±0.03 15.80±0.07 11.49±0.05 25.14±0.01
96 5.25±0.03 1.23±0.03 1.64±0.02 14.83±0.02 11.17±0.05 14.66±0.05
120 4.85±0.02 1.24±0.03 1.68±0.03 10.59±0.01 8.97±0.01 8.64±0.07
144 4.65±0.03 1.34±0.07 1.68±0.07 6.25±0.03 4.70±0.02 7.74±0.02
168 4.45±0.01 1.43±0.03 1.70±0.01 5.75±0.04 4.24±0.01 5.72±0.06
192 4.35±0.04 1.45±0.03 1.73±0.03 3.21±0.07 3.09±0.04 4.03±0.03
216 4.7±0.2 1.62±0.02 1.79±0.02 1.91±0.05 1.37±0.05 3.37±0.05
240 4.95±0.02 1.63±0.06 1.82±0.02 0.60±0.05 0.31±0.05 1.18±0.05
149
Table-5.110: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% (NH4)2SO4 at 30 ± 2 ºC and pH was
adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.8±0.2 1.11±0.04 1.34±0.03 21.37±0.06 14.46±0.04 7.46±0.02
48 5.4±0.3 1.19±0.06 1.37±0.04 19.82±0.05 13.90±0.07 9.98±0.02
72 4.85±0.03 1.25±0.03 1.42±0.05 16.92±0.02 12.14±0.04 21.46±0.06
96 4.25±0.02 1.31±0.01 1.44±0.04 14.28±0.05 11.96±0.03 13.72±0.07
120 5.15±0.02 1.35±0.03 1.49±0.07 12.69±0.01 9.16±0.01 8.72±0.03
144 5.45±0.01 1.39±0.03 1.56±0.03 10.36±0.03 8.12±0.06 8.14±0.01
168 5.75±0.02 1.39±0.01 1.68±0.02 7.24±0.03 6.36±0.05 4.36±0.03
192 5.05±0.01 1.41±0.01 1.71±0.02 5.12±0.02 4.84±0.07 3.11±0.05
216 5.1±0.1 1.42±0.05 1.71±0.01 2.18±0.06 1.74±0.01 2.20±0.03
240 5.3±0.1 1.44±0.04 1.65±0.03 0.53±0.05 0.39±0.06 1.17±0.07
Table-5.111: P. lilacinum was grown on mineral medium supplemented with 5%
sucrose, 5% molasses and 0.4% (NH4)2SO4 at 30 ± 2 ºC and pH was adjusted at 6.5.
Time
Hours
Final pH Biomass
g/50 ml Broth
Total
Proteins (mg/ml)
Total Sugar
(mg/ml)
Reducing
Sugars (mg/ml)
Pectinase
Activity (U/ml)
24 5.80±0.02 0.91±0.04 1.76±0.03 21.82±0.06 13.82±0.05 7.41±0.08
48 6.15±0.02 1.04±0.03 1.82±0.02 18.75±0.04 12.88±0.07 9.39±0.03
72 5.85±0.01 1.20±0.06 1.91±0.08 14.76±0.03 11.93±0.07 23.13±0.03
96 5.65±0.01 1.21±0.04 1.85±0.05 11.32±0.02 8.54±0.03 12.87±0.05
120 5.50±0.01 1.26±0.04 1.84±0.02 9.83±0.02 6.94±0.01 8.91±0.04
144 5.60±0.03 1.29±0.04 1.75±0.02 7.18±0.03 5.82±0.02 8.36±0.01
168 5.75±0.02 1.34±0.03 1.74±0.06 5.42±0.04 4.94±0.03 6.83±0.06
192 4.95±0.03 1.46±0.03 1.73±0.03 3.93±0.03 3.47±0.05 4.38±0.03
216 4.30±0.02 1.62±0.04 1.72±0.06 2.67±0.05 2.44±0.01 2.59±0.01
240 4.20±0.01 1.64±0.02 1.69±0.02 1.09±0.06 0.73±0.05 1.84±0.04
150
In this study various nitrogen sources with two concentrations
(0.2 and 0.4%) were used to check the effect on the growth and production of
pectinase as results shown in Tables 5.112-5.113. Maximum biomass and
pectinase production 1.19 g/50 ml and 25.14 U/ml respectively recorded, when
A.niger grown on 0.4% ammonium sulphate in comparison to other nitrogen
sources used in this study. The ammonium ion taken up as ammonia, thereby
releasing a proton into the medium and causing a decrease in pH, a proton is taken up
from the medium when nitrate is transported into the cell, and this causes the pH to
increase as reported by (Prior et al., 1992); Torrado et al., (1998) and Gokhale et al., (1992).
Effect of various nitrogen sources were also checked when Penicillium lilacinum grown
on 5% corn steep liquor with addition to sucrose and molasses produced maximum
mycelial biomass and pectinase activity 1.30 g/50 ml and 23.23 U/ml respectively.
Present findings are in agreement with (Gupta et al., 1997) where nitrogen from
inorganic sources activated the production of polygalacturonase. Shastri et al., (1988)
have also showed similar results. The nitrogen source can play an important role in
affecting the pH changes in the substrate during the fermentation. A combination of
these two nitrogen sources can be used to reduce the pH changes during the fermentation
(Prior et al., 1992; Torrado et al., 1998 and Gokhale et al., 1992) also reported the capacity
of urea to prevent the drop in pH during fermentation system. These findings
support the present study also.
During optimizing the nitrogen source in the fermentation experiments, it
is very clear that in the presence of molasses and sucrose, as substrate appeared a
best carbon source. The ammonium sulphate was selected as a best nitrogen
source and Aspergillus niger was selected as best organism, which has produced
higher amount of pectinase in comparison to Penicillium lilacinum on same
optimal conditions as results shown in Fig-5.11
151
Table-5.112 Effect of nitrogen sources on growth and Pectinase production by Aspergillus niger
Nitrogen sources Biomass Pectinase Activity
% g/50 ml Broth U/ml
Corn Steep liquor 0.2 1.27±0.04 21.19±0.06
0.4 1.20±0.03 23.39±0.06
Urea 0.2 1.16±0.01 21.29±0.08
0.4 1.193± 0.003 23.94±0.04
NaNO3 0.2 1.24±0.02 21.11±0.05
0.4 1.09±0.06 22.90±0.05
KNO3 0.2 1.11±0.07 19.22±0.04
0.4 1.17±0.05 20.67±0.03
NH4NO3 0.2 1.14±0.03 20.35±0.05
0.4 1.24±0.04 22.40±0.07
Peptone 0.2 1.07±0.04 21.80±0.01
0.4 1.18±0.06 23.98±0.01
NH4)2SO4 0.2 1.32±0.05 23.19±0.02
0.4 1.19±0.08 25.14 ±0.03
152
Table-5.113 Effect of nitrogen sources on growth and pectinase production by Penicillium lilacinum
Nitrogen sources Biomass Pectinase Activity
% g/50 ml Broth U/ml
Corn steep liquor 0.2 1.30±0.01 21.172±0.006
0.4 1.30±0.06 23.23±0.04
Urea 0.2 1.21±0.01 20.14±0.04
0.4 1.14±0.03 22.16±0.04
NaNO3 0.2 1.21±0.02 20.39±0.03
0.4 1.17±0.01 22.50±0.04
KNO3 0.2 1.21 ±0.02 18.18±0.03
0.4 1.9±0.03 19.14±0.03
NH4NO3 0.2 1.33±0.01 20.11±0.01
0.4 1.15±0.04 21.27±0.05
Peptone 0.2 1.10±0.05 20.57±0.04
0.4 1.15±0.03 22.97±0.01
(NH4)2SO4 0.2 1.25±0.05 21.46±0.05
0.4 1.20± 0.07 23.13±0.01
153
vii- Selection of the organism:
A.niger was selected for further studies on the basis of results of carbon
and nitrogen sources used for the growth and pectinase synthesis through
consecutive experiments. An overview of the results obtained that 5% sucrose,
5% molasses and 0.4% (NH4)2SO4 are the best substrate component for the
production of pectinase by A .niger. The production of the enzymes from agro-
wastes by fungi in submerged fermentation system could not only be cost
effective but it could also offer several process merits. It is suggested that
microorganisms need sugar and nitrogen sources, which are essential for the
growth of microorganism and production of enzymes.
Fig 5.11. Comparison of Pectinase production produced by Aspergillus niger and
Penicillium lilacinum
25.14
23.23
22
22.5
23
23.5
24
24.5
25
25.5
Aspergillus niger Penicillium lilacinum
Pe ctina se Activity U/mL
154
viii- Effect of pH on pectinase production:
Microorganisms have their own individual pH for growth and the production of enzymes
but it is also dependent on the pH of the medium. pH plays a very important and is a
critical role in the synthesis of microbial enzymes.
Table-5.114 reveals the result of pectinase synthesis by A. niger grown in
optimized cultured conditions with different initial pH values. The maximum
production of pectinase 26.87 U/ml was observed at an initial pH 6.0 at 72 hrs
incubation, than its production, was decreased with the increase of pH values.
The concentration of total, reducing sugars and total protein content was
different in different pH values. The pH optimum of 6.0 for pectinase production
by A. niger is in contrast with the reported values of Penicillium sp. As reported
by Martin et al., (2004) and also with A. niger (Jyothi et al., 2005; Díaz-Godínez, et
al., 2001). It is reported that low pH values are favorable for high pectinase
production in Penicillium italicum (Alana et al., 1989). The optimum pH of
mesophilic pectinase has been established to range in-between 4.0-5.5 as reported
by Favela-Torres et al., (2006). The influence of pH on the culture medium may be
directly related with the stability of enzymes (Ueda et al., 1982). Studies with
Pectin Lyase of P. expansum showed that in spite of the optimum activity pH of
Pectin Lyase to be 7.0, the enzyme can be kept stable between the pH 6.5 to 8.0
(Santiago, 1993).
155
Table-5.114 : Effect of pH on Biosynthesis of Pectinase by A. niger grown on
mineral medium supplemented with 5% sucrose, 5% molasses
and 0.4% (NH4)2SO4 at 30 ± 2 ºC for 72 hours.
Initial pH
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
U/ml)
4.0 4.45±0.02 0.84±0.04 1.78±0.05 13.27±0.05 9.12±0.05 13.13±0.03
4.5 4.75±0.01 1.11±0.03 1.75±0.04 12.12±0.02 8.96±0.03 17.94±0.03
5.0 5.9±0.1 1.14±0.03 1.87±0.04 12.99±0.06 9.47±0.02 20.42±0.04
5.5 6.35±0.02 1.59±0.06 1.89±0.04 12.76±0.01 9.52±0.02 23.27±0.06
6.0 6.85±0.02 1.36±0.04 1.94±0.04 13.92±0.02 9.30±0.05 26.87±0.02
6.5 7.6±0.2 1.29±0.04 1.82±0.01 12.92±0.06 9.54±0.03 16.93±0.04
7.0 7.95±0.02 1.26±0.03 1.77±0.04 13.01±0.01 8.98±0.04 11.81±0.04
7.5 8.8±0.1 1.34±0.04 1.73±0.05 13.86±0.03 9.73±0.05 4.96±0.04
8 8.5±0.2 1.22±0.05 1.56±0.04 11.4±0.2 8.90±0.03 4.87±0.04
In the present study, it was noticed that the pH of the culture media
assorted over a broad range on the pH scale. Usually fungi amend the pH of the
medium in which they nurture, due to uptake of the cations or anions in the
medium (Moore-Landecker 1996; Griffin, 1994). Therefore, the different changes
noted in the pH of the culture media may be due to a result of the consumption
of some compounds in the media Juwon and Emmanuel (2012).
The researchers like Uenojo and Pastore, (2007), Cordeiro and Martins,
(2009) investigated that the reason of decrease of pH value is might be due to the
synthesis of galactouronic acid in the medium by the pectinase when acts on
pectin and that may be affects the more pectinase production. The nature of
microorganism and initial pH value for the production of pectinases is significant
because it varies organism to organism as a higher initial optimal pH of 8.5 for
the synthesis of pectinase has been reported by Sharma and Satyanarayana,
(2006). In contrast, to above researchers the optimum initial pH for pectinase
production was found as 7.0 by the thermophilic fungus S. thermophile (Kaur et
al., 2004). According to Young et al., (1996) the genes involved in the production
of certain enzymes in at least some microorganisms may be pH regulated.
156
ix- Effect of Temperature:
Temperature is very important parameter for the growth and other vital activities
of organisms. It is directly connected to the metabolic activities of the
microorganism and it affects the growth and product synthesis by the
microorganisms (Lonsane et al., 1985). Every organism shows its individual optimal
temperature at which it grows well and produces the desired products maximally.
Hence maintenance of optimal temperature is an important and critical factor.
Thermal conditions for the maximum production of pectinase enzyme were studied in
Aspergillus niger . In Table-5.116 enzymes showed maximal activity at 35 °C. The
enzymetic activity was almost similar at 20 °C and 45 °C i.e just below and
above optimal temperature.
The result is in agreement with Said et al., (1991) regarding production of
pectinase by Penicillium frequentans at 30 – 35 °C. The present finding is also
supported by Lonsane et al., (1985), while (Bailey and Pessa , 1990) studied the effect
of temperature on pectinase enzyme production by Aspergillus niger and the optimum
temperature was found to be 30 °C. The results are in contrast with Soni and Bhatia,
(1981) reported 21°C for Fusarium oxysporum. Gummadi and Kumar (2007) and
Nakajima et al., (1999) reported an optimum temperature 30 °C and 37 °C for
pectin lyase and pectin pectate lyase production by A. niger NCIM 548 and
Clostridium butyricum-beijerineki, respectively.
157
Table-5.115: Effect of temperature on biosynthesis of pectinase by A. niger grown
on mineral medium containing 5% molasses 5% sucrose and 0.4% (NH4)2SO4
while pH was adjusted 6.00 for 72 hours.
Initial Tem
Final pH Biomass g/50 ml
Broth
Total Proteins
(mg/ml)
Total Sugar
(mg/ml)
Reducing Sugars
(mg/ml)
Pectinase Activity
(U/ml)
20ºC 6.9±0.2 1.09±0.06 1.94±0.03 22.73±0.03 19.38±0.04 9.84±0.04
25ºC 6.8±0.2 1.11±0.05 2.00±0.1 22.85±0.03 19.12±0.02 17.99±0.02
30ºC 6.1±0.1 1.12±0.02 1.97±0.04 22.63±0.02 19.77±0.02 26.13±0.02
35ºC 6.0±0.3 1.19±0.04 1.99±0.03 21.79±0.04 19.43±0.02 28.25±0.03
40ºC 5.8±0.3 1.02±0.02 1.95±0.03 21.64±0.02 19.36±0.03 18.17±0.02
45ºC 5.7±0.1 1.01±0.01 1.96±0.05 21.58±0.01 19.28±0.02 11.91±0.03
B- Characterization of crude Pectinase Enzyme: The Pectinase produced by
Aspergillus niger when it was grown on 5% molasses, 5% sucrose and 0.4%
(NH4)2SO4 with the initial pH adjusted 6.0 and incubated for 72 hours.The
enzyme was characterized on the basis of time of incubation, substrate
specificity, substrate concentration, enzyme volume, buffer, pH, pH stability,
temperature, thermo stability, activators and inhibitors.
i- Effect of time of incubation on crude Pectinase: The Pectinase activity was
observed at various time periods (10-60 minutes) and result shown in Figure-
5.12, the results indicate that 15 minutes incubation period basis of the highest
activity, because increase and decrease in time reduces the pectinase activity
more or less same results are presented by Akhilesh et al., (2010) who reported
maximum activity of polygalacturonase from M. circinelloides after 20 minutes
and 30 minutes incubation period for highest pectinase activity was reported by
Roosdiana et al., (2013).
The declined activity after 15 minutes is may be due to the presence of other
enzymes in crude sample or the reason behind the decrease in Pectinase activity
might be inactivation of enzyme on prolongs incubation or self digestion or
158
product inhibition. Similar results are reported from earlier studies by (Dahot,
(1992) and Weil et al., (1966)
Figure-5.12: Effect of time of incubation on crude Pectinase
ii- Effect of substrate concentration on crude Pectinase: The effect of substrate
concentration was investigated for the rate of enzymatic reaction of Pectinase by
using pectin with different concentrations ranging from 0.5-4.0%. The rate of
reaction of enzyme directly increases with the increase in substrate concentration
till a certain optimum point is appeared and the substrate saturated with enzyme
present in reaction mixture. The hydrolytic activity of petinase enzymes was
investigated and results are represented in Figure-5.13 shows that 1.5% Pectin
had given optimum pectinase activity while below and above this concentration
activity declines the rate of reaction. The substrate concentratation (1.5 mg /ml)
in this study are in full accordance with NitinKumar and Bhushan, (2010) while
Afifi and Foaad (2002) reported the optimum concentration of substrate for
Pectin lyase as 1.1% citrus pectin. Deshmukh et al., (2012) reported 0.5 substrate
concentration for highest activity of pectinase produced by various strains of
159
Aspergillus. The declination of pectinase activity may be due to alteration in
enzyme and substrate ratio Gillard, (1971) and Price and Stevens (1999).
Figure-5.13: Effect of substrate concentration on crude Pectinase
iii- Effect of enzyme volume on crude Pectinase: The effect of enzyme volume
(0.2-1.4 ml) on the rate of enzyme reaction was studied as shown in Figure-5.14
that an increase in enzyme volume increases the pectinase activity while addition
of above 1.0 ml lowers the activity. The higher pectinase activity was found at 1.0
ml crude enzyme while low pectinase activity was observed when less amount of
enzyme volume was used. Deshmukh et al., (2012) accomplished highest
polygalacturonase activity by using 0.5 ml of crude enzyme isolated from
Aspergillus strains.
0
5
10
15
20
25
30
35
0.5 1 1.5 2 2.5
Pec
tin
ase
Act
ivit
y U
/mL
Substrate concentration %
160
Figure-5.14: Effect of enzyme volume on crude Pectinase
iv- Effect of different buffers on pectinase activity:
Figure-5.15 shows the effect of different buffers 0.1 M (sodium phosphate,
sodium citrate and universal buffer) on Pectinase activity. The 100% relative
activity was determined by using sodium citrate buffer, which was used
throughout the study. The use of sodium citrate buffer in pectinase activity is
reported by Banu et al., (2010) and Akhilesh et al.,(2010)
Figure-5.15: Effect of different buffers on crude Pectinase
0
5
10
15
20
25
30
35
phaosphate citrate universal
Buffers (pH 5.5)
Pec
tinas
e A
ctiv
ity IU
/ml
161
v- Effect of pH on crude pectinase
The pectinase activity was checked at various pH values (3.0 to 10.0) and
Figure-5.16 shows that the optimum pH for the crude pectinase was noted 5.0.
The catalytic activity of many enzymes is markedly dependent on pH. pH can exerts its
effect in different ways, on the ionization of groups in the enzyme's active site,
either on the ionization of groups in the substrate or by affecting the conformation of
either the enzyme or the substrate. These effects are influencing to the changes in
kinetic constants, (Dennison, 2003). In the present study Pectinase from A. niger
showed its maximum activity at pH 5.0 which is against the results of Marcia et al.,
(1999) when worked with polygalacturonase the highest activity was found at 6.0.
More or less same results were reported by Banu et al., (2010) that maximum
pectinase activity from P. chrysogenum was found at pH 6.5 using sodium citrate
buffer . The results of this study are in ful agreement with Afifi and Foaad (2002)
who reported that the maximum activity of Pectin lyase was found at pH 5.0 .The
result are also according to the results of Obi and Moneke (1985) and Moharib et
al. (2000) who reported that the pectinase enzyme was highly active at pH 5.0
and 4.5 respectively.
Martos et al.,(2013) reported that polygalcturonase isolated from
Wickerhanomyces anomalus showed maximum activity at pH 4.5-5.0, similar
observations for ploygalacturonase were reported for Rhizopus spp. by Elegado
and Fujio, (1994), for A. niger CH4 Acuña-Argüelles et al., (1995), for Lentinus
edodes by Zheng and Shetty, (2000), for A. awamori and A. japonicas by Jayani et
al.,( 2005). Damásio et al., (2011) reported highest activity of polygacaturonase
from Rhizopus microspores var. rhizopodiformis was recorded at pH 3.5 .
Fungal polygalacturonases from other sources show different optimum pH
conditions like verticillium albo atrum, 6.5 (Wang and Keen, 1970), Ganoderma
lucidum, 5.5 (Kumari and Sirsi. 1971), Pencillum capsulaturn, 4.68 (Gillespie et al.,
1990) and Penicillium frequentans, 4.0-4.7 (Borin et al., 1996). Even though a
marked variation of pH in polygalacturonase was found in different strains, the
162
optimum activity lies in between pH conditions 3.5-6.0. This is the typical
characteristic of fungal polygalacturonases (Rombouts and Pilnik, 1980). This is a
well establish fact that each enzyme has a characteristic pH optimum for its
activity (Lehninger et al. 1992), Wei-Chen et al., (1998) has observed the
maximum pectin layse ctivity at pH 8.0. Favela-Torres et al., (2006) observed that
the optimum pH for exo-Polygalacturonase was higher than the majority of
fungal PGs described, and they are considered as acidic enzymes.The results of
present study are also in accordance to Freitas et al., (2006) who obtained the
maximum activity for exo-PG at pH 5.5 when working with Monascus sp. and
Aspergillus sp. Pectinase. P. viridicatum RFC3 showed an optimum pH 6.0 (Silva et
al., 2007), from Moniliella sp. SB9 at pH 4.5 and Penicillium sp. EGC5 at pH 4.5 -
5.0 (Martin et al., 2004). The optimum activity for PL was pH 5.0, at pH 6.0; PL
activity decreased by 93% but was inactivated in neutral and basic pH. The pH
optima of the previously reported PL have been found to be acidic for Penicillium
canescens (5.5), (Sinitsyna et al., 2007), neutral for Penicillium expansum (Silva et al.,
1993) and basic for Aspergillus flavus (8.0) (Yadav et al., 2008) and Aspergillus
terricola (8.0) (Yadav et al., 2009).
Figure-5.16: Effect of pH on crude Pectinase
163
vi- Effect of pH stability on crude pectinase : Crude enzymatic extract was
diluted (1:1) in different buffers (pH 3- 10 ) incubated at 37 °C and pectinase
activity was checked at various pH values (3.0 to 10.0) Figure-5.17 shows that the
the crude pectinase was stable over a range of 3.0 to 6.0 pH retain more than
70% activity up to pH 8.0.
These results are in accordance with Freitas et al., ( 2006) who observed that exo-
polygalacturonase from Monascus sp. was stable in between pH 4.5 - 6.0, while
that from Aspergillus sp. was stable at pH 4.0..These results are not in agreement
with Silva et al., (2007) who worked with P. viridicatum RFC3 and reported
stability of exo-polygalacturonase in a pH of range 7.0 - 10.0. Marcia et al., (1999)
reported the the stability of in a pH range of 6-8 while Martin et al., (2004)
reported that polygalacturonase was stable at the pH range between 3-8 obtained
from Penicillium sp. and maintained 70% of its initial activity but pectin lyase
was stable in acidic to neutral range of pH (4-8 ) produced from same oraganism.
polygalacturonase from A. sojae ATCC 20235 was stable at pH 5.0 and retained
60 % and 70 % of its activity at pH 3.0 and 7.0 respectively Tari et al., (2008).
polygalacturonase of Penicillium viridicatum RFC3 was observed stable at pH 5.0-
8.0 Silva et al., (2002) and maintaining 80% of its activity at pH 9.0. pectin lyase
was more sensitive to pH variation, presenting maximum stability at pH 3.5 - 4.5
which declined to 80% at pH 5.0. Martin et al., (2004) reported that pectin lyase
produced by Moniliella sp SB9 and Penicillium sp EGC5, and was stable in acidic
to neutral pH (4.0-7.0). However, the results of Yadav et al., (2008 and 2009)
indicated that the stability of pectin lyase was noted in a pH range of 4.0 - 10.0
and 4.0 - 9.0 with A. flavus and A. terricola, respectively. According to Pedrolli
and Carmona, (2010) polygalacturonases of fungal origin are typically stable in
acidic medium, yet, polygalacturonase produced by A. giganteus proved to be
more stable over a neutral and alkaline pH range. It seems that pectinase
produced through A. niger works better in acidic environment.
164
Figure-5.17: Effect of pH stability on crude Pectinase
vii- Effect of temperature on crude Pectinase: The A. niger Pectinase activity was
investigated on different temperatures. The influence of temperature to pectinase
activity was displayed in Figure 5.18. Pectinase activity increased between the
temperature 30 °C and 50 °C and the 40 °C was found optimum temperature.
The decreased activity of the enzyme at temperatures above 40 °C could be due
to protein denaturation at higher temperatures.
Banu et al., (2010) reported the optimum temperature was found to be at 50 °C
when highest activity was recorded for the polygalcturonase obtained from
P. chrysogenum. Alana et al., (1990) also reported the similar results the for pectin
lyase enzyme from P. italicum which showed an increase of activity up to 50 °C.
El-Batal et al., (2013) reported that highest activity of polygalacturonase from P.
citrinum achieved at 40 °C, similar results were shown by Palaniyappan et al.,
(2009) and Arotupin et al., (2012) who reported a decline in the enzyme activity
with a temperature more than 40°C. Arotupin, (2007) investigated that
polygalacturonase produced by A. Flavus, A. fumigatus and A. repens showed
highest activity at 35 °C, 40 °C and 45 °C respectively. According to Arotupin
(1991) who explained these differences in the optimum temperature of fungal
polygalacturonase imply a wide range of temperature, In addition the
0
20
40
60
80
100
120
3 4 5 6 7 8 9 10
% R
ela
tiv
e A
ctiv
ity
mg
/ml
pH
165
environment, sources and differences in the physiological activities of the fungi
may be dependable for this phenomenon.
Temperature °C
Figure-5.18: Effect of temperature on crude Pectinase
166
viii- Effect of temperature stability on crude Pectinase: The activity was
investigated on different temperatures by heating for 10 minutes from 20 -100 °C
with an interval of 10 °C and the remaining activity was checked by adding
substrate according to standard method.
Enzymes are specific to temperature, every enzyme is optimally active and stable
up to a certain temperature and gets denatured at higher temperatures while
crude Pectinase was 100 % stable up to 40 °C and then activity declines slowely,
pectinase and retains more than 30 % activity up to 80 °C as shown in Figure
5.19. These results are in accordance with those obtained by Phutela et al., (2005),
and in contrast with Martin et al., (2004) who reported that polygalactouranase
from Penicillium sp. was stable in temperature lower than 40 °C
polygalactouranase activity of Rhizopus microsporus var. Rhizopodiformis was
stable up to 55 ºC Damásio et al., (2011). An endo- polygalactouranase of A. niger,
produced in solid state fermentation, was stable up to 40 ºC and presented 60 %
of its maximum activity after about 60 min of incubation at 50 ºC Hendges et al.,
(2011).
Temperature °C
Figure-5.19: Effect of temperature stability on crude pectinase
0
20
40
60
80
100
120
20 30 40 50 60 70 80 90 100
% R
ela
tiv
e A
ctiv
ity
167
ix- Effect of metal ions or compounds on crude pectinase: Effect of various
metal ions and compounds on pectinase activity was investigated by adding the
enzyme with 5 mM of each reagent in 0.1 M Sodium citrate buffer pH 5.5
alongwith enzyme and incubated at optimum temperature for 10 minutes prior
to addition of substrate and their remaining activities were determined.
Figure.5-20 shows the effect of various metal ions and compounds (5mM) on
Pectinase activity produced by A. niger. Among the metal ions tested, the
addition of 5 mM CaCl2 enhanced the activity of pectinase enzyme produced by
A. niger while MnSO4 , AgNO3 , HgNO3 CoCl2 inhibited pectinase enzyme
activity to the level of 40 % to 60 %. There is a significant influence by Ca2+
on the
activity and stability of enzymes (Shevchik et al., 1999). Cabanne and Doneche,
(2002) reported the partial inhibition of polygalacturonase by 1mM of CaCl2 might be
due to the chelating effect of calcium on the substrate (PGA) of the enzyme. Hg had no
effect on Polygalacturonase on Neurospora crassa (Polizeli et al., 1991). ZnSO4 was
an activator of polygalacturonase produced by Rhizopus sp. Elegado and Fujio,
(1994). Afifi and Foaad, (2002) reported that enzyme was inhibited with the
addition of Co++, Mn++, Zn++ and showed complete inhibition with the
addition of Ag++ and Hg++, similar results were produced by Chen et al.,
(1998). The change in electrostatic bonding could change the tertiort structure of
enzyme and may be metal ions change the electrostatic bonding Palmer,
(1991).According to Afifi and Foaad, (2002) reported that it could be due to
participation of sulphydryl groups in the active site of the enzymes. Afifi and
Foaad, (2002) reported that EDTA and L-cysteine did not affect enzyme activity
(Starr and Moran, 1962; Whilaker, 1972; Delgado et al., 1992).
168
Figure-5.20: Effect of metal ions/ compounds on pectinase activity
x- Effect of different concentration of CaCl2 as activator: After investigating that
CaCl2 was best activator for pectinase activity and there fore the effect of
different concentrations CaCl2 were tested and it was observed that increase in
CaCl2 concentration increases Pectinase activity upto 15 mM. As described in
Figure- 5.21.This was also observed that with the increase of CaCl2 concentration
decreased the enzyme activity may be due to the higher amount of activator acts
as inhibitor. However, in some cases the activity was inhibited, in other cases it was
stimulated by CaCl2 Perley and Page, (1971). Some of the polygalacturonases require
Ca+2 whether Ca+2 performs a role in binding and/or catalysis, in maintaining the
conformation of the enzyme, or whether it masks the car boxylic groups of the substrate
is not clear. Polygalacturonase was found to be inhibited by Ca+2 in the cases of,
Neurospora crassa Polizeli et al., (1991) and Rhizopus sp. Elegado and Fujio, (1994)
pectinase and was activated by Ca+2 in Geotrichum candidum (Shastri et al., 1988).
Banu et al., (2010) and Hla et al., (2005) have reported that normal optimal
activity was observed by Clostridium stercorarium at 0.05 mM of CaCl2 and the
enzyme was more or less activated throughout a range of CaCl2 concentrations
0
20
40
60
80
100
120
140%
of
Acti
vati
on
& in
hib
itio
n
169
from 0.05 to 0.2 mM but addition of 0.2 mM EDTA inhibited activity to less than
5% of maximum.
Figure-5.21: Effect of different concentrations of CaCl2
xi- Effect of thermostability with and without activator: The thermostability of
pectinase was checked by incubating the crude pectinase with and without
activator (15 mM CaCl2) in 0.1 M Sodium citrate buffer of pH 5.5 at two different
temperatures (60 ºC and 70 ºC) for 60 minutes and remaining activity was
determined under standard assay conditions. Figure-5.24 shows the temperature
profile that crude pectinase retained 81.28% and 74.32% relative activities with
and without activator respectively and lost about 18.72%and 25.68% activity
with and without activator respectively, when heated at 60°C for 60 minutes
respectively as shown in Figure- 5.22.
Similarly in Fig 5.23, The thermostability of pectinase was checked by incubating
the crude pectinase with and without activator (15 mM CaCl2) in 0.1 M Sodium
citrate buffer of pH 5.5 at 70 ºC for 60 minutes and remaining activity was
determined under standard assay conditions. Figure-5.25 shows the temperature
profile that crude pectinase retained 75.32 % and 67.2 % relative activities with
and without activator respectively and lost about 24.28 % and 32.2% activity with
84
86
88
90
92
94
96
98
100
102
0.5 1 15 2 25
% of Re le tive a ctivity
Concentration of CaCl2
mM
170
and without activator respectively, when incubated at 70 °C for 60 minutes
respectively.
Figure-5.22: Effect of themostability at 60 °C on different time periods with and
without activator CaCl2 (15mM) on pectinase activity produced by Aspergillus niger
Figure-5.25: Effect of themostability at 70 °C on different time periods with and without activator CaCl2 (15mM) on pectinase activity produced by
Aspergillus niger
100 97.6493.53
85.278.46 75.34
10096.53
89.56
80.34
71.3467.2
0
20
40
60
80
100
120
10 20 30 40 50 60
Time period (minute)
% o
f R
leti
ve A
ctiv
ity
with activator without activator
100 99.1 97.3794.11
89.56
81.28
100 98.2395.32
89.23
82.21
74.32
0
20
40
60
80
100
120
10 20 30 40 50 60
% o
f R
elat
ive
Act
ivit
y
With Activator without Activator
171
C- Purification of enzyme: In present study pectinase from Aspergillus niger was
purified and characterized. Purification of microbial enzymes is not easy because of the
removal of contaminating substances and also the separation of the enzyme from other
pectinolytic enzymes produced by the microorganisms. Normally one cell or tissue
homogenate contain thousands of different proteins and the purification seems to be
difficult. However in practice four different steps are involved in purification of proteins
and some times a single chromatographic step is enough based on the purpose of
purification.
For structural and functional studies 100% purification is not necessary and
enzymes can be purified several fold but the yield of the enzymes may be very poor.
Industrial enzymes are purified as little as possible i.e. for the removal of
interfering materials. Since additional stages are costly in terms of equipment,
manpower and loss of enzyme activity. As a result, some commercial enzyme
preparations consist especially of concentrated fermentation broth, plus
additives to stabilize the enzyme's activity Wilson and Walker, (2000). It is
important to retain maximum activity of the enzyme during its preparation.
The main objective of the first stage for the recovery of an
extracellular product and the removal of large solid particles as well as
microbial cells usually by centrifugation or filtration. In the next stage, the broth
is fractionated or extracted into major fractions using ultra filtration, reverse
osmosis, adsorption/ion exchange/gel filtration, two phase aqueous
extraction or precipitation. Afterwards, the product-containing fraction is
purified by fractional precipitation. Further more precise chrornatographic
techniques and crystallization are done to obtain a product, which is free from
impurities. Purification of enzyme is very important procedure. Enzyme test while
it is crude does not exhibit isolated action or the presence of a system of multi
enzyme working together to degrade a substrate Pedrolli et al., (2009). It is also
very important line of research that purified enzymes may be characterized as it
differentiates between the enzymic complex components for substrate
172
degradation mechanism, optimum activity conditions and enzyme synthesis
regulation Pedrolli et al., (2009).
i- Removal of microbial cells and other solid matter: Microbial cells and other
insoluble materials are normally separated from the harvested broth by filtration or
centrifugation Stanbury et al., (1997) . Filtation separates particles simply on the basis
of their size. Centrifugation separates on the basis of the particle size and density difference
between the liquid and solid phases Chaplin and Bucke, (1990).
ii- Concentration by precipitation: Enzymes can be concentrated by
precipitation, and this is generally used as the initial step of purification.
Salting out by ammonium sulphate is the best-known method for concentration
and purification of the enzyme. Some enzymes do not survive with
ammonium sulphate precipitation. In such case organic solvents such as ethanol,
propanol and acetone can be used as the alternative. For further purification, the
ammonium sulphate present in the protein precipitate is to be removed. This can be
achieved by dialysis through 10,000 Da cut off membranes (dialysis membrane/bag)
iii- Ammonium sulphate fractionation: The cultured medium of fermentation
was centrifuged at 10,000 rpm for 20 minutes in a refrigerated condition. In this
study enzymatic protein from culture supernatant was precipitated with solvents
such as acetone, ethanol, and methanol with different concentration of
ammonium sulphate (40-80%). The experiments show that more pectinase
activity has been recovered with 60% solid ammonium sulphate precipitation.
The low pectinase enzyme activities were recovered in acetone, ethanol and
methanol may be due to the change of pH or temperature, which denatures the
proteins during the process (Holme and Peck, 1994 and Doi and Nojima, 1971).
Solid ammonium sulphate (GR grade MERCK) was slowly added to the
supernatant of crude enzyme preparation so as to reach 60% saturation.
Addition of ammonium sulphate was carried out with continuous stirring at
4.0 °C, and then it was kept at 4.0 °C for overnight. The precipitated protein was
removed by centrifugation at 10, 000 rpm for 30 minutes at 4.0 °C. The precipitated
173
protein was dissolved in 15 ml Sodium citrate buffer pH 5.0. The protein content
of the fraction was determined by the method of Lowry et al., (1951).
iv- Dialysis: The precipitate obtained after treatment with ammonium sulphate was
dialyzed against Sodium citrate buffer, pH 5.0 for overnight at 4.0°C with
occasional changes of the buffer. Cellulose membrane dialysis tubes was used for
dialysis (Cut off 10,000 Da).
D- Chromatography: Chromatographic techniques are used in the final
stages of purification. For enzyme purification, two types of chromatographic
principles are used.
Gel permeation chromatography: Gel permeation separates molecules on the
basis of pore size. The smaller molecules diffuse into the gel more rapidly than the
larger ones. The most widely used gels include cross-linked dextrans -
sephadex and sephacryl, and cross-linked agarose - sepharose with various pore
sizes Stanbury et al., (1997).
Ion exchange chromatography: Ion exchange can be defined as the reversible
exchange of ions between a liquid and solid phase (ion exchange resin), which is
not achieved by any radical change in the salt structure. Ion exchange materials
are generally water insoluble polymers containing cationic and anionic groups.
Carboxy methyl cellulose is a common cationic exchanger and diethyfaminoethyl
cellulose is a common anionic exchanger.
i- Gel filtration chromatography: The dialyzed sample of pectinase produced
from Aspergillus niger under submerged fermentation was purified by
ammonium sulphate precipitation 10 ml gel filtration chromatography on
sephadex G-100 chromatographic column. The Pectinase was eluted with 0.1M
sodium citrate buffer pH (5.5). Elution pattern is shown in Figure-5-24. Pectinase
activity and protein contents were determined for each fraction. All active
fractions were pooled and assayed for enzyme activity and protein. A recovery
of 9917 U of Pectinase activity with specific activity of 34.55U was obtained. This
gave 2.5 fold purification of the enzyme shown in Table-5.116.
174
Fig.5.24: Gel Chromatography
ii- Ion exchange Chromatography: The pooled and dialyzed fractions (F-3a and
F-3b) were separated on ion exchange DEAE Sephadex A-50 chromatographic
column. Equilibrium and elution shown in Figures 5.25 and were performed first
with 0.1 M Sodium citrate buffer (pH 5.0) to remove unbound proteins and then
with a linear salt gradient from 0.0 to 1.0 N NaCl. Active fractions were pooled
and specific activity of the pooled active fractions was measured as shown in
Table -1.116
175
Fig.5.25: Purifiction of Pectinase (F-3) on ion exchange chromatography
Table-5.116: Purification steps of Pectinase produced by Aspergillus niger
Sample Pectinase Activity Units
Total protein mg
Specific Activity
Purification Fold
% Yield
Culture broth (Crude sample)
13760 995 13.82 1 100
Dialyzed sample 11630 539 21.57 1.56 84.52
G – 100 Column Chromatography
9917 287 34.55 2.5 72.07
Fraction 1 3125 88.2 35.43 2.56 22.71
Fraction 2 2937 71.5 41.0 2.96 21.34
Fraction 3 1750 57.4 30.48 2.19 12.66
Fraction 4 2150 70.3 30.2 2.19 15.44
A – 50 Column Ion exchange
Chromatography
1750 57.4 30.48 2.19 12.66
Fraction 3-a 923 32.2 28.66 2.07 6.70
Fraction 3-b 827 25.2 32.81 2.37 6.0
176
iii- Homogeneity: SDS-polyacrylamide gel electrophoresis (Sambrook and
David 2001) of pectinase revealed multiple protein bands on staining (F-3) while
Fractions F-1, F-2 and F-4 shows single bands when staining with Coomassie
brilliant blue (Figure-5.26). When purified fractions were electrophoresed on
native 10% polyacrylamide gel were observed as different activity bands.
iv- Molecular weight: Electrophoretic mobilities of purified pectinase and
reference proteins on SDS-polyacylamide gel electrophoresis were plotted versus
their molecular weight. The mobility of the purified pectinases; Fraction F-I, F-2,
F-4, F-3a and F-3b corresponded to a molecular weight of 40,000 ± 5000 and of
35,000±3000 Da respectively. Figure-5.26-27. Fractions exhibiting pectinase
activity were pooled and used as purified enzymes indicates that two types of
pectinase are produced by Aspergillus niger. It has been reported by Buga et al.,
(2010) that SDS-PAGE of the active fractions showed two different bands on the
gel with visible molecular weights of 35 KDa and 40 KDa. The presence of
different bands implies the presence of a dimeric protein consisting of both
‘endo and exo’ polygalacturonase activities.
Khairnar et al., (2009) also reported the molecular weight of the pectinase
in his study was found to be 40 KDa. Kester et al., (1996) reported a pectinase
with a molecular weight of 20,000 Da while deVries and Visser (2001) isolated a
pectinase having a range of molecular weight as 35,000 – 80,000 Da. This range is
in agreement with the obvious molecular masses of the pectinase purified in this
study.
177
Figure-5.26: SDS-PAGE (10% Polyacrylamide) of the purified enzymes. Lane 1, low Mw
Marker; Lane 2, Fraction 1; Lane 3, Fraction 2; Lane 4, Fraction 3; Lane 5, Fraction 4; Lane
6, Crude enzyme
Figure-5.27: SDS-PAGE (10% Polyacrylamide) of the purified enzymes. Lane 1, low Mw
Marker; Lane 2, Fraction 3a; Lane 3, Fraction 3b; Lane 4, Crude enzyme
178
E- Characterization of purified Pectinase
i- Effect of Substrate specificity:
Pectins are complex high molecular mass glycosidic macromolecules in
the primary cell wall the major components of the middle lamellae, an adhesive
thin extracellular layer between the walls of adjacent juvenile cells and are
basically responsible for the structural uprightness and consistency of plant
tissues. (Rombouts et al., 1980, Alkorta et al., 1998). There are three major pectic
polyssacharides groups are renowned, to a greater or a lesser extent all contain
D-galacturonic acid. Pedrolli et al., (2009). It is reported by Kabli, (2007) that
pectinase enzyme was treated to check hydrolyzing activity with peels of Mango,
orange, lemon ,grape fruit and with beet pulp.Enzyme activity was exhibited
according to the type of substrate low activity was shown with beet pulp. Singh
and Appu, (2002) also reported that pectinase enzymes preferred their specific
substrate irrespective of the source.
The purified Pectinase enzyme was most active with pure pectin as
shown in Fig -5.28, while crude pectin, Lemon pectin and orange peel given
lower activity as compared to (control) i-e pure pectin. Similar results were
presented by Pedrolli and Carmona, (2010) who described that substrate
specificity for polygalacturonases isolated from Aspergillus giganteus as for citrus
pectin was 100%, polygalactouranic acid 94.9%, citrus pectin 51.9%, citrus
pectin 25.5% and apple pectin 23.9%. The present work is supported by Fahmy et
al., (2008), enzyme from A. niger NRRL3 shown citrus pectins with different
esterification 41 to 97%) Mohamed et al., 2009 reported that pectinase enzyme
isolated from T. harzianum shown citrus pectins with different esterification to
187%. Pectinase from Mucor circinelloides ITCC 6025 hydrolyze citrus pectin 11.0
% and apple pectin 22 % Thakur et al., (2010). More or less similar findings were
reported by Al - Najada et al.,(2012).
179
Figure-5.28: Effect of substrate specificity on pectinase produced by
Aspergillus niger
ii- Effect of substrate concentration on pectinase activity:
Effect of initial reaction rates of pectin hydrolysis by pectinase was estimated
with purified enzyme. Highest pectinase enzyme activity was observed when
1.5% pectin in 0. 1 M sodium citrate buffer pH (5.0) was used as a substrate in the
reaction mixture and incubated for 15 minutes at 37 °C.
Results were shown in Figure-5.29 indicates that significant increase in
pectinase activity was achieved with the increase of substrate concentration.
After the maximum concentration pectinase activity started to declined in
fractions FI, F2 , , F3-b, and F 4 where as the maximum substrate concentration F3-a,
was noted to 1.0 of purified pectinase enzymes. The findings of (Nitinkumar et
180
al., 2010 ) are in agreement with the present study that when 1.5% substrate was
used the enzyme activity was highest.
Fig.5.29: Effect of substrate concentration on Pectinase activity produced by
Aspergillus niger
iii- Effect of pH on pectinase activity produced by Aspergillus niger:
The pectinase activity was investigated at various pH values to check the
optimum pH for the purified enzyme produced by A. niger .The fig-5.30 shows a
graphical representation which exhibits that with the increase in pH, the activity
of the enzyme increased up to an optimum pH. However, further increase in pH
has shown a gradual decrease in pectinase activity in all the fractions like (F1, F2 ,
F3-a, F3-b and F4, ) which was between pH 5.0 and 6.0 and these results are higher
than those generally evident for fungi in the range of 3.0 and 5.0 (Kojima et al.,
1999, Martins et al., 2002, Niture and Pant, 2004, Martin et al., 2004) but the
observations of present study are in full agreement with the results of Pedrolli
et al., (2008) who reported optimal pH for polygalacturonase activity as 5.5–
6.0.In literature fungal pectinase is reported to show maximum activity at a pH
4.0-5.0 ( Jayani et al., 2005, Niture et al., 2008), and mostly more active in acidic or
neutral pH Pedrolli et al., (2009). According to Pedrolli and Carmona, (2010)
0
1
2
3
4
5
6
7
8
0.5 1 1.5 2 2.5 3
Substrate Concentration
Pec
tin
ase
Act
ivit
y IU
/mL
F-1 F-2 F-3a F-3b F-4
181
Polygalacturonase isolated from A.gigantus shows optimum activity at pH 6.0–
6.5. Kashyap et al., (2001) , Gummadi and Panda (2003), Jyothi et al., (2005), Díaz-
Godínez et al.,(2001) and Tari et al.,(2007) reported that acidic pectinases are
mostly fungal, especially from Aspergillus niger with optimum pH of 4.5-6 . The
pectinase isolated from Kluyveromyces wickerhamii showed optimum pH of 5.0
according to Moyo et al., (2003).
The results of optimum pH of purified pectinase are comparable with
the results of Al-Najada et al., (2012) who has reported that acidic pH optima
was found 4.0 for PGase from F. oxysporum ,while PH optima for PGase I and
PGaseII was found as 4.5 and 6.0 respectively from A. tubingensis. Similar results
were reported by Damasio et al., (2010) for polygalacturonase obtained by P.
variotii pH 4.0, where as Schnitzhofer et al., (2007) reported pH 4.5 to 5.0 for
enzyme isolated from Scleroderma rolfsii. Saad et al., (2007) presented the findings
for pH optima as 4.5 for pectinase from Mucor rouxii NRRL 1894 .
Pectinolytic enzyme shown optimum pH 3.8 from A. niger was reported by
Khairnar et al., (2009), from Mucor circinelloides ITCC 6025 with pH 5.5 Thakur et
al., 2010, by A. tubingensis with pH 4.2 (Gewali et al., 2007) and Aspergillus oryzae
with pH 5.0 , (Riou et al., 1998), Alana et al., (1989) have also reported low pH
values as being favorable for high pectinase production in Penicillium italicum.
The optimum pH of mesophilic pectinases has been established to range
between 4.0-5.5 (Favela-Torres et al., 2006).
Cylindrocarpon destructans pH 5.0 (Sathiyaraj et al., 2011) and typical
characteristic of fungal polygalacturonase according to Rombouts et al., (1980)
182
Fig.5.30: Effect of pH on pectinase activity produced by Aspergillus niger
iv- Effect of pH stability on pectinase activity produced by Aspergillus niger:
The effect of pH stability on Pectinase activity was investigated at various pH
values for the purified enzyme produced by A. niger. The result shown in
Figure-5.31 state that all the Fractions (F1, F2 , F3-a, F3-b and F4, ) are stable up to pH
range 3.0-8.0 . More that 30 % activity was retained when purified Pectinase was
incubated with pH 8.0 The results are in strong accordance to Pedrolli and
Carmona (2010) who isolated a pectionase from A. giganteus which shown
stability over a neutral and alkaline pH range. Yet, fungal pectinase are usually
stable in the acidic range of pH as reported by Devi and Rao (1996), Niture and
Pant, (2004), Jayani et al., (2005), and Dinu et al., (2007).
The results presented by Siddiqui et al., (2012) shown low range of pH stability as
compared to present stuy. He has presented the results of pectinase enzyme
from R. pusillus and the stability of the purified polygalacturonase was recorded
at pH 4.0-5.0. beyond this pH range enzyme stability began to decrease. At pH
8.0 after 4th hour the residual activity was 40.21% of that of the control while no
activity was recorded at pH 9.0 at 4th hour. Martos et al., (2013) reported about a
PG isolated from A. niger which shown highest activity at a pH range between
4.5 to 5.0. At pH 4.0 and 5.5, polygalacturonase activity values were 35 % and 38
0
20
40
60
80
100
120
3 4 5 6 7 8 9 10
pH
% o
f R
elat
ive
Act
ivit
y
F-1 F-2 F-3a F-3b F-4
183
% respectively. The enzyme was stable at 4ºC for 24 h over a pH range between
2.5 to 5.5. At pH 7.5, the residual activity was 54 % (Tari et al., 2008) and A. sojae
ATCC 20235 was stable at pH 5.0 and retained 60 % and 70 % of enzymetic
activity at pH 3.0 and 7.0 respectively
On the basis of above result, it could be predicted and recommended that
A. niger is the best candidate for Pectinase production as compared to other
fungi. The enzyme has a significant future in commercial use especially in food
industries.
Fig.5.31: Effect of pH stability on pectinase activity produced by Aspergillus niger
v- The Effect of temperature on pectinase activity produced by Aspergillus
niger:
The effect of temperature on pectinase activity was investigated at various
temperatures for the purified enzyme produced by A. niger. The result shown in
Figure-5.32 reveals that all the Fractions (F1, F2 , F3-a, F3-b and F4, ) are showing
maximal activity at 40 and 50 °C respectively. These optimal temperature results
were similar to those observed for pectinase enzyme obtained from Streptomyces
erumpens at 50°C ( Karl and Ray, 2011). Dogan and Tari, (2008) presented that
pectinase isolated from Aspergillus sojae (55 °C) shown slightly higher optimum
temperature. The present results are in agreement with the results of Nabi et al.,
0
20
40
60
80
100
120
3 4 5 6 7 8 9 10
pH Stability
% o
f R
elat
ive
Act
ivit
y
F-1 F-2 F-3a F-3b F-4
184
(2003) and Fahmy et al., (2008) for Pectinase enzymes isolated from
Trichoderma harzianum (40 °C) and A. niger NRRL3 (40 °C) respectively. While
Favela-Torres et al., (2006) reported that Pectinase obtained from Streptomyces sp
QG-11-3 have optimal activity at 60 °C. Pectinase from fungi have optimum
activity at 50 °C while yeast pectinase has a wide range of optimum temperature
from 40 °C to 60 °C. Acoording to Pedrolli and Carmona, (2010) purified
polygalacturonase showed highest at 55-60 °C. Pedrolli et al., (2009) reported
that that optimum temperature of PG from fungi was repeatedly recorded at 40-
60 °C. Siddiqui et al., (2012) reported the optimum temperature (55 °C) of the
purified pectinase enzyme produced by R. pusillus. While Martins et al., (2002)
reported high temperature optima 60 °C for the enzyme obtained from
Thermoascus aurantiacus. Kaur et al., (2004) reported little higher temperature
optima for Sporotrichum thermophile apinis at 55 °C . While Thakur et al., (2010)
reported temperature optima for Mucor circinelloides at 42 °C. Andrade et al.,
(2011) reported the high optimum temperature for polygalacturonase enzyme
between 60–70°C which is contrast with the present study.
Fig.5.32: Effect of temperature on pectinase activity produced by
Aspergillus niger
0
20
40
60
80
100
120
20 30 40 50 60 70 80 90 100
Temperature
% o
f R
elat
ive
Act
ivit
y
F-1 F-2 F-3a F-3b F-4
185
vi- Effect of temperature stability on pectinase activity produced by
Aspergillus niger :The effect of temperature stability on pectinase activity was
examined at various temperatures for the purified enzyme isolated from A. niger.
The result shown in Figure-5.33 state the graphical presentation of
thermostability of all the Fractions (F-1, F-2 , F-3a, F-3b and F-4,) the enzyme is
maximally stable upto 60 to 70 °C and then decreased as the incubation
temperature was increased. The decrease in temperature stability is probably due
to enzyme denaturation at higher temperatures. More or less same observations
were reported by Pedrolli et al., (2008). Generally Pectinase activity in many
filamentous fungi is stable in a range of 50–60 °C as reported by Kaur et al.,
(2004), Kapoor et al., (2000), Kojima et al., (1999), Silva et al., (2002) and Ortega et
al., (2004). The results having similarity with the results of Siddiqui et al., (2012)
that purified pectinase enzyme from R. pusillus which was found stable at 50 °C
and this study is fully agreed with Martins et al., (2002). The activity of Pectinase
enzymes is depended on thermostability Chaudhri and Suneetha, (2012).
Thermal stability was reported by Damasio et al., (2010) for pectinase enzyme
obtained from P. variotii as 45 to 55 °C. Nabi et al., (2003) reported that pectinase
from T. harzianum was stable upto 60 °C and similar results were presented by
Dogan and Tari, (2008) who reported that thermal stability of pectinase by A.
niger was noted 45 to 55 °C. Kaur et al., (2004) reported temperature stability at
65°C for the pectinase enzyme obtained from Sporotrichum thermophile apinis.
Thakur et al., (2010) reported that the enzyme was stable at 42 °C.
186
Fig.5.33: Effect of thermostability on pectinase activity produced by Aspergillus niger
vii- Effect of activators and inhibitors: The Effect of different metal ions and
compounds at 5mM concentration was tested with purified pectinase activity as
shown in Figure-5.34-5.38. It was observed that 30 to 40% inhibiting effect was
shown on pectinase activity by 1, 10 Phenonthroline in all Fractions (FI, F2,, F3-a,
F3-b and , F4). The Pectinase activity was stimulated in the presence of CaCl2 in all
fractions in the effect of 10-30%, which is contrast to Pedrolli and Carmona (2010)
who reported that no significant effect was shown by Ca2+ on polygalacturonase
activity. ZnSO4 , MnSO4 and Mg SO4 shown higher activity in fractions ( F3-a,
F3-b and F4), while in fractions F1 and F2 ZnSO4 and MnSO4 shown slight
inhibition effect on pectinase activity which is in agreement with Pedrolli and
Carmona (2010) who reported slight inhibition of Zn2+ on prctinase activity Al
Najada et al., (2012 ) reported that Zn 2+ caused inhibition in activity of pectinase
enzyme isolated from F. oxysporum , while CoCl2 , AgNO3, HgNO3, and EDTA
have strong inhibition effect on the pectinase activity in all fractions (FI, F2,, F3-a,
F3-b and F4) which is in partial agreement with Al Najada et al., (2012) who
reported that Hg2+ caused inhibition in pectinase activity isolated from
F. oxysporum , while Co2+ found to enhance the enzyme activity. Pedrolli and
0
20
40
60
80
100
120
20 30 40 50 60 70 80 90 100 100
Temperature Stability
% o
f R
ela
tiv
e A
ctiv
ity
F-1 F-2 F-3a F-3b F-4
187
Carmona, (2010) reported that Co2+ showed an enhancing effect on pectinase
activity but Hg2+ showed total inhibition while EDTA inhibited enzyme
activity.
Zn2+, Pb2+ and Hg2+ caused 12, 39 and 32% inhibition, while Ca2+, Co2+ and
Ni2+ were found to activate the enzyme by 56, 38 and 32%, respect-tively
for F.oxysporum polygalacturonase activity. A. tubingensis PGaseI and PGaseII
were activated by Zn2+, Ca2+, Co2+, where Ni2+ only activated PGaseII. The other
cations Cu2+, Hg2+ and Pb2+ had partially inhibitory effect. In P. viridicatum,
Ca2+ was foundto enhance the stability of polygalacturonase, while Hg2+,
Zn2+ and Cu2+ were strongly inhibited (Gomes et al., 2009). Co2+ and Cu2+ had no
inhibitory effects on the polygalacturonase activity of P. variotii ( Damasio et al.,
2010). Polygalacturonases from M. rouxii (Saad et al., 2007) and A. niger NRRL3
(Fahmy et al., 2008) were partially inhibited by Ca2+, Zn2+, Cu2+, Co2+, Ni2+ and
Hg2+. These results also shown a significant role of the cysteine in the catalysis
and/or substrate binding by the purified pectinase of A. niger and the results
show agreement with the results of Nelson and Cox, (2004), Scopes, (1994) and
Pedrolli and Carmona, (2010) Kaur et al., (2004) , Kapoor et al., (2000), and
Semenova et al., (2003). Kashyap et al., (2000) reported that some pectinases are
dependent on cations like Ca2+. Pedrolli et al., (2008) described that complexes
are formed between anions and cations like Ca2+ or any other which are required
for the activity of the enzyme.
188
Fig.5.34: Effect of activators & inhibitors (F-1)
Fig.5.35: Effect of activators & inhibitors (F-2)
0
20
40
60
80
100
120
140
Con
trol
Cys
teine
Met
hion
ene
EDTA
1,10
Phe
nont
hrol
ine
ZnSO
4
CaC
l2
MnS
O4
AgNO
3
CoC
l2
MgS
O4
HgN
O3
% o
f A
ctiv
atio
n &
in
hib
itio
n
0
20
40
60
80
100
120
140
Con
trol
Cys
teine
Met
hion
ene
EDTA
1,10
Phe
nont
hrol
ine
ZnSO
4
CaC
l2
MnS
O4
AgN
O3
CoC
l2
MgS
O4
HgN
O3
% o
f A
cti
vati
on
& in
hib
itio
n
189
Fig.5.36: Effect of activators & inhibitors (F-3a)
Fig.5.37: Effect of activators & inhibitors (F-3b)
0
20
40
60
80
100
120
140
Con
trol
Cys
teine
Met
hion
ene
EDTA
1,10
Phe
nont
hrol
ine
ZnSO
4
CaC
l2
MnS
O4
AgN
O3
CoC
l2
MgS
O4
HgN
O3
% o
f A
cti
vati
on
& in
hib
itio
n
0
20
40
60
80
100
120
140
Con
trol
Cys
teine
Met
hion
ene
EDTA
1,10
Phe
nont
hrol
ine
ZnSO
4
CaC
l2
MnS
O4
AgNO
3
CoC
l2
MgS
O4
HgN
O3
% o
f A
cti
vati
on
& in
hib
itio
n
190
Fig.5.38: Effect of activators & inhibitors (F-4)
CONCLUSION
The major objective of the present study was to extract the important
bioproduct that is extracellular fungal pectinase. From the studies on pectinase
production from Aspergillus niger, it would be appealing to develop this strain
for the production of pectinase enzyme. Pectinase from this strain can be
recommended for the commercial production because of its constitutive and
less catabolically repressive nature, thermostability (upto 50°C), wide range of
pH (4.0-5.5), utilization of agro-industrial waste as well as cost effective
production could be a best opportunity to for industrial use and applications of
this enzyme. However, scale-up studies are needed for the better output for
commercial production. It is also concluded that the pectinase produced by
Aspergillus niger can be used in juice and beverage industry, which save huge
amount of foreign exchange to purchase and import pectinase enzyme.The
0
20
40
60
80
100
120
140
Con
trol
Cys
teine
Met
hion
ene
EDTA
1,10
Phe
nonthr
oline
ZnSO4
CaC
l2
MnS
O4
AgN
O3
CoC
l2
MgS
O4
HgN
O3
% o
f A
cti
va
tio
n &
in
hib
itio
n
191
enzyme is found to be thermostable and has wide pH stability and optimum pH
in the acidic range. These factors establish its importance in fruit juice industries.
The acid tolerant property of the polygalacturonase from Aspergillus niger makes
the enzyme an ideal candidate for tissue maceration, juice extraction and
clarification in the fruit and vegetable processing industry.The result revealed
that in comparison to other organisms Aspergillus niger is found best for pectinase
production.The maximum pectinase production was achieved with 5% molasses
along with 5.0% sucrose as carbon source and Ammonium sulphate0.4% as
nitrogen source with initial pH 6.0 and 35 °C temperature at 72 hours.
The crude enzyme characterization proved that enzyme is thermostable
and pH stable. .The enzyme is found to be thermostable and has wide pH
stability and optimum pH in the acidic range. The pectinase enzyme produced
by A. niger was purified by gel chromatography on Sephadex G-100 and Ion
Exchange DEAE A-50. with NaCl gradient concentration 0.0 to 1.0N. The
molecular weight of the purified fractions were found to be 33000 ± 2000 and
38000 ± 2000 Dalton by SDS-PAGE. All fractions show homogeneity on SDS-
PAGE.The purified enzyme characterization proved that enzyme is thermostable
and pH stable, which is suitable for industrial use.
Pectninase from Aspergillus niger could convert pectins from different
sources successfully into simple sugars and thus the enzyme could not only act as
an agent for bioconversion but also could replace the use of highly expensive
commercial pectin in food industry.
192
Further Suggesstions :
The acid tolerant property of the Pectinase from Aspergillus niger makes the
enzyme an ideal candidate and may be used for tissue maceration, juice
extraction and clarification in the fruit and vegetable processing industry.
Pectninase from Aspergillus niger could convert orange peel pectin successfully,
and thus the enzyme could not only act as an agent for bioconversion but also
could be used to replace the use of highly expensive commercial pectin in food
industry.
Present study could open an avenue for the analysis of various other factors,
which can influence the production of pectinase.
The work can be done to analyze active centre, analysis of amino acids and
sequencing of pectinase, which can help to understand its structure.
193
REFERENCES
Abbott, E.V. (1923), The occurrence and action of fungi. Soil Sci. 16: 207–216.
Abbott, E.V. (1926), Taxonomic studies on soil fungi. Iowa State College Journal of
Sci. 1 (1): 15-36.
Abdullah, R., Ashraf, H and Haq, I. (2003), Optimization and kinetic analysis of
carbon sources on the production of alpha amylase by Saccharomyces
cerevisiae. Journal of food Technology 1(14): 187-190.
Acuña-Argüelles, M. E., Gutierrez-Rojas, M., Viniegra-Gonza´lez G., Favela-
Torres, E. (1995), Production and properties of three pectinolytic activities
produced by Aspergillus niger in submerged and solidstate fermentation.
Applied and Microbiology Biotechnology, 43, 808-814.
Afifi, A. F and Foaad, M. A. (2002), Purification and characterization of pectin
lyase produced by Curvularia inaequalis NRRL 13884 on orange peels
waste, solid state cultivation. Annal. Microbiol. 52:287−297.
Aguilar, G. and Huitron, C. (1986), Application of fed-batch treatments in the
production of extracellular pectinases by Aspergillus sp. Enzyme Microb.
Technol. 8: 541-545.
Aguilar, G. and Huitron, C. (1987) Stimulation of the production of extracellular
pectinolytic activities of Aspergillus sp. by galacturonic acid and glucose
addition. Enzyme Microbial. Tecnhol. 9: 690-696.
Akhilesh, T., Roma Pahwa, R., Singh, S and Gupta, G. (2010), Production,
purification, and characterization of polygalacturonase from Mucor
circinelloides ITCC 6025. Enzyme Research, Pp: 7 Article ID 170549.
194
Alaea, A., Gabilondo, A., Hernando, F., Moragues, M. D., Dominguez, J. B.,
Llama, M.J., and Serra, J.L . (1989), Pectin Lyase production by a
Penicillium italicum strain. Appl. Environm. Microbiol. 55:1612-1616.
Alana, A., Gabilondo, A., Hernando, F., Moragues, M.D., Dominguez, J.B.,
Llamam, M.J and Serra, J.L. (1989), Pectin Lyase pro-duction by a
Penicillium italicum strain. Appl. Environ. Microbiol. 55:1612-1616
Alana, A., Alkorta, I., Dominguez, J.B., Liama, M.J and Serra, J.L. (1990), Pectin
lyase activity in a Penicillium italicum strain. Appl. Environ. Microbiol. 56
(12): 3755-3759.
Alazard, D and Raimbault, M. (1981), Comparative study of amylolytic enzymes
production by Aspergillus niger in liquid and solid-state cultivation. Eur.
J. Appl. Microbiol 12: 113-1 17.
Al-Farsi, M., Alasalvar. C, Al-Abid. M, Al-Shoaily. K, Al-Amry. M and Al-
Rawahy, F. (2007), Compositional and functional characteristics of dates,
syrup, and their by-products. Food Chem. 104:943-947.
Al-Hooti, S. N., Sidhu, J.S., Al-Saqer, J. M and Al-Othman, A. (2002), Chemical
composition and quality of date syrup as affected by pectinase / cellulase
enzyme treatment. Food Chem. 79:215-220.
Alkorta, I., Garbisu, C., Llama, M.J and Serra, J.L. (1998), Industrial applications
of pectic enzymes. Process Biochem. 33: 21-28.
Al-Najada, A.R., Al-Hindi, R.R and Mohamed, S.A. (2012), Characterization
of Polygalacturonases from fruit spoilage Fusarium oxysporum and
Aspergillus tubingensis. African Journal of Biotechnology, 11 (34) : 8527-
8536.
Alvarez, S., Alvarez, R., Riera, F. A. and Coca, J. (1998),Influence of
depectinization on apple juice ultra filtration. Colloids and Surfaces A:
Physicochemical and Engineering Aspects 138(2): 377–382.
195
Andrade, M.V.V., Delatorre, A.B., Ladeira, S.A and Martins, M.L.L. (2011),
Production and partial characterization of alkaline polygalacturonase
secreted by thermophilic Bacillus sp. SMIA-2 under submerged culture
using pectin and corn steep liquor. Cienc. Technol. Aliment. Campinas
31(1):204-208.
Apel, P.C., Panaccione, D.G., Holden, F.R and Walton, J.D. (1993) Cloning and
targeted gene disruption of XYL1 a β-1, 4 xylanase gene from the maize
pathogen Cochlobolus carbonum. Mol Plant-Microbe Interac 6:467-473
Arijit, D., Sourav,B., Reddy Naimisha, V., and Sundara Rajan, S. (2013),
Improved Production and Purification of Pectinase from Streptomyces sp.
GHBA 10 isolated from Valapattanam mangrove habitat, Kerala, India Int.
Res. J. Biological Sci. 2(3): 16-22
Arotupin, D.J. (1991), Studies on the microorganisms associated with the
degradation of sawdust, M.Sc. Thesis, University of IIorin, IIorin, Nigeria.
Arotupin, D. J. (2007), Effect of different carbon sources on the growth and
Polygalacturonase activity of Aspergillus flavus isolated from cropped
Soils. Research Journal of Microbiology 2(4): 362-368.
Arotupin, D. J., Akinyosoye, F. A., and Onifade, A. K. (2008), Purification and
characterization of pectin methylesterase from Aspergillus repens isolated
from cultivated soil. African Journal of Biotechnology 7 (12): 1991–1998.
Arotupin, D. J., Akinyosoye, F.A and Onifade, A. K. (2012), Malaysian Journal of
Microbiology 8(3), 175-183.
Bahkali, A, H. (1995), Production of Cellulase, Xylanase and Polygalacturonase
by Verticillium tricorpus on different Substrates.Bioresources Technology
51:171-174.
196
Bailey, J., Bailey, P and Poutanen, K. (1992), Interlaboratory testing of methods
for assay of xylanase activity. Journal of Biotechnol. 23: 257-70
Bailey, M.J and Pessa, E. (1990), Strain and process for production of
polygalacturonase. Enzyme Microbiol.Technol. 12: 266-271.
Banu, A.R., Devi, M.K., Gnanaprabhal, G.R., Pradeep, B.V and Palaniswamy, M.
(2010), Production and characterization of pectinase enzyme from
Penicillium chysogenum. Indian Journal of Science and Technology 3(4):
377 – 381
Baracat-Pereira, M.C., Coelho, J.L.C., Silva, D.O. (1994), Production of pectin
lyase by Penicillium griseoroseum cultured on sucrose and yeast extract for
degumming of natural fibers. Lett. Appl. Microbiol. 3: 127-129.
Barnby, F.M., Morpeth, F. F and Pyle, D.L. (1990), Endopolygalacturonase
production from Kluyveromyces marxianus. I. Resolution, purification and
partial characterization of the enzyme. Enzyme Microbial Technol. 12:
891–7.
BBC Research (2011) In Report BIO030F - Enzymes in Industrial Applications:
Global Markets.
Beg, Q.K., Kapoor, M., Tiwari, R. P and Hoondal, G.S. (2001), Bleach-boosting
of eucalyptus kraft pulp using combination of xylanase and pectinase
from Streptomyces sp. QG-11-3. Res Bull Panjab University 57: 71–8.
Blanco, P., Sieiro, and Villa, T.G. (1999), Production of pectic enzymes in yeasts,
FEMS Microbiol Lett., 175 (1) :1-9.
Blandino, A., Dravillas, K., Cantero, D., Pandiella, S.S and Webb, C. (2001)
,Utilization of whole wheat flour for the production of extracellular
pectinases by some fungal strains. Process Biochemistry 37:497-503.
197
Botella, C., Diaz, A., de-Ory, I., Webb. C and Blandino, A. (2007) , Xylanase
and pectinase production by Aspergillus awamori on grape pomace in
solid state fermentation. Process Biochem.42 (1): 98-101.
Borin, M.F., Said, S and Fonseca, M.J.V. (1996), Purification and biochemical
characterization of an extracellular endopolygalacturonase from
Penicillium frequentans.J.Agric.Food Chem. 44 (6):1616-1620.
Brasil, I.M., Maia, G. A. and Figuiredo, R. W. (1995), Physical–chemical changes
during extraction and clarification of guava juice. Food Chemistry 54(1):
383–386.
Buga, M. L, Ibrahim, S and Nok, A. J.(2010), Partially purified polygalacturonase
from Aspergillus niger (SA6). African Journal of Biotechnology, 9 (52):8944-
8954.
Burrel, R.G., Clayton, C.W., Gallegely, M.F and Lilly, V.D. (1966), Factors
affecting the antigencity of the mycelium of three species of Phytopathorea.
Phytopathalogy, 56: 422.
Cabanne, C and Doneche, B. (2002), Purification and characterization of two
isozymes of polygalacturonase from Botrytis cinerea. Microbiol. Res. 16:
1183–1195.
Castilho, L.R., Medronho, R.A and Alves, T.L.M. (2000), Production and
extraction of pectinases obtained by solid state fermentation of
agroindustrial residues with Aspergillus niger. Bioresour. Technol 71: 45-
50.
Ceci, L. and Lozano, J. (1998), Determination of enzymatic activities of
commercial pectinases for the clarification of apple juice. Food Chemistry
61(1): 237–241.
198
Chaplin, M. F and Bucke, C. (1990), In: Enzyme Technology Cambridge
University Press. Cambridge.
Chaudhri, A and Suneetha, V. (2012), Microbially Derived Pectinases: A Review.
IOSR J. Pharm. Biol. Sci. 2(2):1-05.
Chen, W.C., Hsieh, H.J and Tseng, T.C. (1998), Purification and characterization
of a pectinlyase from Pythium splendens infected cucumber fruits. Bot. Bull.
Acad. Sin. 39:181-186.
Cordeiro, C.A.M and Martins, M.L.L. (2009), Produção de poligalacturonases
pelo termofílico Bacillus sp SMIA-2 e algumas propriedades daenzima.
Crotti, L.B., Terenzi, T.H.F., Jorge, J.A and Polizeli, M.L.T.M. (1998),
Characterization of galactose induced extracellular and intra- cellular
pectolytic activities from the exo-1 mutant strain of Neurospora crassa. J.
Ind. Microbiol. Biotech. 20: 238-243.
Dahot, M. U. (1992), Alkaline protease activity of some plant seeds. Proc. All Pak. Sci.
Conf. (1): 102-104.
Dalal, S., Sharma, A and Gupta, M.N. (2007), A multipurpose immobilized
biocatalyst with pectinase, xylanase and cellulase activities. Chemistry
Central Journal 1: 16.
Damasio, A.R.L., da Silva, T.M., Maller, A., Jorge, J.A., Terenzi, H.F and Polizeli,
M.L.T.M. (2010), Purification and partial characterization of an
exopolygalacturonase from Paecilomyces variotii liquid cultures. Appl.
Biochem. Biotechnol. 160: 1496-1507.
Damasio, A,M.R.L., daSilva, T.M., Maller, A., Jorge, J.A., Terenzi, H.F and
Polizeli, L.T.M. (2011), Biotechnological potential of alternative carbon
sources for production of pectinases by Rhizopus microsporus var.
199
rhizopodiformis. In Brazilian Archives of Biology and Technology 54(1): 41-
148.
Delgado. L., Blanca, A.T., Huitron, C and Aguilar, G. (1992), Pectin lyase from
Aspergillus sp.CH-Y-1043. Appl. Microbiol. Biotechnol. 39: 515-519.
Dennison, C. (2003), In: A Guide to Protein Isolation 2nd Edition. Kluwer
Academic Publishers London.
Deshmukh, N., Talkal,R., Jha, K., Singh, P.G and Prajapati, D.C. (2012),
Production, Purification, Characterization and Comparison of
Polygalacturonase from various strains of Aspergillus International
Journal Of Scientific & Technology Research 1( 9): 85-91.
Devi, N.A and Rao, A.G.A. (1996), Fractionation, purification, and preliminary
characterization of polygalacturonases produced by Aspergillus
carbonarius. Enzyme Microb Technol 18:59–65.
de Vries, R.P and Visser, J. (2001), Aspergillus enzymes involved in degradation
of plant cell wall polysaccharides. Microbiology and Molecular Biology
Reviews 65(4) : 497-522.
Díaz-Godínez, G., Soriano-Santos, J., Augur, C and Viniegra- Gonzalez, G.
(2001), Exopectinases produced by Aspergillus niger in solid state and
submerged fermentation: a comparative study. Journal of Industrial
Microbiology and Biotechnology 26 (5): 271-275.
Dinu, D., Nechifor, M.T., Stoian, G., Costache, M and Dinischiotu, A. (2007)
,Enzymes with new biochemical properties in the pectinolytic complex
produced by Aspergillus niger MIUG 16. J. Biotechnol. 131:128–137.
Dogan, N and Tari, C. (2008), Characterization of three-phase partitioned exo-
polygalacturonase from Aspergillus sojae with unique properties. J.
Biochem. Eng. 39: 43-50.
200
Doi, O and Nojima, S. (1971), Phospholipase C from Bacillus cereus Biochim.
Biophys. Acta, 248: 234-244.
Dominguez, R., Pacho, D and Gonzalez, H. (2002), Bioconversion of industrial
effluents of the tortilla industry for the production of hydrolytic enzymes.
In: Annual Meeting Archive, American Institute of Chemical Engineers.
Dubey, J.P. (2000), Prevalence of Sarcocystis species sporocysts in wild caught
opossums (Didelphis virginiana). Journal of Parasitology 86: 705–710.
El-Batal, A. I.1., Osman, E. M and Ibrahim, A. M. S. (2013), Optimization and
characterization of polygalacturonase enzyme produced by gamma
irradiated Penicillium citrinum. Journal of Chemical and Pharmaceutical
Research 5(1):336-347.
Elegado, P.B and Fujio, Y. (1994), Purification and some properties of
endopolygalacturonase from Rhizopus sp LKN. World Journal of
Microbiology and Biotechnology 10, 256-259.
Fadel, M. (2000), Production physiology of cellulases and β-glucosidase enzyme
of A.niger grown under SSF conditions. Online Journal of Biological
Sciences 5: 401 –411.
Fahmy, A.S., El-beih, F.M., Mohamed, S.A., Abdel-Gany, S.S and Abd-Elbaky,
E.A. (2008), Purification and characterization of an exo-polygalacturonase
from Aspergillus niger. Appl. Biochem. Biotechnol. 149: 205-217.
Favela-Torres, E., Volke-Sepúlveda, T and Viniegra González,G. (2006),
Production of hydrolytic depolymerising pectinases. Food Technology
and Biotechnology 44(2): 221–227.
Fawole, O. B and Odunfa, S. A. (2003), Some factors affecting production of
pectic enzymes by Aspergillus niger. International Biodeterioration and
Biodegradation 52:223–227.
201
Federici, F and Petruccioli, M. (1885), Growth and polygalacturonase production
by Aureobasidium pullulans on orange peel waste. Microb. Alim. Nutri.
3: 39-46.
Fernandes-Salomão, T. M., Amorim, A.C.R., Chaves-Alves, V. M., Coelho, J.L.C.,
Silva, D.O and Araújo, E.F. (1996), Isolation of pectinase hyperproducing
mutants of Penicillium expansum. Rev. Microbiol. 27: 15-18.
Fogarty, W.M and Kelly, C.T. (1983), Pectic enzymes In: Microbial enzymes and
biotechnology. Edited by W.M. Fogarty, Applied Science Publishers,
ISBN: 0853341850, London, England (Ed.) Pp. 131-181.
Fonseca, M.J.V and Said, S. (1995), The pectinase produced by Tubercularia
vulgaris in submerged culture using pectin or orange-pulp pellets as
indutor. Appl. Microbiol. Biotechnol. 42: 32-35.
Fraissinet-Tachet, L and Fevre, M. (1996), Regulation by galacturonic acid of
pectinolytic enzyme production by Sclerotinia sclerotiorum Current
Microbiology 33: 49–53.
Freitas, P.M., Martin, N., Silva, D., Silva, R and Gomes, E. (2006), Production and
partial characterization of polygalacturonases produced by thermophilic
Monascus sp N8 and by thermotolerant Aspergillus sp N12 on solid-state
fermentation. Braz. J. Microbiol. 37: 302-306.
Freixo, M.R., Karmali, A and Arteiro, J.M. (2008a), Production and
chromatographic behavior of polygalacturonase from Pleurotus ostreatus
on immobilized metal chelates. Process Biochem. 43:531-539.
Fujio, Y and Eledago, F.B. (1993), Polygalacturonase production by Rhizopus
species. J. Gen. Appl. Microbiol. 39: 409-418
Geetha, M., Saranraj, P., Mahalakshmi, S and Reetha, D. (2012), Screening of
pectinase producing bacteria and fungi for its pectinolytic activity using
202
fruit wastes. International Journal of Biochemistry & Biotech Science 1: 30-
42.
Gewali, M.B., Maharjan, J., Thapa, S and Shrestha, J.K. (2007), Studies on
polygalacturonase from Aspergillus flavus. Sci. World 5: 19-22.
Giese, E.C., Dekker, R.F.H and Barbosa, A.M. (2008), Orange Bagasse as substrate
for the production of pectinase and laccase by Botryoshpaeria rhodina
MAMB - 05 in submerged and solid state fermentation. Bioresour. 3(2):
335-345.
Gillard, T. (1971), Enzymatic deacylation of lipids in plants. Eur. J. Biochem.
21: 90-98.
Gillespie, A., Cook, K and Coughlan, M.P. (1990), Characterization of an
endopolygalacturonase produced by solid-state cultures of the aerobic
fungus Penicillium capsulatum. Journal of Biotechnology 13: 279-292.
Gilman, J.C. (1957), A Manual of soil fungi, second edition Academic Press
London Pp. 34.
Gilman, J.C. (1998), A Manual of soil Fungi. First Indian edition published by
Biotech Book lds. 1123/74, Deva Rama Park Tri Nagar, Delhi 110035 Pp.
25-30 and Pp. 173-176.
Gokhale, D.V., Patil, S.G and Bastawde, K.B. (1992), Protection of Aspergillus
niger cellulases by urea during growth on glucose or glycerol
supplemented media. Appl. Biochem. Biotechnol. 37: 11-17.
Global Industry Analysts, Inc (2011) In: Report - Global Strategic Business.
Gomes, E., Leite, R.S.R., da Silva, R and Silva, D. (2009). Purification of an
exopolygalacturonase from Penicillium viridicatum RFC3 produced in
submerged fermentation. International Journal of Microbiology Pp:1-8
203
Griffin, D.H. (1994), Fungal physiology. Wiley-Interscience, New York, Pp 458
Guessous, Z., Lebbar S., Ouhssine M., Mokhatari A and Yachioui, M.E.l. (2001),
Endo and exopolygalacturonase from Geotrichum candidum: partial
purification and characterization. Science Letters 3(1): 1–5.
Gummadi, S.N and Panda, T. (2003), Purification and biochemical properties of
microbial pectinases-a review. Process Biochem. 38: 987-996.
Gummadi, S.N and Kumar, D. S. (2007), Batch and fed batch production of
pectin lyase and pectate lyase by novel strain Debaryomyces nepalensis in
bioreactor. Bioresource Technology 99: 874-881.
Gupta, R. and Mukerji, K.G. (2001), Fungi as a major group of organisms. In:
Microbiol Technology A.P.H. Publishing corporation S, Ansari Road
Darya gang, New Delhi.1-6.
Gurung, N., Ray, S., Bose, S., Rai, V. (2013), A broader view: microbial enzymes
and their relevance in industries, medicine, and beyond. Hindawi
Publishing Corporation, Bio Med Research International. 2013: 18 Pp.
Hames, B.D and Rickwood, D. (1986), Gel Electrophoresis of Proteins (eds
Hames, B. D and Rickwood, D.). Oxford: IRL Press.
Hasan, F., Shah, A.A and Hameed, A. (2006), Industrial applications of microbial
lipases. Enz. Microb.Technol. 39:235–51.
Hendges, D.H., Montanari, Q., Malvessi, E and da Silveira, M.M. (2011),
Production and characterization of endo polygalacturonase from
Aspergillus niger in solid-state fermentation in double-surface bioreactor.
Braz. Arch. Biol. Technol. 54(2): 253 – 258.
204
Hla, S.S., Kurokawa, J., Suryani Kimura, T., Ohmiya, K and Sakka, K. (2005), A
novel thermophilic pectate lyase containing two catalytic modules of
Clostridium stercorarium. Biosci. Biotechnol. Biochem. 69: 2138-2145.
Hoa, B.T and Hung, P.V. (2013), Optimization of nutritional composition and
fermentation conditionsfor cellulase and pectinase production by A.
oryzae using response surface methodology. International Food Research
Journal 20(6):3269-3274 .
Hölker, U., Höfer, M and Lenz, J. (2004), Biotechnological advan-tages of
laboratory-scale solid-state fermentation with fungi. Appl. Microbiol.
Biotechnol. 64: 175-186.
Holme, D. J and Peck, H. (1994), Analytical chemistry. 2nd Ed. Longman
Scientific & technical, Burnt Mill.
Hours, R. A., Voget, C. E and Ertola, R. J. (1988), Some factories affecting
pectinase production from apple pomace in solid states
cultures. Biological wastes 24:147-157.
Hunter, S.H. (1972), Inorganic nutrition. Annu Rev Microbio. 26: 313-346.
Jacob, N., Niladevi, K.N., Anisha, G.S. and Prema, P. (2008), Hydrolysis of
pectin: An enzymatic approach and its application in banana fiber
processing. Microbiological Research, 163:538-544.
Janani, L., Karthik, G., Kumar, K.V and Rao, B. (2011), Screening of Pectinase
Producing Microorganisms from Agricultural Waste Dump Soil. Asian J.
Biochem. Pharm. Res. 2(1):2231-2560.
Jayani, R.S., Saxena, S and Gupta, R. (2005), Microbial pectinolytic enzymes: a
review. Process Biochem. 40(9):2931-2944.
205
Joshi, V.K., Parmar, M and Rana, S. (2006), Pectin esterase production from apple
pomance in solid state and submerged fermentation. Food Technology
Biotechnology 44 (2): 253-256.
Joshi. V. K., Parmar, M and Rana, N. S. (2011), Purification and characterization
of pectinase produced from Apple pomace and evaluation of its efficacy in
fruit extraction and clarification. IJNPR 2(2):189-197.
Junwei, C., Lianshuang, Z and Shuyun, C. (1992), Screening of pectinase
producer from alkalophilic bacteria and study on its potential application
in degumming of ramie. Enzyme Microb. Technol. 14(12):1013-1016.
Junwei, C., Weihua, S., Yong, P and Shuyun, C. (2000), High producers of
Polygalacturonase selected from mutant resistant to Rifampin in
alkalophilic Bacillus sp. NTT3. Enzyme Microb. Technol. 27:545-548.
Juwon, A.D and Emmanuel, O.F. (2012), Experimental Investigations on the
Effects of carbon and nitrogen sources on concomitant amylase and
Polygalacturonase Production by BITRS-1001 in Submerged Fermentation
Biotechnology Research International Article ID 904763, Pp. 1-8.
Jyothi, T.C., Singh, S.A and Rao, A.G.A. (2005), The contributionof ionic
interactions to the conformational stability and function of
polygalacturonasefrom A. niger. International Journal of Biological
Macromolecule 36: 310-317.
Kabli, S. A. (2007), Purification and Characterization of Protopectinase Produced
by Kluyveromyces marxianus. Journal King Abdul Aziz University Sci. 19:
139-153.
Kapoor, M., Beg, Q. K., Bhushan, B., Dadhich, K.S and Hoondal, G. S. (2000),
Production and partial purification and characterization of a thermo-alkali
stable polygalacturonase from Bacillus sp. MG-cp-2. Process Biochem. 36:
467-473.
206
Karl, S and Ray, R.C. (2011), Purification, characterization and application of
thermostable exo-polygalacturonase from Streptomyces erumpens MTCC
7317. J. Food Biochem. 35: 133-147.
Kashyap, D.R., Chandra, S., Kaul, A and Tewari, R. (2000), Production,
purification and characterization of pectinase from a Bacillus sp. DT7.
World Journal of Microbiology and Biotechnology 16: 277-282.
Kashyap, D. R., Vohra, P.K., Chopra, S and Tewari, R. (2001), Applications of
pectinases in commercial sector. Bioresour Technol. 77(3): 215–227.
Kashyap, D. R., Soni, S. K. and Tewari, R. (2003), Enhanced production of
pectinase by Bacillus sp. DT7 using solid state fermentation. Bioresource
Technology 88(3): 251–254.
Kaur, G., Kumar, S and Satyanarayana, T. (2004), Production, characterization
and application of a thermostable polygalactouronase of a thermophilic
mould Sporotrichum thermophile Apinis. Bioresour. Technol. 94:239-234.
Khairnar, Y., Vamsi, K.K., Boraste, A., Gupta, N., Trivedi, S., Patil, P., Gupta, G.,
Gupta, M., Jhadav, A., Mujapara, A., Joshi, B and Mishra, D. (2009), Study
of pectinase production in submerged fermentation using different strains
of Aspergillus niger. Int. J. Microbiol. Res.1: 13-17.
Kester, H.C.M., Kusters-van Someren, M.A., Müller, Y and Visser J. (1996),
Primary structure and characterization of an exopolygalacturonase from
Aspergillus tubingensis. Eur. J. Biochem. 240: 738-746.
Koffi, E. K, Sims, C. A. and Bates, R. P. (1991), Viscosity reduction and prevention
of browning in the preparation of clarified banana juice. Journal of Food
Quality 14: 209–218.
Kojima, Y., Sakamoto, T., Kishida, M., Sakai, T and Kawasaki, H. (1999), Acidic
condition-inducible polygalacturonase of Aspergillus kawachii Journal of
Molecular Catalysis B: Enzymatic 6: 351–357.
207
Kotzekidov, P. (1991), Production of polygalacturonases by Byssachlamys fulva.
Journal of Industrial Microbiology 7: 53–56.
Kumar, A and Sharma, R. (2012), Production of alkaline pectinase by bacteria
(Cocci sps.) isolated from decomposing fruit materials. Journal of
Phytology 4(1): 01-05.
Kumari, H.L and Sirsi, M. (1971), Purification and proper- ties of endo-poly-
galacturonase from Gzmoderma lucidum. J. Gen. Microbiol. 65: 285-290.
Kunte, S and Shastri, N.V. (1980), Studies on extracellular production of
pectolytic enzymes by a strain of Alternaria alternata. Ind. J. Microbiol.
20(3):211-214.
Lee, S. M. and Koo, Y. M. (2001), Pilot-scale production of cellulose using
Trichoderma reesei Rut C-30 in fed-batch mode. Journal of Microbiology
and Biotechnology 11:229-233.
Lee, W. C., Yusof, S., Hamid, N. S. A. and Baharin, B. S. (2006), Optimizing
conditions for enzymatic clarification of banana juice using response
surface methodology (RSM). Journal of Food Engineering 73: 55-63.
Lehninger, A.L., Nelson, D.L and Cox (1992), Principles of Biochemistry (2nd
ed.) Worth Publishers, Inc., New York, Pp. 222.
Leone, G and Van den Heuvel, J. (1987), Regulation by carbohydrates of the
sequential in vitro production of pectic enzymes by Botrytis cinerea. Can. J.
Bot. 65: 2133-2142.
Li, Z., Jin.B, Zhang. H, Bai.Z, Xue. W and Li, H. (2008), Purification and
characterization of three alkaline endopolygalacturonases from a newly
isolated Bacillus gibsonii. The Chinese Journal of Process Engineering
8 (4): 768 - 773
208
Loera, O., Aguirre, J and Viniegra-González, G. (1999), Pectinase production by
a diploid construct from two Aspergillus niger overproducing mutants,
Enzyme and Microbial Technology 25(1-2): 103–108.
Lonsane, B.K.,Ghildyal , N.P., Budiatman, S. and Ramkrishna, S.V. (1985),
Engineering aspects of solid state fermentation. Enzyme and Microbial
Technology 7,258-265.
Lowry, O.H., Rosebrough, N. J., Farr, A. L and Randall, R.J. (1951), Protein
measurement with the Folin phenol reagent. J. Biol. Chem.193: 265-75.
Luangsa-ard, J., Houbraken, J., van Doorn, T., Hong, S.B., Borman, A.M., Hywel-
Jones, N.L., Samson, R.A. (2011), Purpureocillium, a new genus for the
medically important Paecilomyces lilacinus. FEMS Microbiology
Letters 321 (2): 141–9.
Macfarlane, G.T., Hay, S., Macfarlane, S and Gibson, G.R. (1990), Effect of
different carbohydrates on growth, polysaccharidase and glycosidase
production by Bacteroides ovatus, in batch and continuous culture. J. Appl.
Bacteriol: 68 (2):179-87.
Maldonado, M.C., Saad, A.M.S and Callieri, D. (1989), catabolic repression of the
synthesis of inducible poligalacturonase and pectinesterase by Aspergillus
niger sp. Curr. Microbiol. 18:303-306.
Maldonado, M.C.S and Strasser de Saad, A.M. (1998), Production of
pectinesterase and polygalacturonase by Aspergillus niger in submerged
and solid state systems. J. Ind. Microbiol. Biotechnol. 20(1): 34-8.
Maller, A., Damásio, A.R.L., Silva, T.M., Jorge, J.A., Terenzil, H.F and Polizeli,
M.L.T.M. (2011), Biotechnological Potential of Agro- Industrial Wastes as
a Carbon Source to Thermostable Polygalacturonase Production in
Aspergillus. Enzyme Research 5: 1 – 6.
209
Maller, A., Silva, T.M., Damásio, A.R.L., Jorge, J.A., Terenzi, H.F and Polizeli,
M.L.T.M. (2007), Partial purification of pectinases produced by the
filamentous fungus Aspergillus niveus. In: 36th Annual Meeting of the
Brazilian Society for Biochemical and Molecular Biology (SSBq) and 10th
International Union of Biochemistry and Molecular Biology (IUBMB)
Conference, Salvador.
Malvessi, E and da Silveira, M.M. (2004), Influence of Medium Composition and
pH on the Production of Polygalacturonases by Aspergillus oryzae. Braz.
Arch. Biol. Technol. 47(5): 693 -702.
Marcia, M.C.N., Roberto da Silva, S and Gomes, E. (1999), Screening of bacterial
strain for pectinolytic activity: Characterization of the polygalacturonase
produced by Bacillus sp. J. Microbiol. 30: 299-303.
Maria, H.A., Galba, M.C.T., Ana Lúcia, F.P and Adauto. (2002), Screening of Mucor sp.
for the production of amylase, lipase, polygalacturonase and protease. Braz. J.
Microbiol. 33:325-330.
Margesin, R., Fauster, V and Fonteyne, P.A. (2005). Characterization of
coldactive pectate lyases from psychrophilic Mrakia frigida. Lett. Appl.
Microbiol. 40:453-459.
Marques, M.R., Buckeridge, M.S., Braga, M.R. and Dietrich, S.M.C. (2006), Characterization
of an extracellular endopolygalacturonase from the saprobe Mucor ramosissimus
samutsevitsch and its action as trigger of defensive response in tropical plants.
Mycopathologia 162:337-346.
Martin.N., De Souza, S. R., de Silva, R and Gomes, E. (2004), Pectinase
production by fungal strains in solid-state fermentation using agro-
industrial bioproduct. Brazilian Archives of Biology and Technology.
47(5): 813-819.
210
Martin, N., Guez, M.A.U., Sette, L.D., Silva Da, R and Gomes, E. (2010), Pectinase
production by a Brazilian thermophilic fungus Thermomucor indicae-
seudaticae N31 in solid- state and submerged fermentation. Microbiology
79(3):306-313.
Martínez-Trujillo, A., Aranda,J. S.,Gómez-Sánchez, C., Trejo-Aguilar, B and
Aguilar-Osorio, G. (2009), Constitutive and inducible pectinolytic
enzymes from Aspergillus flavipes Fp-500 and their modulation by ph and
carbon source. Brazilian Journal of Microbiology 40: 40-47.
Martins, E.S., Silva, D., Silva, R and Gomes, E. (2002), Solid state production of
thermostable pectinases from thermophilic Thermoascus aurantiacus.
Process Biochem. 37:949-954.
Martos, M.A., Zubreski, E.R., Garro, O.A and Roque A. Hours, R.A. (2013),
Production of Pectinolytic Enzymes by the Yeast Wickerhanomyces
anomalus Isolated from Citrus Fruits Peels. Biotechnology Research
International Volume Article ID 435154, Pp:1-7.
McKay, A. M. (1988), A plate assay method for the detection of fungal
polygalacturonase secretions. FEMS Microbiol. Lett. 56:355-358.
Miller, G.L. (1959), Use of dinitrosalicylic acid reagent for determination of
reducing sugar. Anal. Chem. 31(3): 426-428.
Minussi, R.C., Coelho, J.L.C., Baracat-Pereira, C.M and Silva, D.O. (1996), Pectin
lyase production by Penicillium griseoroseum: Effect of tea extract, caffeine,
yeast extract and pectin. Biotechnol. Lett., 18:1283-1286.
Mohamed, S.A., Al-Malki, A. L and Kumosani, T.A. (2009), Characterization of a
polygalacturonase from Trichoderma harzianum grown on citrus peel with
application for apple juice. Aust. J. Basic Appl. Sci. 3: 2770-2777.
211
Moharib, S.A., El-Sayed, S.T and Jwanny, E.W. (2000), Evaluation of enzymes
produced from yeast. Nahrung. 44: 47-51.
Mohsen, S.M., Bazaraa, W.A and Doukani, K. (2009), Purification and
characterization of Aspergillus niger U-86 polygalacturonase and its use
in clarification of pomegranate and grape juices. Procedings 4th
Conference on Recent Technologies in Agriculture Pp 805-817.
Montgomery, R. (1961), Phenol sulfuric acid reagent for carbohydrates. Biochem.
Biophys. Acta 48: 591.
Moore-Landecker, E. (1996 ), Fundamentals of Fungi, Prentice Hall, Upper
Saddle River, NJ, USA, 4th edition.
Moyo, S., Gashe, B.A., Collison, E.K and Mpuchane, S. (2003), Optimising
growth conditions for the pectinolytic activity of Kluyveromyces
wickerhamii by using response surface methodology. Int. J. Food
Microbiol. 85: 87–100.
Mrudula. S and Anitharai, R. (2011), Pectinase production in solid state
fermentation by Aspergillus niger using orange peel as substrate.G J
Biotechnol Biochem . 6:64-71.
Murad, H.A and Azzaz, H.H. (2011), Microbial pectinases and ruminant
nutrition. Res. J. Microbial. 6: 246-269.
Nabi, N.G., Asgher,M., Shah, A.H., Sheikh, M.A and Asad, M.J. (2003),
Production of pectinase by Trichoderma harzianum in solid state
fermentation of citrus peel .Pak. J. Agric. Sci. 40(3-4):193-201.
Naidu, G .S. N and Panda, T. (1998), Production of pectolytic enzymes – a review.
Bioprocess Eng. 19: 355-61.
212
Nakajima, N., Ishihara, K., Tanabe, K and Matsubara, K. (1999), Degradation of
pectic substances by two pectate lyases from a human intestinal bacterium,
Clostrdium butyricum-bijerinkii group. J Biosci Bioeng. 88:331-333.
Narasimha, G., Sridevi,A., Buddolla,V., Subhosh Chandra,M and Rajasekhar
Reddy, B.(2006), Nutrient effects on production of cellulolytic enzymes
by Aspergillus niger. African Journal of Biotechnology 5 (5):472–476.
Neeta, R. S., Sasankan, A., Singh, A and Soni, G. (2011), Production of
polygalacturonase and pectin methyl esterase from agrowaste by using
various isolates of Aspergillus niger. Insight Microbiology 1 (1) : 1-7.
Negi, S and Banerjee, R. (2010), Optimization of culture parameters to enhance
production of amylase and protease from Aspergillus awamori in a single
fermentation, African Journal of Biochemistry Research 4(3): 73–80.
Nelson, D.L and Cox, M.M. (2004), Lehninger principles of biochemistry, 4th edn.
WH Freeman, New York.
Nitinkumar, P. P and Bhushan, L. C. (2010), Microbiology production and
purification of pectinase by soil isolate Penicillium sp and search for better
agroresidue for its ssf. Recent Research in Science and Technology 2(7): 36-
42.
Niture, S.K and Pant, A. (2004), Purification and biochemical characterization of
polygalacturonase II produced in semi-solid medium by a strain of
Fusarium moniliforme. Microbiol. Res. 159: 305-314.
Niture, S.K., Kumarb, A.R., Parabc, P.B and Pant, A. (2008), Inactivation of
Polygalacturonase and pectate lyase produced by pH tolerant fungus
Fusarium moniliforme NCIM 1276 in a liquid medium and in the host
tissue. Microbiol. Res. 163: 51-62.
213
Norus, J. (2006), Building sustainable competitive advantage from knowledge in
the region: the industrial enzymes industry. European Planning Studies
14: 681–696.
Obi, S.K and Moneke, N.A. (1985), Pectin lyase and polygalacturonase of
Aspergillus niger pathogenic for yam tubers. Int. J. Food Microbiol. 1:277-
289.
Ögle, Z.B., Yarangümeli, K., Dürdar, D and Ifrij, I. (2001), Sub-merged cultivation
of Scytalidium thermophilum on complex lignocellulosic biomass for
endoglucanase production. Enzyme and Microbial. Technol. 28: 689-695.
Ogunlade and Oluwayemisi, A. (2012), Effect of blanching, ripening and other
treatments on the production characteristics of pectinolytic enzymes from
banana peels By Aspergillus niger. Global Journal of Science Frontier
Research Chemistry 12 (2): 37 – 46.
Ortega, N., de Diego, S., Perez-Mateos, M and Busto, M. D. (2004), Kinetic
properties and thermalbehavior of polygalacturonase used in fruit juice
clarification. Food Chemistry 88(2): 209–217.
Oudemans, C.A and Koning, C.J. (1902), Prodrome d’une flore mycologique
obtenu par la culture sur gelatione pr’epar’ee de al terre humeuse du
spanderswound pre’ Bussum. Arch. Neerl. Sci. Nat. Ser. 2 (7): 286-298.
Palaniyappan, M., Vijayagopal, V., Renuka, V and Viruthagiri, T. (2009),
Screening of natural substrates and optimization of operating variables on
the production of pectinase by submerged fermentation using Aspergillus
niger MTCC 281. Afr. J. Biotechnol. 8 (4):682-686.
Palmer T. (1991), Extraction and purification of enzymes. In: Understanding
Enzymes, Ellis Horwood. Ltd., England, pp: 301-317.
214
Palomäki, T and Saarilahti, H.T. (1997), Isolation and characterization of new
Cterminal substitution mutation affecting secretion of polygalacturonases
in Erwinia carotovora ssp.carotovora. FEBS Letters 400: 122- 126.
Panda, T., Nair, S. R and Kumar, M. P. (2004), Regulation of synthesis of the
pectolytic enzymes of Asperggillus niger. Enzyme and Microbial
Technology 34(5):466–473.
Panda, S.S., Sahoo, K., Das, R and Dhal, N.K. (2012), Pectinolytic and cellulolytic
activity of soil fungal isolates from similipal bioreserve forest. World
Environment 2(2): 1-3.
Pandey A. (1991), Effect of particle size of substrate of enzyme production in
Solid state fermentation. Biores. Technol. 37: 169–172.
Pardo, C., Lapeña, M.A and Gacto, M. (1991), Purification and characterization of
an extracellular exopolygalacturonase from Geotrichum lactis. Can J.
Microbiol. 37: 974-977.
Patil, S.R and Dayanand, A. (2006a), Exploration of regional agrowastes for the
production of pectinase by Aspergillus niger. Food Technol. Biotechnol. 44
(2): 289–292.
Patil, S.R and Dayanand, A. (2006b), Production of pectinase from deseeded
sunflower head by Aspergillus niger in submerged and solid-state
conditions. Bioresour. Technol. 97 (16): 2054-2058.
Patil, N.P and Chaudhari, B.L. (2010), Production and purification of pectinase
by soil isolate Penicillium sp and search for better agro-residue for its ssf.
Recent Research in Science and Technology 2(7): 36-42
Patil, R.C., Murugkar, T.P and Shaikh, S.A. (2012), Extraction of pectinase from
pectinolytic bacteria isolated from carrot waste. International Journal of
Pharmaceutical and Bio-Sciences 3 (1): 261-266.
215
Pedrolli, D.B., Gomes, E., Monti, R and Carmona, E.C. (2008), Studies on
productivity and characterization of polygalacturonase from Aspergillus
giganteus submerged culture using citrus pectin and orange waste. Appl
Biochem Biotechnol. 144(2): 191-200.
Pedrolli, D.B., Monteiro, A.C., Gomes, E and Carmona, E.C.(2009), Pectin and
Pectinases: production, characterization and industrial application of
microbial pectinolytic enzymes. Open Biotechnol. J. 3: 9-18.
Pedrolli, D.B and Carmona, E.C. (2010), Purification and characterization of the
exopolygalacturonase produced by Aspergillus giganteus in submerged
cultures. J. Ind. Microbiol. Biotechnol. 37: 567-573.
Perley, A.F and Page, O.T. (1971), Differential induction of pectolytic enzymes of
Fusarium roseum (LK.) emend. Snyder and Hansen. Canadian Journal of
Microbiology 17:415-420.
Phutela, U., Dhuna, V., Sandhu, S and Chadha, B.S. (2005), Pectinase and
polygalacturonase production by a thermophilic Aspergillus fumigatus
isolated from decomposting orange peels. Brazilian Journal of
Microbiology 36 (1):63-69.
Polizeli, M. L. T., Jorge, J.A and Terenzi, H.F. (1991), Pectinase production by.
Neurospora crassa: purification and biochemical characterization of
extracellular polygalacturonase activity, J. Gen. Microbiol. 137: 1815-1823.
Poonpairoj, P., Peerapatsakul, C and Chitradon, L. (2001), Trend in using fungal
enzymes lignin- and pectin-degrading enzymes, in improvement of the
paper mulberry pulping process. Proc. Int. Symp. Pap. Pulp, Bangkok,
Thailand, Pp 179-199.
Praveen, K.D., Thangabalan, B., Venkateswara, R.P and Yugandhar, N.M. (2011)
,production of pectinase enzyme by Aspergillus niger using ficus religiosa
216
leaves in soild state fermentation. International Journal of Pharmacy &
Technology 3(1): 1351-1359
Price, N.C and Stevens, L. (1999), In: Fundamentals of Enzymology: The Cell and
Molecular Biology of Catalytic Proteins. Third Edition. Oxford University Press
Oxford.
Prodanović, J.M and Antov, M.G. (2008), The influence of molecular weight of
polyethyleneglycol on separation and purification of pectinases from
Penicillium cyclopium in aqueous two-phase system. Acta Periodica
Technologica 39(39): 193-199.
Ramachandran, S and Kurup G.(2013), Screening and isolation of pectinase from
fruit and vegeable wastes and the use of orange waste as a substrate
for pectinase production. Int. Res. J. Biological Sci. 2 (9), 34-39.
Rajendran, R., Karthik, S.S., Radhai, R., Rajapriya, P and Balakumar, C. (2011),
Fusarium sp. International Journal of Current Research 33 (4): 254-258.
Rashmi, R., Siddalinga Murthy, K.R., Sneha, G., Shabana, S., Syama, A and
Radhika, V.S. (2008), Partial purification and biochemical characterization
of extra cellular pectinase from Aspergillus niger isolated from groundnut
seeds. Journal of Applied Bioscience 9(1): 378-384.
Reda, A.B., Hesham, M and Yassin, M. (2008), Production of bacterial pectinase
(s) from agro-industrial wastes under solid state fermentation conditions.
Journal of Applied Science Research. 4(12): 1708-1721.
Rexová-Benková, L and Marcovic, O. (1976), Pectic enzymes. Advances in
Carbohydrate Chemistry 33: 323-385.
Riou, C., Salmon, J-M., Vallier.M-J., Gunata, Z and Barre, P. (1998), Purification,
characterization, and substrate specificity of a novel highly
217
glucosetolerant β-glucosidase from Aspergillus oryzae. Appl. Environ.
Microbiol. 64: 3607-3614.
Rodríguez-Fernández, D.E., Rodríguez-León, J. A., Carvalho, J. C., Sturm, W and
Soccol, C. R. (2011), The behavior of kinetic parameters in production
ofpectinase and xylanase by solid-state fermentation. Bioresource
Technology 102: 10657-10662.
Rombouts, F.M and Pilnik, W. (1980), Pectic enzymes. In: Rose, A. H. (Ed.).
Economic Microbiology. London: Academic Press. 5: Pp. 227-282.
Runco, R., Navarro, A. R and Maldonado, M. C. (2001), Regulation of the
production of poligalacturonase by Aspergillus terreus Journal of
Microbiology and Biotechnology 17:487–491.
Ronne, H. (1995), Glucose repression in fungi. Trends Genet. 11:12–17.
Ros, J.M., Saura, D., Salmerón, M.C and Laencina, J. (1993), Production of pectic
enzymes from Rhizopus nigricans cultures with different sources of carbon.
Ann. Microbiol. Enzimol. 43: 71- 76.
Roosdiana, A., S. Prasetyawan, C. Mahdi and S. Sutrisno, (2013, Production and
characterization of Bacillus firmus pectinase. J. Pure Applied Chem. Res. 2:
35-41.
Roque, A.H and Takuo, S. (1994), Protopectinase production in solid state
culture of Aspergillus awamori. Biotechnol Lett . 7:721–6.
Saad, N., Briand, M., Gardarin, C., Briand, Y and Michaud, P. (2007), Production,
purification and characterization of an endopolygalacturonase from
Mucor rouxii NRRL 1894. Enzyme Microb. Technol. 41(6-7): 800-805.
Said, S., Fonseca, M.J.V and Siersera, V. (1991), Pectinase production by
Pencillium frequentans. World Journal of Microbiology and Biotechnology
7: 607-608.
218
Sakai, T. (1992), Degradation of pectins In: Winkelmann, G. (ed), Microbial
degradation of natural products. Weinheim, VCH Pp. 57- 81.
Sakellaris, G., Nikolaropoulos, S and Evangelopoulos, A.E. (1988),
Polygalacturonase biosynthesis by LactoBacillus plantarum: effect of
cultural conditions on enzyme production. Journal of Applied
Bacteriology 65: 397-404.
Sambrook, J. R. and David, W. (2001), Molecular cloning, A Laboratory Manual,
3rd. edn. Cold Spring Harbor Laboratory Press, New York, A8.32.
Sampriya, S., Rishi, P.M and Jitender, S. (2012), Utilization of agro-industrial
resideues for pectinase production by the novel strain Pseduozyma sp. SPJ
under solid state cultivation. Ann. Microb. 62:169-176.
Samson. RA., (1974). Paecilomyces and some allied hyphomycetes. Studies in
Mycology (Baarn: Centralbureau voor Schimmelcultures) 6: 58
Santiago, M.F. (1993), Produção e propriedades de pectina liase de um
isolado Penicillium expansum. M.S Dissertation, Universidade Federal de
Viçosa, Viçosa, Brazil
Sathiyaraj, G., Srinivasan, S., Kim, H.B., Subramaniyam, S., Lee, O.R., Kim, Y.J
and Yang, D.C. (2011), Screening and optimization of pectin lyase and
polygalacturonase activity from ginseng pathogen Cylindrocarpon
destructans. Braz. J. Microbiol. 42:794-806.
Saxena, R.K., Sheoran, A., Giri, B and Davidson, W.S. (2003), review purification
strategies for microbial lipases Journal of Microbiological Methods 52, 1 –
18.
Schnitzhofer, W., Weber, H.J., Vrsanska, M., Biely, P., Cavaco-Paulo, A and
Guebitz, G.M. (2007), Purification and mechanistic characterisation of two
polygalacturonases from Sclerotium rolfsii. J. Enzyme Microbiol. Technol.
40: 1739–1747.
219
Scopes, R.K. (1994), Protein purification: principles and practice, 3rd edn.
Springer, New York, USA.
Scopes, R. (1985), Methods of protein purifications. M. Mir.Pp 358 (Russian)
Semenova, M.V., Grishutin, S.G., Gusakov, A.V., Okunev, O.N and Sinitsyn, A.P.
(2003), Isolation and properties of pectinases from the fungus Aspergillus
japonicus. Biochem (Moscow) 68(5): 559-69.
Sharma, D.C and Satyanarayana, T. (2006), A marked enhancement in the
production of a highly alkaline and thermostable pectinase by Bacillus
pumilus dcsr 1 in submerged fermentation by using statistical methods.
Bioresour. Technol. 97:727-733.
Shastri, P.N., Patil, M and Shastri, N.V. (1988), Production, purification and
properties of Geotrichum candidum polygalacturonase: regulation of
production by pyruvate. Indian J. Biochem Biophys. 25(4):331–335.
Shevchik, V.E., Condemine, G., Robert-Baudoy, J and Hugouvieux-Cotte-Pattat,
N. (1999), The exopolygalacturonase lyase PelW and the
oligogalacturonate lyase Ogl, two cytoplasmic enzymes of pectin
catabolism in Erwinia chrysanthemi 3937. J. Bacteriol. 181: 3912-3919.
Shivakumar, P.D and Krishnanand, S.S. (1995), Anaerobic degradation of pectin
by mixed consortia and optimization of fermentation parameters for higher
pectinase activity. Lett. Appl. Microbiol. 20: 117–119.
Shubakov, A.A and Elkina, E.A. (2002), Production of polygalacturonases by
filamentous fungi Aspergillus niger acm f-1119 and Penicillium dierckxii
acim f-152. Chemistry and Computational Simulation Butlerov
Communications 2 (7): 65-68.
220
Siddiqui, M.A., Pande, V and Arif, M. (2012), Production, Purification and
Characterization of Polygalacturonase from Rhizomucor pusillus Isolated
from Decomposting Orange Peels. Enzyme Research. Pp.1-8
Siddiqui, M.A., Pande, V and Arif, M. (2013), Polygalacturonase production from
Rhizomucor pusillus isolated from fruit markets of Uttar Pradesh. African
Journal of Microbiology Research 7(3): 252-259.
Sieiro C., Garcia-Fraga, B., Lopez-Seijas, J., Silva, A.F and Villa, T.G. (2012).
Microbial Pectic Enzymes in the Food and Wine Industry. In: Food
Industrial Processes - Methods and Equipment, pp 201-218
Silva, D.O., Attwod, M.M and Tempest, D.W. (1993), Partial purification and
properties of pectin lyase from Penicillium expansum. World J. Microbiol.
Biotechnol. 9(5): 574-578.
Silva, D., Martins, E.S., Da Silva, R and Gomes, E. (2002), Pectinase production
by Penicillium viridicatum RFC3 by solid state fermentation using
agricultural wastes and agro-industrial by-products. Braz. J. Microbiol. 33:
318-324.
Silva, D., Tokuioshi,K., Martins, E.D.S., Silva, R.D and Gomes, E. (2005),
Production of pectinase by solid-state fermentation with Penicillium
viridicatum RFC3. Process Biochem. 40:2885–2889.
Silva, D., Martins, E.S., Leite, R.S.R., Da Silva, R., Ferreira,V and Gomes. E. (2007),
Purification and characterization of an exo-polygalacturonase produced
by Penicillium viridicatum RFC3 in solid-state fermentation. Process
Biochem. 42: 1237-1243.
Silley, P. (1986), The production and properties of a crude pectin lyase from
Lachnospira multiparus. Lett. Appl. Microbiol. 2: 29-31.
Singh, R.R and Appu Rao, A.G. (2002), Reductive unfolding and oxidative
refolding of a Bowman-Birk inhibitor from horsegram seeds (Dolichos
221
biflorus): evidence for “hyperreactive” disulfide bonds and rate-limiting
nature of disulfide isomerization in folding. Biochim. Biophys. Acta 1597:
280-291.
Singh, S and Mandal, S.K. (2012), Enhanced production of pectinolytic Enzymes
from immobilized of mixed Asergillus Species. International Journal of
Applied Biology and Pharmaceutical Technology 3(4): 20-27.
Sinitsyna, O.A., Fedorova, E.A., Semenova, M.V., Gusakov, A.V., Sokolova, L.M.,
Bubnova, T.M. (2007), Isolation and characterization of extracellular pectin
lyase from Penicillium canescens. Biochemistry, (Moscow) 72: 565-567.
Soares, M.M.C.N., Da Silva, R., Gomes, E. (1999), Screening of bacterial strains for
pectinolytic activity: characterization of the polygalacturonase produced
by Bacillus sp. Revista de Microbiologia 30(4): 299-303.
Solís –Pereyra, S., Favela Torres, E., Viniegra -González. G and Gutiérrez- Rojas,
M. (1993), Effects of different carbon sources on the synthesis of pectinase
by Aspergillus niger in submerged and solid state fermentations. Appl.
Microbiol. Biotechnol. 39: 36-41.
Solis-Pereyra, S., Favela-Torres, E., Gutierrez-Rojas, M., Roussos, S., Saucedo-
Castan˜eda, P., Gunasekaran, P and Viniegra-Gonza´lez, G. (1996),
Production of pectinases by Aspergillus niger in solid state fermentation at
high initial glucose concentrations. World J. Microbiol. Biotechnol. 12:
257–260.
Solís, S., Jacinto, L., Graciella, S., Jorge ,T., Nohemí ,R., Felipe, R and Carlos, H.
(2009), Hydrolysis of orange peel by a pectin lyase-overproducing hybrid
obtained by protoplast fusion between mutant pectinolytic Aspergillus
flavipes and Aspergillus niveus CH-Y-1043. Enzyme Microb. Technol.
44:123-128.
222
Soni, G.L and Bhatia, I.S. (1981), Studies on pectinases from Fusarium oxysporum.
Indian J .Exp. Biol. 19: 547-550.
Southerton, S.G., Osbourn, A. E., Dow, J. M and Daniels, M. 1. (1993), Two
xylanases from Gaeumannomyces graminis withidentical N-terminal amino
acid sequences. Physiol Mol Plant Pathol. 42:97-107.
Sreekantian, K. R., Jaleel, S.A and Rao, T.N.R. (1971), Utilization of fungal
enzyme in soft fruit and extraction and clarification of fruit juice. J. Food
Sc. Technol., 8: 201-203.
Stanbury, P.F., Whitaker, A and Hall, S.J. (1997), In: Principles of Fermentation
Technology. Indian Reprint. Aditya Books (P) Ltd.New Delhi. Strategies
for microbial lipases. Journal of Microbiological Methods 52: 1-18.
Starr M., Moran F. (1962). Eliminative split of pectic substances by
Phytopathogenic soft-rot. Science, 135: 920-921.
Stressler, K.D and Joslyn, M.A. (1971), Fruits and vegetable Processing
Tecnology, West-Port, AVI Publishing Co. Inc. 2ª rf
Stutzenberger, F. (1992), Pectinase production. In: J Lederberg, ed. Encyclopedia
of Microbiology. Vol III, San Diego: Academic Press, Pp 327–337
Sunnotel, O and Nigam, P. (2002), Pectinolytic activity of bacteria isolated from
soil and two fungal strains during submerged fermentation. World
Journal of Microbiology and Biotechnology.18: 835-839.
Suresh, B and Viruthagiri, T. (2010), Optimization and kinectics of pectinase
enzyme using Aspergillus niger by solid-state fermentation. Indian J.
Sci.Technol. 3(8): 867-870.
Taragano, V., Sanchez, V.E and Pilosof, A.M.R. (1997), Combined effect of water
activity depression and glucose addition on pectinases and protease
production by Aspergillus niger. Biotechnol. Lett. 19(3): 233-236.
223
Tari, C., Gögus, N and Tokatli, F. (2007), Optimization of biomass, pellet size and
polygalacturonase production by Aspergillus sojae ATCC 20235 using
response surface methodology. Enzyme Microb. Technol.40 (5):1108-1116.
Tari, C., Dogan, N and Gogus, N. (2008), Biochemical and thermal
characterization of exo-polygalacturonase produced by Aspergillus sojae.
Food Chemistry. 111:824-829.
Tariq, A.L and Reyaz, A.L. (2012), The influence of carbon and nitrogen sources
on pectinase productivity of Penicillium chrysogenum in solid state
fermentation. International Research Journal of Microbiology (IRJM). 3(5):
202-207 .
Teixeira, M.F.S., José, L.L.F and Durán, N. (2000), Carbon sources effect on
pectinase production from pectinase production from Aspergillus japonicus
586. Braz. J. Microbiol. 31:286-290.
Thakur, A., Pahwa, R., Singh, S and Gupta, R. (2010), Production, purification,
and characterization of polygalacturonase from Mucor circinelloides ITCC
6025, Enzyme Res.Article ID 170549, Pp.1-7.
Thom, C. (1910), Bulletin of the Bureau of Animal Industry US Department of
Agriculture 118: 73.
Tonukari, N. J., Scott-Craig, J. S and Walton, J.D. (2002), Influence of carbon
source on the expression of Cochliobolus carbonum xylan-degrading
enzyme genes. Afr. J. Biotechnol. 1: 64-66.
Torrado, A., Gonzalez, M.P and Murado, M.A. (1998), pH regulation in solid
stateculture through the initial ratio between oxidized and reduced
sources of nitrogen.A model applicable to the amylase production by
Aspergillus oryzae. Biotechnol. Techniques. 12: 411-415.
Tsuymu, S. (1979), Self catabolite repression of pectate lyase in Ervinia caratovora,
Journal of Bacteriology. 137:1035-1036.
224
Ueda, S., Fujio, Y., Lim, J. Y. (1982), Production and some properties of pectic
enzymes from Aspergillus oryzae A3. Journal of Applied Biochemistry.
4:524-532.
Uenojo, M and Pastore, G. (2007), Pectinases: Aplicações industriais
eperspectivas Quimica Nova, Campinas- São Paulo, 30(2):388-394.
Vahidi, H., Kobarfard, F and Namjoyan, F. (2004), Effect of cultivation conditions
on growth and antifungal activity of Mycena leptocephala .African Journal
of Biotechnology. 3(11): 606–609.
Vaillant, F., Millan, P.O., Brien, G., Dornier, M., Decloux, M. and Reynes, M.
(1999), Cross flow microfiltration of passion fruit juice after partial
enzymatic liquefaction. Journal of Food Engineering. 42(4): 215-224.
Vasanthi, N.S and Meenakshisundaram, M. (2012), Optimization of pectinase
enzyme production by using sour orange peels as substrate in solid state
fermentation. Asian Journal of Biochemical and Pharmaceutical Research.
1 (2): 16-26
Vibha, B and Neelam, G. (2010), Exploitation of microorganisms for isolation and
screening of pectinase from environment. 8th International Conference
Making Innovation Work for Society, Linking, Leveraging and Learning
November 1-3, 2010, University of Malaya, Kuala Lumpur, Malaysia.
Vibha, B and Neelam, G. (2011), The utilization of citrus peel for pectinase
Production. Journal of Sustainable Development and Environmental
Protection. 1(3):60 – 66.
Viikari, L., Tenakanen, M and Suurnakki, A. (2001), Biotechnology in the pulp
and paper industry. In: Rehm H. J. (ed), Biotechnology VCH-Wiley Pp.
523 - 546.
225
Viquez, F., Laetreto, C. and Cooke, R. D. (1981), A study of the production of
clarified banana juice using pectinolytic enzymes. International Journal of
Food Science and Technology 16(2): 115-125.
Wang, M.C and Keen, N.T. (1970), Purification and characterization of
endopolygalacturonase from Verticillium alba-actrum. Arch. Biochem.
Biophys. 141:749-757.
Wang, P.C., Vancura, A., Mitcheson, T.G and Kuret, J. (1992), Two genes in
Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-
1. Mol. Biol. Cell. 3(3):275-86.
Ward, O.P. and Fogarty, W. M. (1973), Bacterial growth and enzyme production
in sitka spruce sapwood during water storage. J Inst. Wood. Sci. 6(2):8-12.
Wei-Chen, C., Hsieh, H, J and Tseng, T.C. (1998), Purification and
characterization of a pectin lyase from Pythium splendens infected
cucumber fruits. Bot. Bull. Acad. Sin. 39: 181-186.
Weil, J., Pinsky, A and Grossman, S. (1966), The protease of the soybean. Cereal
Chem. (43): 392.
Whilaker, I.R. (1972). Pectic substances, pectic enzymes and haze formation in
fruit juices. Enz. Microbiol. Technol. 6: 341-349.
Whitaker, J. R. (1990), Microbial pectinolytic enzymes In: Fogarty, W. M. & Kelly,
C. T. (editors), Microbial enzymes and Biotechnology. 2nd ed. London:
Elsevier Science Ltd. Pp. 133 - 176.
Wilson, K and Walker, J. (Eds.) (2000), In: Principles and Techniques of Practical
Biochemistry Cambridge University Press. Cambridge.
Wood, W. A and Kellogg, S. (1988), Methods in Enzymology, Vol. 161: Biomass,
Part B: Lignin, Pectin, and Chitin, Academic Press, New York Pp. 315–322.
226
Yadav, S., Yadav, P.K., Yadav, D and Yadav, K.D.S. (2008), Purification and
characterization of an alkaline pectin lyase from Aspergillus flavus. Process
Biochem. 43: 547-552.
Yadav, S., Yadav, P.K., Yadav, D and Yadav, K.D.S. (2009), Purification and
charac- terization of pectin lyase produced by Aspergillus terricola and its
application in retting of natural fibers. Appl. Biochem. Biotechnol. 159:
270-283.
Yoshikawa , M., Hayashi, M., Sakamoto, T., Hours, R.A., Katsuragi, T and Sakai,
T. (1995), Protopectinase production by Aspergillus awamori in
submerged and solid state fermentation. Division of Biological
Conversion, Osaka Prefecture University, Osaka, Japan. Appl Biol Sci
.1(1):71–82.
Young, T.W., Wadeson., Glover, D.J., Quincey, R.V., Butlin, M.J and Kamei, E.A.
(1996), The extracellular acid protease gene of Yarrowia lipolytica: sequence
and pH-regulated transcription. Microbiology 142:2913- 2921.
Yusof, S and Ibrahim, N. (1994), Quality of sour sop juice after pectinase enzyme
treatment. Food Chemistry 51 (1): 83-88.
Yugandhar, N.M., Kumar, D.V.R., Prasanti, V., Kumar, N.K and Reddy, D.S.R.
(2008), Optimization of pectinase production from Manihot utilissima by
Aspergillus niger NCIM 548 using statistical experimental design. Research
J.Microbiol. 3:9-16
Zeilinger, S., Mach, R.L., Schindler, M., Herzog, P and Kubicek, C.P. (1996),
Different inducibility of expression of the two xylanase genes xyn1 and
xyn2 in Trichoderma reesei. J. Biol. Chem. 271:25624-25629.
Zheng, Z and Shetty, K. (2000), Solid state production of polygalacturonase by
Lentinus edodes using fruit processin wastes. Process Biochem. 35: 825-830.