Enhancement of fungal pectinolytic enzymes production

Enhancement of fungal pectinolytic

enzymes production using gamma

radiation under solid state

fermentation

Thesis

Submitted in Partial Fulfillment for

MSc Degree in Microbiology

Botany and Microbiology Dept Faculty of

Science Helwan University

Presented BY

Shaima Abdel Mohsen Ibrahim

BSc MicrobiologyampBiochemistry (2005)

Under Supervision of

Prof Dr Mohamed E Osman

Prof of Microbiology Faculty of Science Helwan University

ProfDrAhmed Ibrahim ELSayed El-

BatalProf of Applied Microbiologyamp BiotechnologyNCRRT

Faculty of Science

fermentation

Thesis

Presented BY

Ain Shams University

Faculty of Science

Botany and Microbiology Department

جحضيي الاحاج الفطري للازيوات الوحللة للبكحيي باصحخذام

اشعة جاها جحث ظروف الحخور شبه الجافة

رسالة هقدهة هي

شيواء عبذ الوحضي ابراهين

ampكيوياء حيويهيكروبيولوجى ndashريوس العلوم وبكال

(0225)عيي شوشجاهعة

كوحطلب جزئى

للحصول على درجة الواجيضحير فى الويكروبيولوجى

ث إشرافجح

قسن النبات والويكروبيولوجى -أستاذ الويكروبيولوجى

جاهعة حلواى -كلية العلوم

استاذ الويكروبيولوجيا التطبيقية والتكنولوجيا الحيوية

وتكنولوجيا الاشعاع الوركز القوهي لبحوث

جاهعة حلواى ndashكلية العلوم

قسم النبات والميكروبيولوجى

جاهعة حلواى

كلية العلوم

ampكيوياء حيويهيكروبيولوجى ndashبكالوريوس العلوم

(0225) عيي شوشجاهعة

للحصول على درجة الواجيضحير

فى الويكروبيولوجى

Approval Sheet

Title of master thesis

Enahncement of fungal pectinolytic

fermentation

Submitted to

Department of

Botany and Microbiology

Faculty of Science- Helwan University

Supervision Committee

ProfDrAhmed Ibrahim El Sayed El Batal

Prof of Applied Microbiologyamp BiotechnologyNCRRT

ACKNOWLEDGMENT

First and foremost my unlimited thanks are to

our God who guides and sustains

My deepest gratitude and appreciation to

ProfDrMohamed EOsman Prof of Microbiology

Botany and Microbiology Department Helwan

University for his closely supervision and kind help

I am deeply thankful to ProfDrAhmed

Ibrahim El Sayed El-Batal Prof of Applied

MicrobiologyampBiotechnology Drug Radiation

Research Dep National Center for Radiation

Research ampTechnology (NCRRT) for suggesting the

research topic valuable supervision as this thesis is

a part of the ProjectldquoNutraceuticals and

Functional Foods Production by Using

NanoBiotechnological and Irradiation Processesrdquo

that is financially supported by NCRRT

My sincere thanks extended to all the staff

and members of the Microbiology lab in NCRRT

Gratitude is extended to all the staff and

members of the Microbiology lab at the Department

of Botany and Microbiology Faculty of Science

Helwan University

Lastly my thanks go to my family for their

understanding and willingness to assist

Enhancement of Fungal Pectinolytic Enzymes

Production Using Gamma Radiation Under Solid State

Fermentation

(Shaima Abdel Mohsen Ibrahim)

(Botany and Microbiology DepFaculty of ScienceHelwan

University)

Summary

14 fungal species were screened for their ability to

produce pectinases on sugar-beet pulp medium The

highest producer strain was identified as Penicilium

citrinum

The optimum conditions for polygalacturonases

production were achieved by growing the fungus on

sugar beet pulp mineral salts medium and incubation for

7 days at 250C pH 55and 004g Ng dry SBP by using

the conventional method and 12 of nitrogen source

by using the factorial design method and surfactant of

01 Tween 40 The use of gamma irradiation at a dose

of 07 kGy yields the highest increase of production of

PGase Polygalacturonases were precipitated from

culture supernatant using ammonium sulphate then

purified by gel filtration chromatography on sephadex

The optimum pH and temperature of the enzyme

activity production were found to be 60 and 40degC

respectively The enzyme was found to be stable at pH

rang 4 ndash 8 and showed high stability at temperature rang

20degC -60degC Mg+2

and Zn+2

stimulated PGase activity

Contents

No Title Page

1 Introduction 1

2 Review of literature 4

1-Classification of pectic substance 5

15Pharmaceutical uses of pectin 8

2-Classification of pectic enzymes 10

21 Pectic estrases 10

22 Depolarizing pectinases 11

23 Cleaving pectinases 12

3 Production of Pectinases 14

31 Submerged fermentation (SmF) 15

32 Solid substrate fermentation (SSF) 15

4 Uses of Pectinases 23

41Fruit juice industry 23

42 Wine industry 25

43 Textile industry 26

5 Factors controlling the microbial pectinase production 26

51 PH and thermal stability of pectinases 26

52 Carbon Sources 28

53-Nitrogen sources 29

54ndashTemperature 30

55- Incubation period 31

56- Inoculum size 31

57- Surfactants 32

6 Factorial Design 33

7 Gamma Rays 35

71 Ionizing radiation 37

72 Responses of pectinases to gamma radiation 37

8 Purification of microbial pectinases 38

9 Applications of pectinases 39

3- Materials and Methods 40 31Microorganisms 40

32Culture media 40

33 Fermentation substrates 41

4 Culture condition 41

5 Screening for pectinolytic enzymes using Sugar beet

pulp medium

6 Analytical methods 43

61 Pectinases assay 43

62 Assay for pectin lyase 45

63 Protein determination 45

64 Statistical analysis 45

7 Optimization of parameters controlling pectinases

production by Pcitrinum

71 Effect of different natural products 46

72 Effect of different nitrogen sources 47

73 Effect of different inoculum sizes 47

74 Effect of different incubation periods 48

75 Effect of different pH values 48

76 Effect of different temperatures 49

77 Effect of different surfactants 49

78 Application of factorial design for optimization of

pectinase production by Pcitrinum under Solid state

fermentation

79 Effect of different gamma irradiation doses 50

8 Purification of pectinases 51

81 Production of pectinases and preparation of cell-free

filtrate

82 Ammonium sulphate precipitation 51

821 Steps for precipitation by ammonium sulphate 52

83 Dialysis 52

84 Gel filtration chromatography 53

9 Characterization of the purified polygalacturonase

enzyme

93 Effect of different temperatures on the enzyme 57

94 Effect of different metal ions on the activity of the

purified exo-polygalacturonase enzyme produced by

Pcitrinum

10 Bioextraction of pectin from different agro-residues for

different pharmaceutical applications

4- Results 58

41Screening of the most potent fungal pectinase producer 58

411 polygalacturonase activity 58

412 Pectin lyase activity 60

42 Optimization of the fermentation parameters affecting

enzyme production

421 Effect of some agroindustrial by-products as carbon

source on polygalacturonase production by Pcitrinum

under Solid state fermentation

422 Effect of different nitrogen sources on

polygalacturonase production using Penicillium citrinum

423 Effect of inoculum size on polygalacturonases

production by Pcitrinum under solid state fermentation

424 Effect of different incubation periods on extracellular

polygalacturonase enzyme production by Penicillium

citrinum

425 Effect of different pH values on polygalacturonases

426 Effect of different incubation temperatures on

polygalacturonase production by Pcitrinum under solid

state fermentation

427 Effect of some surfactants on polygalacturonase

polygalacturonase production by Pcitrinum under Solid

state fermentation

429 Effect of Gamma irradiation doses on

polygalacturonase production by P citrinum under Solid

state fermentation using optimized conditions of factorial

design

43 Purification and characterization of the enzyme 84

431 Purification steps 84

432 Characterization of the purified enzyme 86

4322Effect of different temperatures 90

purified polygalacturonase enzyme produced by Pcitrinum

44 Extraction and determination of pectic substances 96

5- Discussion 98

6- Concluding remarks 126

7- References 127 7

List of tables

No Title page

1 Composition of pectin in different fruits and vegetables 7 2 Comparison of solid and submerged fermentation for

pectinase production

3 Polygalacturonase activity of the tested fungal species under

solid state fermentation

Effect of some agroindustrial by-products as carbon source

on polygalacturonase production by Pcitrinum under Solid

state fermentation

Effect of different nitrogen sources on polygalacturonase

production using Penicillium citrinum under Solid state

fermentation

6 Effect of inoculum size on polygalacturonase production by

Pcitrinum under solid state fermentation

7 Effect of different incubation periods on production of the

polygalacturonase enzyme by Penicillium citrinum

polygalacturonase production by Penicillium citrinum

10 Effect of some surfactants on polygalacturonase production

by P citrinum under solid state fermentation

Effect of the variables and their interactions in the 25

factorial designs for optimization of polygalacturonase

production by Pcitrinum under Solid state fermentation

ANOVA table for the enzyme activity effect of inoculums

size yeast extract and temperature on the activity of PGase

13 Effect of Radiation Dose on polygalacturonase production

using Penicillium citrinum

14 Purification of PGase secreted by Pcitrinum 85

Effect of different pH values on the activity of the purified

polygalacturonase enzyme produced by Pcitrinum

Effect of different pH values on the stability of the purified

Effect of the temperature on the activity of the purified

Effect of different temperatures on the stability of the

Pcitrinum

19 Effect of different metal ions on the activity of the purified

20 The different weights of pectin extracted from different

agroindustrial by products inoculated with Pcitrinum

List of Figures

No Title page

1 Structure of pectin 8

2 Mode of action of pectinases 14

3 polygalacturonases activity of the tested fungal species

grown under solid state conditions

Effect of different agroindustrial by products as carbon

sources on polygalacturonase production by Pcitrinum

Effect of different nitrogen sources on polygalacturonases

production using Pcitrinum under solid state fermentation

Effect of different incubation periods on polygalacturonase

Effect of different pH values on polygalacturonases

Effect of different incubation temperatures on

Effect of some surfactants on polygalacturonase production

by Pcitrinum

Factorial design for the effects of yeast extract and

inoculums size at pH55 on the polygalacturonase activity of

Penicillium citrinum

Plot of predicted versus actual polygalacturonase

production

Effect of different doses of gamma Radiation on

14 Gel filtration profile of polygalacturonase 86

Effect of different pH values on the stability of the purified exo-

Effect of the temperature on the activity of the purified exo

purified polygalacturonase enzyme produced by Pcitrinu

purified polygalacturonase enzyme produced by

Pcitrinum

Abbreviations and symbols

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

pH negative logarithm of numerical value

` (hydrogen ion exponent)

rpm round per minute

SMF submerged fermentation

sp species

SSF Solid state fermentation

35 DNS 35 Dinitrosalycylic acid

Aim of the study

The present study aimed to investigate some aspects in

relation to enhancement of fungal production of

pectinolytic enzymes using Gamma radiation under Solid

state fermentation

1 Screening of the most potent fungal isolates for the

biosynthesis of extracellular pectinases

2 Optimization of solid state fermentation parameters

for the highest enzyme producion (different carbon

sources nitrogen sources pH temperature duration

time and surfactants)

3 Role of gamma irradiation on pectinase production

4 Characterization of partially purified enzyme

5 Possible applications of microbial pectinases with

extraction of some natural pectin from agrowastes

sources

Introduction

Application of biotechnology in industrial

production holds many promises for sustainable

development but many products still have to pass the test

of economic viability White biotechnology is

biotechnology used for industrial purposes Industries

incorporating white biotechnology use living organisms

organic materials or chemical components of living

organisms such as enzymes in the production process

Applications of white biotechnology currently being used

or researched include manufacturing processes the creation

of biomaterials and alternate energy sources

In addition to purely commercial benefits white

biotechnology is also being researched as a way to make

industry more environmentally friendly by providing less

polluting sources of energy lessening dependence on fossil

fuels and creating industrial processes with fewer polluting

by-products

Biological processes are based on chemical

processes and so white biotechnology is being

incorporated into many production processes and

Introduction

Products that involve chemical reactions Some

chemicals used in industry such as some polymers and

acids can be produced biologically rather than through

conventional means Industrial enzymes can be used in

chemical-intensive processes such as the production of

paper and the treatment of textiles and leather for

clothing Cleaning products made with this kind of

biotechnology such as laundry and dishwashing

detergents use enzymes in the place of conventional

inorganic chemicals

Pectinases are the first enzymes to be used in

homesTheir commercial application was first reported in

1930 for the preparation of wines and fruit juices Only in

1960 the chemical nature of plant tissues became apparent

and with this knowledge scientists began to use enzymes

more efficiently As a result pectinases are today one of the

upcoming enzymes of the commercial sector Primarily

these enzymes are responsible for the degradation of the

long and complex molecules called pectin that occur as

structural polysaccharides in the middle lamella and the

primary call walls of young plant cells Pectinases are now

Introduction

an integral part of fruit juice and textile industries as well

as having various biotechnological applications Microbial

sources have occupied an important place in the pectinases

production Among microbes fungi as enzyme producers

have many advantages since they are normally GRAS

(generally regarded as safe) strains and the produced

enzymes are extracellular which makes it easy recuperation

from fermentation broth (Pushpa and Madhava 2010)

The pectinase class of hydrolytic enzymes is one of several

enzymes that Penicillium sp can produce to utilize a wide

variety of naturally substrates Accordingly a local isolate

of Penicillium sp was chosen to investigate the production

and characterstics of its pectinase yield

Review of literatures

REVIEW OF LITERATURE

Pectinase comprises a heterogeneous group of

enzymes that catalyze the breakdown of pectin-containing

substrates They are widely used in the food industry to

improve the cloud stability of fruit and vegetable

nectarsfor production and clarification of fruit juices and

for haze removal from wines (Cavalitto et al 1996)

Furthermore phytopathologic studies have reported that

fungal endo-polygalacturonase (endoPGase) which is a

major kind of pectinase has been shown to activate plant

defense responses including phytoalexin accumulation

lignification synthesis of proteinase inhibitors and

necrosis (Cervone et al 1989) Further research has

confirmed that endoPGase can degrade the plant cell wall

releasing pectic oligomers which can stimulate a wide array

of plant defence responses (Boudart et al 1998) With the

increasing application of pectinases decreasing its

production cost has become one of the most important

targets For this purpose selection of carbon source and

nitrogen source with low value is a practical consideration

Previous studies reported that many waste products from

the agricultural industry containing pectin such as sugar

beet pulp (SBP) citrus pulp pellets apple pomace pulp

lemon pulp and other related materials have been used as

carbon source for induction of pectinase by many

microorganisms (Said et al 1991)

1 Pectic substances in plant cell walls

Chemically pectic substances are complex colloidal

acid polysaccharides with a backbone of galacturonic acid

residues linked by a (1 4) linkages The side chains of the

pectin molecule consist of L-rhamnose arabinosegalactose

and xylose The carboxyl groups of galacturonic acid are

partially esterified by methyl groups and partially or

completely neutralized by sodium potassium or

ammonium ions

Classification of pectic substances

Based on the type of modifications of the backbone

chain pectic substances are classified into protopectin

pectic acid Pectinic acid and pectin (Miller 1986)

11Protopectin

This is a parent pectic substance and upon restricted

hydrolysis yields pectin or Pectinic acid Protopectin is

occasionally a term used to describe the water-insoluble

pectic substances found in plant tissues and from which

soluble pectic substances are produced (Kilara 1982)

12Pectic acids

These are the galacturonans that contain negligible amounts

of methoxyl groups Normal or acid salts of pectic acid are

called pectates

13Pectinic acids

These are the galacturonans with various amounts of

methoxyl groups Pectinates are normal or acid salts of

pectinic acids (Kilara 1982) Pectinic acid alone has the

unique property of forming a gel with sugar and acid or if

suitably low in methyl content with certain other

compounds such as calcium salts

Table1Amount of pectin in different fruits and

vegetables (Kashyap et al 2001)

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Dry matter 69ndash186

Orange pulp

Dry matter

124ndash280

Fig1 Structure of pectin (Harholt et al 2010)

2 Pharmaceutical Uses of Pectin

1 In the pharmaceutical industry pectin favorably

influences cholesterol levels in blood It has been

reported to help reduce blood cholesterol in a wide

variety of subjects and experimental conditions as

comprehensively reviewed (Sriamornask

2001)Consumption of at least 6 gday of pectin is

necessary to have a significant effect in cholesterol

reduction Amounts less than 6 gday of pectin are not

effective (Ginter 1979)

2 Pectin acts as a natural prophylactic substance

against poisoning with toxic cations It has been shown

to be effective in removing lead and mercury from the

gastrointestinal tract and respiratory organs (Kohn

1982) When injected intravenously pectin shortens the

coagulation time of drawn blood thus being useful in

controlling hemorrhage or local bleeding (Joseph

3 Pectin reduces rate of digestion by immobilizing

food components in the intestine This results in less

absorption of food The thickness of the pectin layer

influences the absorption by prohibiting contact between

the intestinal enzyme and the food thus reducing the

latterrsquos availability (WilsonampDietschy 1974 Dunaifamp

Schneeman 1981 Flourie et al 1984)

4 Pectin has a promising pharmaceutical uses and is

presently considered as a carrier material in colon-

specific drug delivery systems (for systemic action or

a topical treatment of diseases such as ulcerative

colitis Crohnrsquos disease colon carcinomas) The

potential of pectin or its salt as a carrier for colonic

drug delivery was first demonstrated by studies of

Ashford et al (1993) and Rubinstein et al (1993)

The rationale for this is that pectin and calcium

pectinate will be degraded by colonic pectinolytic

enzymes(Englyst et al1987) but will retard drug

release in the upper gastrointestinal tract due to its

insolubility and because it is not degraded by gastric or

intestinal enzymes(Sandberg et al1983)

3 Classification of pectic enzymes

Pectinases are classified under three headings

according to the following criteria whether pectin pectic

acid or oligo-D-galacturonate is the preferred substrate

whether pectinases act by trans-elimination or hydrolysis

and whether the cleavage is random (endo- liquefying of

depolymerizing enzymes) or endwise (exo- or

saccharifying enzymes) The three major types of

pectinases are as follows

31 Pectinesterases (PE) (Ec 31111)

Pectinesterases also known as pectinmethyl

hydrolase catalyzes deesterification of the methyl group of

pectin forming pectic acid The enzyme acts preferentially

on a methyl ester group of galacturonate unit next to a non-

esterified galacturonate one

32 Depolymerizing pectinases

These are the enzymes

321-Hydrolyzing glycosidic linkages

They include

3211- Polymethylgalacturonases (PMG) Catalyze the

hydrolytic cleavage of a-14-glycosidic bonds They may

32111 Endo-PMG causes random cleavage of α-14-

glycosidic linkages of pectin preferentially highly

esterified pectin

32112 Exo-PMG causes sequential cleavage of α -1 4-

glycosidic linkage of pectin from the non-reducing end of

the pectin chain

32112- Polygalacturonases (PG) (Ec 32115)

Catalyze hydrolysis of α -1 4-glycosidic linkage in pectic

acid (polygalacturonic acid) They are also of two types

321121 Endo-PG also known as poly (14- α -D-

galacturonide) glycanohydrolase catalyzes random

hydrolysis of α - 14-glycosidic linkages in pectic acid

321122 Exo-PG (Ec 32167) also known as poly

(14- α -D-galacturonide) galacturonohydrolase catalyzes

hydrolysis in a sequential fashion of a-14-glycosidic

linkages on pectic acid

33 Cleaving pectinases

Cleaving α -14-glycosidic linkages by trans-

elimination which results in galacturonide with an

unsaturated bond between C4 and C5 at the non-reducing

end of the galacturonic acid formed These include

331 Polymethylegalacturonate lyases (PMGL)

Catalyze breakdown of pectin by trans-eliminative

cleavage They are

3311 Endo-PMGL (Ec 42210) also known as poly

(methoxygalacturonide) lyase catalyzes random cleavage

of a-14-glycosidic linkages in pectin

3312 Exo-PMGL catalyzes stepwise breakdown of

pectin by trans-eliminative cleavage

3322 Polygalacturonate lyases (PGL) (Ec 42993)

Catalyze cleavage of α -14-glycosidic linkage in pectic

acid by trans-elimination They are also of two types

33221 Endo-PGL (Ec 4222)

Also known as poly (14- α D-galacturonide) lyase

catalyzes random cleavage of α -14-glycosidic linkages in

pectic acid

33222 Exo-PGL (Ec 4229) also known as poly (1 4-

α -D-galacturonide) exolyase catalyzes sequential cleavage

of a-1 4-glycosidic linkages in pectic acid

33 Protopectinase

This enzyme solubilizes protopectin forming highly

polymerized soluble pectinOn the bases of their

applications pectinases are mainly of two types acidic

pectinases and alkaline pectinases

Figure 2 Mode of action of pectinases (a) R = H for PG and CH3 for PMG (b) PE and (c) R = H

for PGL and CH3 for PL the arrow indicates the place where the pectinase reacts with the

pectic substances PMG polymethylgalacturonases PG polygalacturonases PE

pectinesterase PL pectin lyase (Jayani et al 2005)

4 Production of Pectinases

Microbial enzymes are commercially produced either

through submerged fermentation (SmF) or solid substrate

fermentation (SSF) techniques

41 Submerged fermentation (SmF)

SmF techniques for enzyme production are generally

conducted in stirred tank reactors under aerobic conditions

using batch or fed batch systems High capital investment

and energy costs and the infrastructural requirements for

large-scale production make the application of Smf

techniques in enzyme production not practical in a

majority of developing countries environments Submerged

fermentation is cultivation of microorganisms on liquid

broth it requires high volumes of water continuous

agitation and generates lot of effluents

42 Solid substrate fermentation (SSF)

SSF incorporates microbial growth and product

formation on or with in particles of a solid substrate under

aerobic conditions in the absence or near absence of free

water and does not generally require aseptic conditions for

enzyme production (Mudgett 1986 and Sanzo et al 2001)

43Microorganisms commonly used in submerged

and solid state fermentation for Pectinases production

Microorganisms are currently the primary source of

industrial enzymes 50 originate from fungi and yeast

35 from bacteria while the remaining 15 are either of

plant or animal origin Filamentous microorganisms are

most widely used in submerged and solid-state

fermentation for pectinases production Ability of such

microbes to colonize the substrate by apical growth and

penetration gives them a considerable ecological advantage

over non-motile bacteria and yeast which are less able to

multiply and colonize on low moisture substrate (Smith et

al 1988) Among filamentous fungi three classes have

gained the most practical importance in SSF the

phycomycetes such as the geneus Mucor the ascomycetes

genera Aspergillus and basidiomycetes especially the white

and rot fungi (Young et al 1983) Bacteria and yeasts

usually grow on solid substrates at the 40to70 moisture

levels (Young et al 1983) Common bacteria in use are

(Bacillus licheniformis Aeromonas cavi Lactobacillus etc

and common yeasts in use are Saccharomyces and Candida

Pectinase production by Aspergillus strains has been

observed to be higher in solid-state fermentation than in

submerged process (Solis-Pereyra et al 1996)

44 Substrate for fermentation

Medium require presence of bioavailable nutrients

with the absence of toxic or inhibitory constituents

medium Carbon nitrogen inorganic ions and growth

factors are also required For submerged fermentation

besides carbon source nitrogen growth factors media

requires plenty of water The most widely used substrate

for solid state fermentation for pectinase production are

materials of mainly plant origin which include starchy

materials such as grains roots tubers legumes cellulosic

lignin proteins and lipid materials (Smith and Aidoo

1988) Agricultural and food processing wastes such as

wheat bran cassava sugar beet pulp Citrus wastecorn

cob banana waste saw dust and fruit pomace (apple

pomace) are the most commonly used substrates for SSF

for pectinase production (Pandey et al 2002)

33 Table2Comparison of solid and submerged

fermentation for pectinase production (Raimbault

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

Heat sterilization and

aseptic control

Vapor treatment non

sterile conditions

High volumes of water

consumed and effluents

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

45 Pectinases production in solid state fermentation

451 Protopectinases

PPases are classified into two types on the basis of

their reaction mechanism A-type PPases react with the

inner site ie the polygalacturonic acid region of

protopectin whereas B-type PPases react on the outer site

ie on the polysaccharide chains that may connect the

polygalacturonic acid chain and cell wall constituentsA-

type PPase are found in the culture filtrates of yeast and

yeast-like fungi They have been isolated from

Kluyveromyces fragilis Galactomyces reesei and

Trichosporon penicillatum and are referred to as PPase-F -

L and -S respectively B-type PPases have been reported in

Bacillus subtilis and Trametes sp and are referred to as

PPase- B -C and -Trespectively B-type PPases have also

been found in the culture filtrate of a wide range of Bacillus

sp All three A-type PPases are similar in biological

properties and have similar molecular weight of 30

kDaPPase-F is an acidic protein and PPase-L and -S are

basic proteins The enzymes have pectin-releasing effects

on protopectin from various origins The enzymes catalyze

the hydrolysis of polygalacturonic acid they decrease the

viscosity slightly increasing the reducing value of the

reaction medium containing polygalacturonic acid PPase-

B -C and -T have molecular weights of 45 30 and 55 kDa

respectively

452 Polygalacturonases

Endo-PGases are widely distributed among fungi

bacteria and many yeasts They are also found in higher

plants and some plant parasitic nematodes They have been

reported in many microorganisms including

Aureobasidium pullulans Rhizoctonia solani Fusarium

moniliforme Neurospora crassa Rhizopus stolonifer

Aspergillus sp Thermomyces lanuginosus Peacilomyces

clavisporus Endo- PGases have also been cloned and

genetically studied in a large number of microbial species

In contrast exo-PGases occur less frequently They

have been reported in Erwinia carotovora Agrobacterium

tumefaciens Bacteroides thetaiotamicron Echrysanthemi

Alternaria mali Fusarium oxysporum Ralstonia

solanacearum Bacillus spExo-PGases can be

distinguished into two typesfungal exo-PGases which

produce monogalacturonic acid as the main end product

and the bacterial exo-PGaseswhich produce digalacturonic

acid as the main end product Occurrence of PGases in

plants has also been reported Polygalacturonate lyases

(Pectate lyases or PGLs) are produced by many bacteria

and some pathogenic fungi with endo-PGLs being more

abundant than exo-PGLs PGLs have been isolated from

bacteria and fungi associated with food spoilage and soft

rot They have been reported in Erwinia carotovora

Amucala sp Pseudomonas syringae Colletotrichum

magna E chrysanthemi Bacillus sp Bacillus sp Very

few reports on the production of polymethylgalacturonate

lyases (pectin lyases or PMGLs) have been reported in

literature They have been reported to be produced by

Aspergillus japonicus Penicillium paxilli Penicillium sp

Pythium splendens Pichia pinus Aspergillus sp

Thermoascus auratniacus

453 Pectinesterase

PE activity is implicated in cell wall metabolism

including cell growth fruit ripening abscission senescence

and pathogenesis Commercially PE can be used for

protecting and improving the texture and firmness of

several processed fruits and vegetables as well as in the

extraction and clarification of fruit juices PE is found in

plants plant pathogenic bacteria and fungi It has been

reported in Rhodotorula sp Phytophthora infestans

Erwinia chrysanthemi B341 Saccharomyces cerevisiae

Lachnospira pectinoschiza Pseudomonas solanacearum

Aspergillus niger Lactobacillus lactis subsp Cremoris

Penicillium frequentans E chrysanthemi 3604

Penicillium occitanis A japonicus and othersThere are

many reports of occurrence of PE in plants viz Carica

papaya Lycopersicum esculentum Prunus malus Vitis

vinifera Citrus sp Pouteria sapota and Malpighia glabra

46 Advantages of Solid-State Fermentation

For several products Solid-State Fermentation offer

advantages over fermentation in liquid brothssubmerged

fermentation ( Cook 1994)

middot Higher product yield

middot Better product quality

middot Cheaper product recovers

middot Cheaper technology middot

middot Higher substrate concentration

middot Less probability of contamination

middot Lower capital investment

47Disadvantages

Despite solid-state fermentation being both

economically and environmentally attractive their

biotechnological exploitation has been rather limited

(Pandey 1992 Aidoo et al 1982)

middot Limitation on microorganism

middot Medium heterogeneity

middot Heat and mass transfer control growth measurement and

monitoring

middot Scale up problems

5 Uses of Pectinases

51Fruit juice industry

511 Fruit juice clarification

Addition of pectinase lowers the viscosity and causes

cloud particles to aggregate to larger units (break) so easily

sedimented and removed by centrifugation Indeed

pectinase preparation was known as filtration enzymes

Careful experiments with purified enzyme have shown that

this effect is reached either by a combination of PE and

Polygalacturonase or by PL alone in the case of apple juice

which contains highly esterified pectin (gt80) (Ishii and

Yokotsuka 1972)

512 Enzymes treatment of pulp for juice extraction

In early periods of pectinase uses for clarification it

was found first for black currents that enzyme treatment of

the pulp before pressing improved juice and color yield

(Charley 1969) Enzymatic pectin degradation yields thin

free run juice and a pulp with good pressing characteristics

(Beltman and Plinik 1971) In case of apples it has been

shown that any combination of enzymes that depolymerize

highly esterified pectin (DEgt90) can be successfully used

(Pilnik and Voragen 1993)

513 Liquefaction

It is process in which pulp is liquefied enzymatically

so pressing is not necessary Viscosity of stirred apple pulp

decreases during treatment with pectinases cellulase and a

mixture of the two-enzyme preparation Cellulase alone had

little effect on pectin and solubilized only 22 of cellulose

Combined cellulase and pectinase activities released 80

of the polysaccharide A similar effect has been found for

grapefruit (Pilnik and Voragen 1993)

514 Maceration

It is the process by which the organized tissue is

transformed into a suspension of intact cells resulting in

pulpy products used as a base material for pulpy juices and

nectars as baby foods The aim of enzyme treatment is

transformation of tissue into suspension of intact cells This

process is called enzymatic maceration (The so called

macerases are enzyme preparation with only

Polygalacturonase or PL activity) A very interesting use of

enzymatic maceration is for the production of dried instant

potato mash Inactivation of endogenous PE is important

for the maceration of many products (Pilnik and Voragen

52 Wine industry

Pectolytic enzymes are added before fermentation of

white wine musts which are made from pressed juice

without any skin contact in order to hasten clarification

Another application of Pectolytic enzymes during wine

making is associated with the technology of

thermovinification During heating the grape mash to 50degC

for few hours large amounts of pectin are released from the

grape this does not occur in traditional processing It is

therefore necessary to add a Pectolytic preparation to the

heated mash so that the juice viscosity is reduced An

additional benefit from the process is that the extraction of

anthocyanins is enhanced probably due to a breakdown in

cell structure by the enzyme which allows the pigments to

escape more readily and thus helps in color enhancement

(Tucker and Woods 1991)

53 Textile industry

In the textile industry pectinases are sometimes used

in the treatment of natural fibers such as linen and ramie

fibers (Baracet et al 1991)

6 Factors controlling microbial pectinases production

61 PH and thermal stability of pectinases

Enzyme deactivation and stability are considered to be

the major constraints in the rapid development of

biotechnological processes Stability studies also provide

valuable information about structure and function of

enzymes Enhancing the stability and maintaining the

desired level of activity over a long period are two

important points considered for the selection and design of

pectinases The stability of pectinases is affected by both

physical parameters (pH and temperature) and chemical

parameters (inhibitors or activators) PH is also one of the

important factors that determine the growth and

morphology of microorganisms as they are sensitive to the

concentration of hydrogen ions present in the medium The

optimal pH for Rhizopus arrhizus endo-PG has been found

to be in the acidic range of 38-65 Rhizopus stolonifer

endo-PG was stable in the pH range 30 upto50 and this

enzyme is highly specific to non-methoxylated PGA The

two PGs were stable at pH 50 and 75 and at a temperature

of 50 ordmC whereas two PLs exhibited maximum stability at

50 and 75 and at a temperature of 400C It has also been

reported that PL from Aspergillus fonsecaeus was stable at

52 This PL does not react with PGA but it does with PGA

pretreated with yeast PG The optimal pH for A niger PMG

was around 40 Most of the reports studied the pH and

thermal stability by conventional optimization methods (ie

the effect of temperature on pectinase stability was studied

at constant pH and vice versa) The interaction effect

between pH and temperature is another interesting aspect

which alters the stability differently The combined effect

of pH and temperature on stability of three pectinases viz

PMG PG and PL from A niger was studied in this

laboratory using response surface methodology For this

purpose a central composite design was used and a

quadratic model proposed to determine the optimal pH and

temperature conditions at which pectinases exhibit

maximum stability The optimum pH and temperature were

22 and 23 ordmC respectively for PMG 48 and 280C

respectively for PG and 39 and 29 ordmC respectively for

PL PL was more stable than PMG and PG

62 Carbon Sources

The production of food enzymes related to the

degradation of different substrates These enzymes degrade

pectin and reduce the viscosity of the solution so that it can

be handled easily Optimization of physical parameters

such as pH temperature aeration and agitation in

fermenters should be done The different carbon sources on

base as apple pectin and the pressed apple pulp stimulated

the production of pectinolytic enzymes and the growth of

the microorganism (dry biomass) The different carbon

sources showed maximum dry biomass (db) with glucose

and fructose The best carbon source on base for better

production of pectinolytic enzymes was the pressed apple

pulp Biosynthesis of endo-PG and growth of the culture

Aspergillus niger in relation to the carbon sources

Biosynthesis of endo-PG is induced by pectic substances

and inhibited in the presence of easy metabolized

monosaccharides (glucose fructose etc) and some other

compounds Many results were obtained by many authors

who described the use on different inexpensive carbon

sources for better production of pectinolytic enzymes

(Aguilar and Huitron 1987 Maldonado et al 1986

Hours et al 1988 Larious et al 1989 Leuchtenberger

et al 1989 Pericin et al 1992 Shevchik et al 1992

Hang and Woodams 1994 Berovic and Ostroversnik

1997 Alkorta et al 1998 Zheng et al 2000 Kaur and

Satyanarayana 2004 Joshi et al 2006 Zhong-Tao et

al 2009 Tsereteli et al 2009)

63-Nitrogen sources

In most microorganisms both inorganic and organic

forms of nitrogen are metabolized to produce amino acids

nucleic acids proteins and cell wall components

(KumarampTakagi 1999) Different organic and inorganic

nitrogen sources yeast extract peptone tryptone glycine

urea ammonium chloride ammonium nitrate ammonium

sulphate and ammonium citrate were supplemented

separately The purified enzyme retains its full activity after

exposure for 1h at 60 and 700C in the presence of 06 and

18 M ammonium sulphate respectively However in

absence of ammonium sulphate enzyme looses its 60

activity at 60 ordmC while 88 activity is lost at 70 ordmC At

higher temperature (80ndash100 ordmC) ammonium sulphate is not

able to stabilize the activity of pectin lyase Of the various

nitrogen compounds tested for pectinase production high

pectinase activities were obtained with (NH4)2SO4 yeast

extract powder soya peptone soybean pulp powder and the

64ndashTemperature

Incubation temperature has been found to be a

significant controlling factor for enzyme

production(Kitpreechavanich et al 1984)Various

optimum temperature values were reported for

maximum pectinase production maximum enzyme

activity was found at 40ordmC and lower activity was

showed at 30 ordmC by Aspergillus Niger The optimal

temperature of PL was detected at 450C Obi and

Moneke 1985 stated that the maximum activity of their

enzyme was observed at this degree No activity was

recorded after heating the enzyme over 55 ordmC A

significant amount of biomass was produced by

Pclavisporus at temperatures between 20 ordmC and 500 C

The highest growth rates were observed at 300C

Endopolygalacturnase production was detected in

cultures incubated at 20 ordmC 30 ordmC 40 ordmC 50 ordmC with

The highest value was attained at 30 ordmCwhereas no

enzyme production was observed at 10 and 60 ordmC

65- Incubation period

With the respect to the role of incubation period on

pectinase production by microorganisms different

incubation periods were reported for maximum

pectinase production The maximum pectinase activity

was found at 7th

day of incubation by Aspergillus

nigerIt means that pectinase production activity is

correlated with the incubation time which was also

found from other investigations (Venugopal et al

2007and Pereira et al 1992)It can be noticed that the

optimum time of fermentation was found to be 72 h

after which there is decrease in the production of the

enzyme by Aspergillus niger Polygalacturanase

production by Moniliella sp peaked between 3rd

and 4th

day of cultivation when Penicillium sp was used

maximal Pg activity was detected at the 8th

66- Inoculum size

It is known that inoculum size has a marked effect on

enzyme yields in fungal cultures in solid state conditions

(Meyrath ampSuchnex 1972) The inoculum size of

1times10 7

resulted in the maximum production of

endo-and exo-pectinases in solid state fermentation

(Solis-Pereyra et al 1996) with the highest level of

spores (10 6 spores g

-1 about a 10 decrease in the

maximum activity was observed The fact that lower

inoculum sizes do not affect enzyme production is very

important because large production of spores becomes

unnecessary Optimum inoculum density is important

consideration for SSF process since over crowding of

spores can inhibit growth and development (Ghanem et

al 2000)Higher inoculum levels besides increasing

spore density increase water content of the medium as

67- Surfactants

Previous experiments on fungal cell permeability

demonstrated that non-ionic surfactants (NIS surface

active agents) can stimulate the release of enzymes

(Reese and Macguire 1969) The effects of surfactants

have been attributed to at least three causes

i) Action on the cell membrane causing increased

permeability (Reese and Macguire 1969)

ii) promotion of the release of bound enzymes

(Reese and Macguire 1969)

iii) Decrease in growth rate due to reduced oxygen

supply (Hulme and Stranks 1970)

Tween 80 (a surfactant) was used to enhance the SSF

rate Addition of tween-80 into the growth medium of

citrus peel enhanced pectin lyase production and

maximum enzyme yield was noted in SSF medium

receiving 02 of this surfactant Growth media

containing less and more than 02 tween-80 showed

lower activities of the enzyme Higher levels of Tween-

80 increased the penetration of water into the solid

substrate matrix and increase the surface area more than

the requirement of the microbe (Fujian et aI 2001)

Tween-80 has also been shown to increase enzyme

production in fungal species such as T-reesei (Mandel

and Weber 1969) The non-ionic surfactant increases

extracellular protein accumulation in culture filtrates by

enhancing the export of proteins or enzymes through the

cell membrane

7 Factorial Design

A factorial design is often used by scientists wishing to

understand the effect of two or more independent variables

upon a single dependent variable Factorial experiments

permit researchers to study behavior under conditions in

which independent variables called in this context factors

are varied simultaneously Thus researchers can investigate

the joint effect of two or more factors on a dependent

variable The factorial design also facilitates the study of

interactions illuminating the effects of different conditions

of the experiment on the identifiable subgroups of subjects

participating in the experiment (Freedman 2005)

Factorial ANOVA is used when we want to consider the

effect of more than one factor on differences in the

dependent variable A factorial design is an experimental

design in which each level of each factor is paired up or

crossed with each level of every other factor In other

words each combination of the levels of the factors is

included in the design (Rosenbaum 2002)

This type of design is often depicted in a table

Intervention studies with 2 or more categorical

explanatory variables leading to a numerical outcome

variable are called Factorial Designs

A factor is simply a categorical variable with two or

more values referred to as levels

A study in which there are 3 factors with 2 levels is

called a 2sup3 factorial Design

If blocking has been used it is counted as one of the

factors

Blocking helps to improve precision by raising

homogeneity of response among the subjects

comprising the block

Advantages of factorial Designs are

A greater precision can be obtained in estimating the

overall main factor effects

Interaction between different factors can be explored

Additional factors can help to extend validity of

conclusions derived

Procedure used is General Linear Modelling

To determine the effects of different factors (yeast extract

incubation period inoculum size pH temperature) on the

production of pectinase enzymes by Penicillium citrinum

Thus we have a study with 5 factors and 2 levels ndash a 2

Factorial Design

8 Gamma Rays

Radiation is energy in the form of waves (beams) or

particles Radiation waves are generally invisible have no

weight or odor and have no positive or negative charge

Radioactive particles are also invisible but they have

weight (which is why they are called a particle) and may

have a positive or negative charge Some radiation waves

can be seen and felt (such as light or heat) while others

(such as x rays) can only be detected with special

instrumentation Gamma rays alpha particles and beta

particles are ionizing radiation Ionizing radiation has a lot

of energy that gives it the ability to cause changes in

atomsmdasha process called ionization Radio and TV signals

microwaves and laser light are non-ionizing types of

radiation Non-ionizing radiation has less energy than

ionizing radiation When non-ionizing radiation interacts

with atoms it does not cause ionization (hence non-

ionizing or not ionizing) (Taflove and Hagness 2005)

Gamma and X rays (also called photons) are waves

of energy that travel at the speed of light These waves can

have considerable range in air and have greater penetrating

power (can travel farther) than either alpha or beta

particles X rays and gamma rays differ from one another

because they come from different locations in an atom

Gamma rays come from the nucleus of an atom while

Xrays come from the electron shells Even though X rays

are emitted by some radioactive materials they are more

commonly generated by machines used in medicine and

industry Gamma and x rays are both generally blocked by

various thicknesses of lead or other heavy materials

Examples of common radionuclides that emit gamma rays

are technetium-99m (pronounced tech-neesh-e-um the

most commonly used radioactive material in nuclear

medicine) iodine-125 iodine-131 cobalt-57 and cesium-

137 (Tipler and Paul 2004)

81 Ionizing radiation

Ionizing radiation is energy transmitted via X-rays

γ-rays beta particles (high speed electrons) alpha particles

neutrons protons and other heavy ions such as the nuclei

of argon nitrogen carbon and other elements This energy

of ionizing radiation can knock electrons out of molecules

with which they interact thus creating ions X rays and

gamma rays are electromagnetic waves like light but their

energy is much higher than that of light (their wavelengths

are much shorter) The other forms of radiation particles are

either negatively charged (electrons) positively charged

(protons alpha rays and other heavy ions) or electrically

neutral (neutrons)

82 Responses of pectinases to gamma radiation

It has been found that at low doses of gamma

radiation the pectinase enzyme was slightly increased as

this is owed to the induction of gene transcriptions or

proteins has been found after low dose effects until it

reached to high doses the enzyme activity was obviously

decreased and further inhibited this may be due to the

absorbed dose caused rupturing in the cell membrane This

major injury to the cell allows the extracellular fluids to

enter into the cell Inversely it also allows leakage out of

ions and nutrients which the cell brought inside Membrane

rupture may result in the death of a cell

9 Purification of microbial pectinases

Purification of microbial pectinases received a great

attention particularly in recent years In general the

purification procedures included several steps the major

steps include precipitation of the enzyme application on

different chromatographic columns using ion exchange or

gel filtration chromatography and in many cases

performing polyacrylamide gel electrophoresis technique

(PAGE) high performance liquid chromatographic

technique (HPLC) and the electrofocusing technique

Ammonium sulphate widely used for enzyme precipitation

since (i) it has a high solubility in water (ii) characterized

by the absence of any harmful effect on most enzymes (iii)

has stabilizing action on most enzymes and (iv) it is usually

not necessary to carry out the fractionation at low

temperature (Dixon amp Webb 1964) Many

chromatographs were applied in the purification of the

enzyme For example Penicillium sp pectinase was

partially purified with sephadex G-100 column (Patil and

Chaudhari 2010) Furthermore the endo-

polygalacturonases isolated from Penicillum oxalicum was

purified using Sephadex G-100 Gel Filtration (Chun-hui et

al 2009)

10 Applications of pectinases

Over the years pectinases have been used in several

conventional industrial processes such as textile plant

fiber processing tea coffee oil extraction treatment of

industrial wastewater containing pectinacious material etc

They have also been reported to work in making of paper

They are yet to be commercialized

Materials and Methods

3-Materials and Methods

31-Microorganisms

Fungal strains were provided from Pharmaceutical

Microbiology Lab Drug Radiation Research Department

(NCRRT) Nasr City-Cairo-Egypt Fungal colonies were

maintained on potato-dextrose agar medium stored at 4ordmC

and freshly subcultured every four weeksThe strains

included (Alternaria alternata Aspergillus niger 1

Aspergillus niger 2 Aspergillus niger 3 Aspergillus niger 4

Aspergillus oryzae Gliocladium vierns Penicillium brevi-

compactum Penicillium chrysogenum Penicillium

citrinum Pleurotus ostreatus Rhizoctonia solani )

32Culture media

321Potato-dextrose agar meacutedium

According to Ricker and Ricker (1936) this medium

was used for isolation and maintenance of the fungal

strains and it has the following composition (g l)

Potato (peeled and sliced) 200 g

Dextrose 20 g

Agar 17 -20 g

Distilled water 1000ml

33 Fermentation substrates

The sugar beet pulp (SBP) used as a carbon source

has the following composition ( on dry basis) pectin

287 cellulose 200 hemicellulose 175 protein 90

lignin 44 fat 12 ash 51 (Xue et al 1992) The high

pectin content could be very helpful for pectinase

production

4 Culture condition

The used fermentation has the following contents

Ten grams of sugar beet pulp (SBP) were placed in

flasks and moistened with 20ml of distilled water

containing (04g Na2HPO4+ 008g KH2PO4+ 04g yeast

extract) and autoclaved for 30 min pH has been

adjusted to 59 using HCl and NaOH

41 pH adjustment (Sodium acetate-acetic acid buffer

solution pH 59)

Sodium acetate trihydrate powder (247 gram) was

solubilized in 910 ml distilled water

Glacial acetic acid (12ml) has been mixed in 100ml

of distilled water

Ninety ml were taken from the previous step and

mixed with the first step

5 Screening for pectinolytic enzymes using Sugar

beet pulp medium

The tested fungi have been maintained on potato

glucose agar slants and kept in the refrigerator and

subcultured monthly The solid state fermentation

medium was mixed and inoculated with 18 times 105

spores

per gram of wet substrate The flasks were placed in a

humid cultivation chamber with a gentle circulation of

air at 30 degC under static conditions for 7 days Triplicate

flasks were used for each fungal species and the end of

incubation period the crude pectinase was extracted

using the following procedure

Five grams of the fermented materials were mixed with

50 ml of sodium acetate buffer and shacked for 1 hour

then squeezed filtered through a cloth filterand stored

at 40C till measuring its pectinolytic activity The

polygalacturonase and pectin lyase activities were taken

as a measure to the pectinolytic enzymes

The activity of the polygalacturonase (PGase) was

assayed by measuring the reducing groups released from

polygalacturonic acid using the 3 5-dinitrosalicylic acid

method with glucose as the standard One unit of PGase

activity was defined as that amount of enzyme which

would yield 1 micromol reducing units per minute

6 Analytical methods

61 Pectinases assay

611 Assay for pectinases (polygalacturonase) activity

in the cell ndashfree filtrate

6111Reagents

1) 35-Dinitrosalicylic acid (DNS)

One g DNS dissolved by warming in 20 ml (2 N NaOH)

Thirty g Pot Sod tartarate dissolved by warming in 50 ml

distilled water After cooling the two solutions combined

together and make up to 100 ml with distilled water

2) 1 pectin solution

1- One hundred of sodium acetate buffer solution were

taken and then warmed in a water bath

2- One gram of pectin powder was added slowly to the

buffer solution on the stirrer until it was homogenous

3) 1g 10ml of standard glucose

1- One gm of glucose powder was dissolved in 10 ml

distilled water

6112 Procedure

The assay was carried out using 025 ml of 1 pectin

025 ml of culture filtrate The resulting mixture was

incubated at 50 ordm C for 10 minutes Polygalacturonase

activity was measured by quantifying the amount of

reducing sugar groups which had been liberated after

incubation with pectin solution using the method of

Miller (1959) 05 ml 3 5 ndashDinitrosalisyclic acid DNS

and 05 ml of reaction mixture were placed in a test tube

and boiled for 5 min used glucose as a standard The

enzyme activity (Ugdfs) was calculated as the amount of

enzyme required to release one micromole (1μmol)

equivalent of galactouronic acid per minute

The absorbance has been measured at 540 nm

determinations were carried out in triplicates

62 Assay for pectin lyase

PL activity was determined by measuring the

increase in absorbance at 235 nm of the substrate solution

(2 ml of 05 citric pectin in 01 M citrate-phosphate

buffer pH 56) hydrolysed by 01ml of the crude enzymatic

extract at 25degC for 2 minutes One enzymatic unit (U) was

defined as the amount of enzyme which liberates 1 μmol of

unsaturated uronide per minute based on the molar

extinction coefficient (ε235 = 5550 M-1

) of the

unsaturated products (Albershein 1966 Uenojo and

Pastore 2006) The enzymatic activity was expressed in

63 Protein determination

The protein content of the crude enzyme was

determined by the method of Lowry et al (1951) using

Bovine Serum Albumin (BSA) as the standard

64 Statistical analysis

Statistical analysis of data was carried out by using

one way analysis of variance (ANOVA) Followed by

homogenous subsets (Duncun) at confidence levels of 5

using the Statistical Package for the Social Science (SPSS)

version 11

7 Optimization of parameters controlling

polygalacturonases production by Pcitrinum

Penicillium citrinum has been chosen for further

studies Factors such as temperature pH incubation period

and others may affect polygalacturonases production So

the effect of such factors was investigated to determine the

optimum conditions for the enzyme production

71 Effect of different natural products

Conical flasks (250 ml) each contain 20 ml of the

production medium autoclaved for 20 minutes after

cooling the flasks were inoculated with 1ml of spore

suspension (18 times105 ) and incubated at 25 ordmC with different

raw materials ( 10g Sugar beet pulp 5g sugar beet pulp

+5g wheat bran 10g wheat bran 5g sugar beet pulp +5g

banana 10g banana 5g sugar beet pulp + 5g vicia faba

10g vicia faba ) for 7days At the end of incubation period

samples were collected extracted and centrifugated

respectivelyThe filtrates used as the crude enzyme extract

were analyzed for enzyme activity to determine the

optimum natural nutrient

72 Effect of different nitrogen sources

The effect of different nitrogen sources on

polygalacturonases production was carried out by

supplementing the production media with equimolecular

amount of nitrogen at concentration of (004 g g dry SBP)

for each nitrogen source Inorganic nitrogen sources such

as (NH4)2 HPO4 NH4NO3 and NaNO3 were investigated

Organic nitrogen sources such as urea yeast extract

peptone tryptone and malt extract were also tested All

culture conditions which obtained in the previous

experiments were adjusted Samples were collected and

analyzed as mentioned

73 Effect of different inoculum sizes

Different concentrations of spore suspension of the

highest producer fungus were used The following

concentrations were applied viz 18 36 54 times105

spores

ml and 9times104

sporesml per each flask (250 ml) At the end

of incubation period polygalacturonase activity was

determined for each concentration after incubation period

as previously mentioned

74 Effect of different incubation periods

cooling the flasks were inoculated with 1 ml of spore

suspension (18times105) and incubated at 25 ordmC at different

incubation periods (2 3 4 5 6 7 8 9 and 10 days) at the

end of incubation periods samples were collected

extracted and centrifuged respectively The filtrates were

used as the crude enzyme extract and analyzed for enzyme

activity and protein content to determine the optimum

incubation period

75 Effect of different pH values

This experiment was carried out by dissolving the

component of the production medium in different pH buffer

solutions pH values from 3 to 75 were examined using

Citric acid-Na2HPO4 buffer solutions Previous optimized

conditions were adjusted samples were collected and

76 Effect of different temperatures

Flasks containing 20 ml of sterilized production

medium were inoculated with 1 ml spore suspension The

flasks were then incubated at different temperatures (20

25 30 35 and 400C) At the end of the incubation period

the cell free filtrates were used to investigate the enzyme

activity

77 Effect of different surfactants

This experiment carried out to investigate the

production of polygalacturonases in the presence of some

surfactants Production media was supplemented with

different surfactants ( Tween 40 olive oil Tween 60

Tween 80 soybean oil sunflower oil Tween 20 maize

oil and triton x 100 ( 01) All surfactants were tested for

their induction or inhibitory effect on polygalacturonases

production compared to the control which carried out

without surfactant addition Production process with all the

above mentioned conditions was carried out to detect the

best conditions for yield improvement Samples were

collected and analyzed as usual

state fermentation

A full factorial two-level design(25) was performed

to confirm the optimization of independent factors level by

taking incubation period (7 and 8 days) pH (50 and 55)

inoculum size (18times105and 36times10

5 sporesml) temperature

(25 and 30ordmC) and nitrogen content(05 and 12) in this

study The level of independent factors were optimized by

studying each factor in the design at two different levels(-1

and +1)Table 12)The minimum[coded as(-1)] and

maximum [coded as(+1)] range of experimental values of

each factor used A set of 32 experiments was performed

The quality of fitting the first-order model was expressed

by the coefficient of determination R2 and its statistical

significance was determined by F-test The sugar beet pulp

had been used as the sole carbon source

79 Effect of different gamma irradiation doses

All irradiation processes were carried out at the

National Center for Radiation Research and Technology

(NCRRT) Nasr City-Cairo-Egypt Irradiation facility was

Co-60 Gamma chamber 4000-A India The source gave

average dose rate 3696 kGyhr during the period of

samples radiation The fungal strain was grown on PDA for

8days and subjected to gamma radiation at doses (01 02

05 07 1 15 and 2 kGy) The tested cultures have been

investigated for its enzyme activity

8 Purification of polygalacturonases

81 Production of polygalacturonase and preparation of

cell-free filtrate

Fungal cultures were grown in conical flasks of

250ml capacity on the optimized medium and incubated at

the optimum temperature At the end of incubation period

the supernatant (500 ml) was harvested by extraction

followed by centrifugation at 5000rpm for 15 minutes at

40C and the supernatant was used as crude enzyme extract

82 Ammonium sulphate precipitation

The cell free filtrate was brought to 75 saturation

by mixing with ammonium sulphate slowly with gentle

agitation and allowed to stand for 24 hrs at 4ordmC After the

equilibration the precipitate was removed by centrifugation

(5000 rpm at 4degC for 15 min)The obtained precipitate has

been dissolved in 50ml of 02M sodium acetate buffer pH

(59) to be dialyzed

821 Steps for precipitation by ammonium sulphate

1- Crude extract was poured in to a beaker with a

magnetic bar in it Beaker volume was chosen 25-3

times larger than the volume of the sample

2- The beaker was placed on the stirrer to mix solution

with a speed which allowed a vortex to form in the

middle of the sample

3- The amount of ammonium sulphate powder that

needed to precipitate the protein was determined and

weighed then added to the sample (with stirring) in

small portions

4- Stirrer was turned off when all salts had dissolved

and sample was left for 24 hrs at 4degC

5- Pellets were collected by centrifugation for 20

minutes at 5000 rpm at 4degC then dissolved in the

appropriate buffer

83 Dialysis

According to Karthik et al (2011) the precipitate

was desalted by dialysis by the following protocol

10cm dialysis bag was taken and activated by rinsing in

distilled water One end of the dialysis bag is tightly tied

and the obtained precipitate is placed into the bag Then

the other end of the dialysis bag is tightly tied to prevent

any leakage After that dialysis bag has been suspended

in a beaker containing 02M sodium- acetate buffer (pH

55) to remove low molecular weight substances and

other ions that interfere with the enzyme activity

84 Gel filtration chromatography (Wilson and

Walker 1995)-

841- Packing of the column-

(a)- 10 grams of sephadex G-75 (sigma) was

weighed and added into 500 ml acetate buffer (05 M

pH6) and allowed to swell for at least 3 days in the

fridge

(b)- Degassing process was carried out by placing the

beaker containing the matrix ( Sephadex G-75 ) into

boiling water bath for several hours with occasional

gentle knock on the beaker wall (to get rid of air

bubbles)

(c) The gel was allowed to cool to the room

temperature then packed in the column by pouring

carefully down the walls of the column (22 cm times 65

-The column tap must be kept open during the bed

settling to allow the formation of one continuous bed

also the bed must not to be allowed to precipitate so that

when more gel is poured it will not lead to the

formation of 2 beds over each others

-The bed which was formed was 22 times 45 cm

(d) The sorbent was allowed to reach the equilibrium

by passing 2 column volume of the used buffer before

the application of the sample

The column was connected to the buffer reservoir and

the flow rate of the buffer was maintained at a constant

rate of approximately 5 ml per 75 min

8-4-2-loading of the sample-

3-7 ml of the enzyme sample was applied carefully

to the top of the gel

8-4-3-Fractionation-

The protein band was allowed to pass through the

gel by running the column Forty fractions each of 5 ml

were collected and separately tested for both the protein

content (at 280 nm) and for the pectinase activity The

active fractions that have the highest pectinase activity

were collected together and concentrated by dialysis

against sucrose then tested for pectinase activity and

protein content This concentrated partially purified

enzyme solution was stored in the refrigerator and used

for the further characterization and application study

844 Calculation of specific activity purification

fold and yield of the enzyme

Specific activity (Umg) Activity of the enzyme (U)

Amount of protein (mg)

Yield of enzyme () Activity of fraction activity of

crude enzyme times100

Purification fold Specific activity of the fraction

specific activity of the crude enzyme

9 Characterization of the partially purified

polygalacturonase enzyme

Several factors have been studied to

investigate their effects on the partially purified

enzyme activity

911 On the enzyme activity

The activity of PGase was determined in the

presence of different buffers using sodium acetate buffer

(pH 40 50) sodium citrate buffer (pH 60 70) and

sodium phosphate buffer (pH 80)The relative activities

were based on the ratio of the activity obtained at certain

pH to the maximum activity obtained at that range and

expressed as percentage

912 On the enzyme stability

The pH stability of the enzyme was determined by

exposing the purified enzyme first to various pH values

(4 to 8) using the different pH buffer solutions

mentioned above for a period of 2 hours Afterwards

aliquots of the mixtures were taken to measure the

residual polygalacturonase activity () with respect to

the control under standard assay conditions

93 Effect of different temperatures on the enzyme

The optimum temperature was determined by

incubating each reaction mixture at variable temperatures

(20-70ordmC) The relative activities (as percentages) were

expressed as the ratio of the purified polygalacturonase

obtained activity at certain temperature to the maximum

activity obtained at the given temperature range

Thermal stability of the enzyme was investigated

by measuring the residual activity after incubating the

enzyme at various temperatures ranging from 20 to

70degC for 30 min

Pcitrinum

For determination the influence of Ca+2

and Co+2

on PGase activity The

listed ions were added to the reaction mixture at

concentration (1mM) Activity without added metal ions

was taken as 100 activity

10 Bioextraction of pectin from different agro-residues

for different pharmaceutical applications

Pcitrinum was cultivated in 50ml aliquots250ml

Erlenmeyer flasks of the following media containing any

of the different wastes Sugar beet pulp 10 Orange peel

waste 10and Banana peel waste 10 yeast extract 1

pH 6 and inoculated with 1ml of spore suspension (about

18times105 sporesml) incubated at 30degC for 8 days under

static conditions These favored maximum pectin

bioextraction At the end of fermentation time the filtrate

was separated by centrifugation at 4000 rpm for 20 min and

poured in 3 volumes of ethanol The precipitated pectin was

collected by centrifugation washed with ethanol dried

under vaccum at 37degC and then weighed accurately(Kabil

and Al-Garni 2006)

Results

4-Results

41Screening of the most potent fungal pectinase

producer

The results showed that Penicillia were the most

potent among the tested genera for enzyme production

(1246) among the tested genera followed by

Sclerotium rolfsii (1157) then Aspergillus niger and

Pleurotus ostreatus (1024) The least enzyme

production was detected in case of Trichoderma viride

(621) Among Penicillia Penicillium citrinum was the

most potent in the production of pectinase (129Ugdfs

so it has been chosen for further studies

411 Polygalacturonase activity

It has been found that polygalacturonase enzyme is

the most potent type in the cell free filtrate by using 35-

Dinitrosalisyclic acid DNS (Miller 1959)

Results

Table (3) Polygalacturonase production by the tested fungal

species under solid state fermentation

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

Not detected for all

tested fungal

species

862plusmn2 Alternaria alternata

862plusmn22 Aspergillus niger 1

968plusmn19 Aspergillus oryzae

957plusmn21 Gliocladium vierns

1232plusmn22 Penicillium brevi-compactum

1214plusmn114 Penicillium chrysogenum

1292plusmn2 Penicillium citrinum

1024plusmn21 Pleurotus ostreatus

831plusmn2 Rhizoctonia solani

1157plusmn19 Scleortium rolfsii

621plusmn21 Trichoderma viride

- gdfs Units of pectinase per gram dry fermented substrate

Results

Fig (3) polygalacturonases production by the tested fungal species grown

under solid state conditions

412 Pectin lyase assay

Pectin lyase enzyme was not detected in the filtrates

of the investigated fungal species

Results

42- Optimization of the fermentation parameters

affecting enzyme production

421 Effect of some agroindustrial by-products as

carbon source on polygalacturonase production by

Pcitrinum under Solid state fermentation

The production medium was inoculated with 1

ml of spore suspension (18times105 sporesml) which

prepared in Tween 80 01 vv The growth medium

was supplemented with different carbon sources at

concentration of ten gram for each treatment (sugar

beet pulpsugar beet pulp+wheat bran wheatbran

sugarbeetpulp + banana sugar beet pulp + broad

beans broad beans) All culture conditions which

obtained in the previous experiments were applied

during the present investigation The results in table (4)

showed that the maximum enzyme production was

achieved when the medium was supplemented with

sugar beet pulp giving activity of (1262 Ugds) while

the addition of other agro by-products gave lower

enzyme production except for sugar beet pulp +wheat

bran (1122 Ugds) There was a significant difference

Results

between all tested by-products Wheat bran exhibited

enzyme activity of 10702 Ugds Beans gave the

activity of 8306 Ugds

Table (4) Effect of some agroindustrial by-

products as carbon source on polygalacturonase

production by Pcitrinum under solid state

fermentation

Carbon source Enzyme activity(Ugdfs)

Sugar beet pulp 1262plusmn 2 a

Sugar beet pulp +wheat

1122plusmn 19 b

Wheat bran 10702plusmn 22 c

Sugar beet pulp +banana 1002plusmn 2 d

Sugar beet pulp + beans 951plusmn 19 e

Beans 8306plusmn 19 f

Banana 7302plusmn12g

Groups with different letters have significant difference between each other

Results

Fig (4) Effect of different agroindustrial by products as carbon

polygalacturonase production using Penicillium

citrinum under Solid state fermentation

Nitrogen sources were supplemented in the

production medium with equimolecular amount of nitrogen

from different nitrogen sources (Yeast extract Malt extract

Urea Peptone Ammonium sulfate Tryptone Ammonium

nitrate Sodium nitrate) All culture conditions were

Results

adjusted according to the optimum condition determined in

the previous experiments The results showed that the

yeast extract was the best nitrogen source in inducing

enzyme production (1292 Ugdfs) Ammonium sulphate as

inorganic nitrogen source was also effective in the

induction of pectinases production (1201Ugdfs) but less

than the activity produced in the presence of yeast extract

as a complex nitrogen source All other nitrogen sources

including organic and inorganic sources produced lower

levels of polygalacturonases compared to the medium

containing the yeast extract

Results

Table (5) Effect of different nitrogen sources on

Nitrogen sources Enzyme activity(Ugdfs)

Yeast extract 1292plusmn 19 a

Malt extract 932plusmn 17 b

Urea 831plusmn 18 c

Peptone 891plusmn 22 d

Ammonium sulfate 1201plusmn 2e

Tryptone 1142plusmn 18 f

Ammonium nitrate 991plusmn 22 b

Sodium nitrate 952plusmn 18 b

-gdfs Units pectinase per gram dry fermented substrate

Groups with different letters have significant between each other

Results

Fig (5) Effect of different nitrogen sources on polygalacturonases

fermentation

(Meyrathamp Suchanex 1972)The results showed that

maximum polygalacturonase production took place using

inoculum size of (18times105sporesml) for solid state

fermentation but decrease subsequently with the increase

in the inoculum size Interestingly with the increase in the

inoculum sizes the enzyme production has been reduced

Results

rather drastically in the SSF Apparently the conditions of

the fermentation were adjusted according to the optimum

conditions determined in the previous experiments

Table (6) Effect of inoculum size on polygalacturonase

fermentation

-gdfsUnits pectinase per gram dry fermented substrate

-Groups with different letters have siginificant between each other

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

Fig (6) Effect of inoculum size on polygalacturonase production by

424 Effect of different incubation periods on

citrinum

The results represented in Table (7) and fig (7)

showed that P citrinum started pectinases production

from the second day of incubation period with enzyme

activity (783Ugds) then started to increase significantly

as the incubation period increased and reached its

maximum activity in the seventh day of the incubation

(1292Ugds) Longer incubation period resulted in a

significance decrease of the enzyme activity especially in

Results

10 days of incubation (942Ugdfs)

Table (7) Effect of different incubation periods on

production of the polygalacturonase enzyme by

Incubation period(Days) Enzyme activity(Ugdfs)

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Groups with same letters are non significant with each other

Groups with different letters are significant with each other

Results

Fig (7) Effect of different incubation periods on polygalacturonase

425Effect of different pH values on

polygalacturonases production by Pcitrinum under

Penicillium citrinum was allowed to grow at

different pH values(3 35 4 45 5 55 6 65 7 75)

under the conditions of the fermentation which adjusted

according to the optimum condition determined in the

previous experiments The results in table (8) and fig (8)

showed that the fungal cultures were able to produce

pectinases at all tested pH values but it was obvious that at

low pH range (3- 45) the production was low and the

determined activities were (802 87 981 1009Ugds

Results

respectively) then began to increase gradually to reach its

maximum production at pH range (5- 6) The maximum

activity was (1261Ugds) at pH 55 then the activity

significantly decreased at pH range ( 60 -75) with the

least recorded activity (905Ugds) was at pH 75

Table (8) Effect of different pH values on

pH Specific activity(Ugdfs)

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

Groups with different letters have significant difference

between each other

Results

Fig (8) Effect of different pH values on polygalacturonases

42 6 Effect of different incubation temperatures on

polygalacturonase production by Pcitrinum under

The temperature is one of the major factors

affecting the process of pectinases production under solid

state fermentation Results in Table (9) and fig (9) showed

that pectinases production started at 20 ordmC with activity

(100Ugds) It increased gradually by the rise in incubation

temperature and reached its maximum activity at 25 ordmC

Results

(1273Ugds) The activity started to decrease with the

increase in the incubation temperature and reached its

minimal value at 40 ordmC (823Ugds)

Table (9) Effect of different incubation temperatures

on polygalacturonase production by Penicillium

citrinum

Temperature(ordmC) Enzyme activity(Ugdfs)

20 ordmC 100plusmn 2 d

25 ordmC 1271plusmn 18 a

35 ordmC 923 plusmn 22 b

40 ordmC 826 plusmn 2 c

Results

Fig (9) Effect of different incubation temperatures on

Table (10) and fig (10) showed the influence of

different surfactants on pectinase production Highest level

of pectinase production has been obtained by the addition

of Tween 40 (01) to the culture medium (1401 Ugds)

While no effect on polygalacturonase production was

observed upon using Triton X-100 Sunflower oil Maize

oil Soybean oil Olive oil and Tween 80Tween 20amp60

produced polygalacturonases in a level similar to that of the

control without surfactants The lowest level of

Results

polygalacturonase has been observed when soybean oil was

added to the fermentation medium (922Ugdfs)

Table (10) Effect of some surfactants on

polygalacturonase production by P citrinum under

surfactants Specific activity (Ugdfs)

Control 1231 plusmn 207 a

Tween 40 1401 plusmn 22 b

Tween 20 1261 plusmn 19 a

Tween 80 1072 plusmn 2c

Olive oil 1109 plusmn 23 d

Soybean oil 922 plusmn 2 e

Maize oil 1042 plusmn 19 c

Sunflower oil 1169plusmn 2 f

Triton x100 1152 plusmn 21 f

Results

Fig (10) Effect of some surfactants on polygalacturonase production

by Pcitrinum

state fermentation

A factorial design has been applied to optimize

polygalacturonase production by Pcitrinum Factorial

design was used to study the effect of 5 variables (yeast

extract pH Inoculum size Incubation period and

Incubation temperature) on enzyme production Only yeast

extract Inoculum size and Incubation temperature had

significant effect on pectinase production under the

Results

conditions of the assay the interaction between them not

being significant So a design of a total 32 experiments

was generated and Table (11) lists the high and low levels

of each variable The 32 experiments were carried out in

triplicate Table (11) (12) show the effect of each variable

and its interactions on the enzyme production As can be

seen high polygalacturonase production was obtained by

using one gram of yeast extract in the fermentation medium

incubated at 30ordmC for 8 days at pH 55 ( 132 Ugds)

Experimentally the obtained PGs yield is 132Ugds A high

degree of correlation between the experimental and

predicted values of the exopolygalacturonase production

was expressed by a high R2 value of 74 (Fig 12)

Results

Table (11) Effect of the variables and their interactions in

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Ugdfs unitgram dry fermented substrat

Results

Fig (11) Factorial design for the effects of yeast extract and

Penicillium citrinum One unit (U) of pectinase activity was

defined as the amount of the enzyme which catalysed the

formation of 1 micromol of galacturonic acid per hour at 30ordmC

Table (12) ANOVA table for the enzyme activity effect of

inoculums size yeast extract and temperature on the activity of

Term Estimate Std Error t Ratio Probgt|t|

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Incubation period(78) 23464844 3822781 061 05485

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Fig (12) Plot of predicted versus actual

polygalacturonase production

Yeast extractIncubation period -0383984 3822781 -010 09213

Yeast extractInoculm size -7427734 3822781 -194 00710

Incubation periodInoculm size -0553516 3822781 -014 08868

Yeast extractpH 58589844 3822781 153 01462

Incubation periodpH 12097656 3822781 032 07560

Inoculm sizepH -3608984 3822781 -094 03601

Yeast extractTemp 17410156 3822781 046 06553

Incubation periodTemp 06777344 3822781 018 08617

Inoculm sizeTemp 63714844 3822781 167 01163

pHTemp -2652734 3822781 -069 04983

Results

solid state fermentation using optimized conditions

of factorial design

Penicillium citrinum fungal spores were irradiated

with increasing doses of gammandashrays and then used for

regular experiment for polygalacturonase production in

sugar beet pulp solid medium Data clearly indicated that

maximum polygalacturonase production was observed

when spores were irradiated at 07 KGy with an activity

1522 Ugds as compared to the wild strain Higher doses

than 1kGy produced significant decrease in

polygalacturonase activity (Table13)

Results

Table (13) Effect of Radiation Dose on

citrinum

Radiation dose

Enzyme activity

(Ugds)

Control (unirradiated) 132plusmn19a

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

-gds Units of pectinase per gram dry fermented substrate

ND not determined

Results

Fig (13) Effect of different doses of gamma Radiation on

43 Purification and characterization of the enzyme

431 Purification steps

Polygalacturonase produced by Pcitrinum was

purified using ammonium sulfate precipitation and then

underwent dialysis and gel filtration Results observed in

Table (14) indicate a decrease in total protein and total

activity whereas specific activity increased Ammonium

sulphate precipitation (salting out) is useful for

concentrating dilute solutions of proteins The ammonium-

dialysate fractionated sample 75 showed purification

Results

fold of 12 and the yield of 91 In contrast elution profile

of the crude enzyme subjected to gel filtration on sephadex

G-100 column chromatography showed purification fold of

16 and yield of 87 Both enzyme activity at 540 nm and

protein content at 280 nm were determined for each

fraction fig (14) The enzyme activity has been detected

between the fractions No16 to the fraction No20

Table (14) Purification of PGase secreted by Pcitrinum

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Absorbance(280nm) Enzyme activity(Uml)

Fig14Gel filtration profile of polygalacturonase

432 Characterization of the purified enzyme

43211 On the activity of the enzyme

The reaction was incubated at various pH range (4 to 8)

using different pH buffers then the activity was measured

under standard assay conditions The effect of pH on the

polygalacturonase activity is presented in Fig 15 As it can

be observed the enzyme was active over a broad pH range

displaying over 60 of its activity in the pH range of 40

Results

up to70 with an optimum pH of 60 Concerning to the

PGase at pH 8 the relative activity decreased down up to

Table (15) Effect of different pH values on the activity

of the partially purified polygalacturonase enzyme

produced by Pcitrinum

pH Relative activity ()

Results

Fig (15) Effect of different pH values on the activity of the partially

43212 On the stability of the enzyme

exposing the purified enzyme firstly to various pH values

(4 to 8) using different pH buffers for 2 hours Then the

activity measured under standard assay conditions The

results presented in table (16) and fig (16) revealed that the

polygalacturonase enzyme was stable at the broad pH range

of pH 4 up to 7 retaining more than 66 of its activity

PGase activity was more stable at pH 60 However the

stability was significantly reduced to 58 at pH 8

Results

Table (16) Effect of different pH values on the stability of the

partially purified polygalacturonase enzyme produced by

Pcitrinum

pH Residual activity ()

Results

Fig (16) Effect of different pH values on the stability of the partially

4322Effect of different temperatures

Different incubation temperatures ( 20 to 70 ordmC) was

investigated for their effect on the purified pectinase

enzyme The results illustrated in table (17) and Fig(17)

showed that the activity of Pcitrinum polygalacturonase

increased gradually at temperature ranged from 20degC up to

C Moreover the optimum temperature was achieved at

Results

C meanwhile the recorded relative activity was 49 at

Table (17) Effect of the temperature on the activity of the

Pcitrinum

Temperature(degC) Relative activity ()

40 100

Results

Fig (17) Effect of the temperature on the activity of the partially

4322 2On the stability of the enzyme

The thermostability of the purified polygalacturonase was

determined by measuring the residual activity of the

enzyme after incubation at different ranges of temperatures

(20degC - 70degC)after 30 minutes Fig 18 showed that the

increase in temperature caused an overall increase in the

stability up to 60degC rising temprature above 60degC caused a

decline in thermostability It is worth mentioned that the

maximum stability of 100 was observed at 50degC

However the residual activity declined to 58 at 70degC

respectively

Results

Table (18) Effect of different temperatures on the

stability of the partially purified polygalacturonase

enzyme produced by Pcitrinum

Residual activity() Temperature(degC)

100 50

Results

Fig (18) Effect of different temperatures on the stability of the

partially purified polygalacturonase enzyme produced by Pcitrinum

4323 Effect of different metal ions on the activity of

the partially purified polygalacturonase enzyme

The effect of metal ions were examined by adding

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

EDTA and nitrate of Ba+2

at concentration of

1mM to the buffer solution Results in table 19 and Fig19

revealed that the enzyme activity was enhanced in the

presence of Mg+2

and Zn+2

to 12 and 5 respectively

whereas Ca+2

resulted in a reduction in the enzyme activity

by 12 Salts such as Ba (NO3) CoCl26H2O CuSO45H2O

and EDTA inhibited enzyme activity up to 50

Results

Table (19) Effect of different metal ions on the activity

Metal ions (1mM) Relative activity ()

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

44 Extraction and determination of pectic substances

Bioextraction of pectin from different agro-residues like

sugar beet pulp Bannana peels wastes and Orange peels

wastes by Pcitrinum was markedly influenced by the

previously mentioned factors obtained by factorial design

system As can be seen SBP contains high amount of

pectin as it weighed 2gm compared to both OPW and BPW

that give 15 and 12gm respectively The raw material

extracted pectin has many applications in the

pharmaceutical industry

Fig (19) Effect of different metal ions on the activity of the partially

Results

Table (20) The different weights of pectin extracted

from different agroindustrial by products inoculated

with Pcitrinum

Agro-residues wastes Dry weight of extracted

pectin(gm)

Sugar beet pulp waste 2

Orange peel waste 112

Banana peel waste 15

Discussion

Increasing population and industrialization has

resulted in sudden increase in pollution Because of the

detrimental effects of pollution on humans animals and

plants the ever inceasing pollution is causing concern all

over the worldThe microbial biodiversity is important on

many grounds ranging from aesthetic considerations to its

usefulness particularly for biotechnologyThe fastest

growing segments are enzymes for feed and fuel

production Abundant amount of waste materials are

produced by agricultural and fruit processing industries

which pose considerable disposal problems and ultimately

leads to pollutionVast varieties of microorganisms are

present in the environment which can be exploited for the

utilization of waste materialsFor example in the processing

of citrus fruits a large proportion of the produced wastes

are in the form of peel pulp and seedsCitrus peel is rich in

carbohydrate protein and pectin Pectic substances are

present in the pimary plant cell wall and the middle

lamella Besides these other fruits like Mango(Mangifera

indica) Avocado Pear (Avocado avocado) Guava (Psidium

guajava) Banana (Musa sapientum) Papaya (Carica

papaya) Cashew Apple (Anacardium occidentale)

Discussion

Garden-egg (Solanum nigrum Linn) Star Apple

(Crysophylum albidium) and Tomato (Lycopersicum

esculentum) also contain substantial amounts of pectin

having a high gelling grade Sugar beet pulp a by- product

of sugar extraction also contains pectinGalacturonic acid

(21) arabinose(~21) glucose(~21) galactose(~5)

and rhamnose(~25) are its main components (Micard et

al1994)They are the constitutive monomers of cellulose

and pectinsPectin is a polymer of galacturonic acid

residues connected by α-1 4 glycosidic linkagesPectin is

hydrolysed by pectinase enzymes produced extracellularly

by microflora available in our natural environmentWith the

help of these pectinase enzyme micro-organisms can

convert citrus wastes into sugars which can be used for

food and value added productsThese micro-organisms can

also be exploited for production of pectinase which is an

industrially important enzyme and have potential

applications in fruit paper textile coffee and tea

fermentation industries

Recently a large number of microorganisms isolated

from different materials have been screened for their

ability to degrade polysaccharides present in vegetable

biomass producing pectinases on solid-state culture (Soares

et al 2001) In the present study fourteen species have

Discussion

been screened for thier pectinolytic activities Penicillium

citrinum has been found to be the best producer of

pectinolytic enzymes (1292plusmn2Ugdfs) Fawole and

Odunfa 1992 reported that Aspergillus Fusarium

Penicillium and Rhizopus showed high pectolytic activities

In a study by Spalding and Abdul-Baki (1973)

Penicillium expansum the causal agent of blue mould rot in

apples was shown to produce polygalacturonase in

artificial media and when attacking apples However

Singh et al 1999 stated that the commercial preparations

of pectinases are produced from fungal sources According

to Silva et al 2002 PG production by P viridicatum using

orange bagasse and sugar cane bagasse was influenced by

media composition Aspergillus niger is the most

commonely used fungal species for industrial production of

pectinolytic enzymes (Naidu and Panda 1998amp

Gummadi and Panda 2003) Pectic substances are rich in

negatively charged or methyl-estrified galacturonic acid

The esterification level and the distribution of esterified

residues along the pectin molecule change according to the

plant life cycle and between different species Thus the

ability of some microorganisms to produce a variety of

pectinolytic enzymes that differ in their characteristics

mainly in their substrate specifity can provide them with

Discussion

more efficacy in cell wall pectin degradation and

consequently more success in the plant infection (Pedrolli

et al 2009)This may explain that Polygalacturonase

enzyme is the most abundant enzyme assayed in this study

In addition Natalia et al (2004) reported that higher

production of PGase depended on the composition of the

medium On the other hand PL production depended on

the strain used More than 30 different genera of bacteria

yeasts and moulds have been used for the production of

PGases In the last 15 years with strains of Aspergillus

Penicillium and Erwinia were reported to be the most

effective in enzyme production (Torres et al 2006)Pectin

lyase (PL) and Polygalacturonase (PG) production by

Thermoascus aurantiacus was carried out by means of

solid-state fermentation using orange bagasse sugar cane

bagasse and wheat bran as a carbon sources(Martins et al

2000) Commercial pectinase preparations are obtained

mainly from Aspergillus and Penicillium (Said et al

1991) Moreover high activities of extracellular pectinase

with viscosity-diminishing and reducing groups-releasing

activities were produced by Penicillium frequentans after

48 h at 350C (Said et al 1991) The selection of substrate

for SSF depends upon several factors mainly the cost and

availability and this may involve the screening for several

Discussion

agro-industrial residues which can provide all necessary

nutrients to the micro organism for optimum function

The main objective of this study was to check the

effect of physical and chemical components of the medium

to find out the activators and inhibitors of pectinolytic

activity from Penicillium citrinum SSF is receiving a

renewed surge of interest for increasing productivity and

using of a wide agro-industrial residue as substrate The

selection of the substrate for the process of enzyme

biosynthesis is based on the following criteria

1) They should represent the cheapest agro-industrial

2) They are available at any time of the year

3) Their storage represents no problem in comparison with

other substrate

4) They resist any drastic effect of environmental

conditions egtemperature variation in the weather from

season to season and from day to night SSF are usually

simple and could use wastes of agro-industrial substrates

for enzyme productionThe minimal amount of water

allows the production of metabolites less time consuming

and less expensive

Solis-Pereyra et al (1996) and Taragano et al (1997)

came to the conclusion that production is higher under solid

Discussion

state fermentation than by submerged one In this field

many workers dealt with the main different factors that

effect the enzyme productions such as temperature pH and

aeration addition of different carbon and nitrogen sources

In order to obtain high and commercial yields of pectinases

enzyme it is essential to optimize the fermentation medium

used for growth and enzyme production Sugar beet pulp

has been shown to be the best used source for pectinase

production from Pcitrinum Pectin acts as the inducer for

the production of pectinolytic enzymes by microbial

systems this is in agreement with the results of Pandey et

al (2001) and Phutela et al (2005) Since pectin can not

enter the cell it has been suggested that compounds

structurally related to this substrate might induce pectic

enzyme productions by microorganisms Also low levels

of constitutive enzyme activities may attack the polymeric

substrate and release low molecular products which act as

inducers Polygalacturonase and pectin transeliminase were

not produced whenever the medium lacked a pectic

substance the production of polygalacturonase and pectin

transeliminase is inductive An adequate supply of carbon

as energy source is critical for optimum growth affecting

the growth of organism and its metabolism Aguilar and

Huitron (1987) reported that the production of pectic

Discussion

enzymes from many moulds is known to be enhanced by

the presence of pectic substrates in the medium Fawole

and Odunfa (2003) found that pectin and polygalacturonic

acid promoted the production of pectic enzyme and they

observed the lack of pectolytic activity in cultures with

glucose as sole carbon source such observations reflect the

inducible nature of pectic enzyme from a tested strain of

Aspergillus niger

nucleic acid proteins and cell wall components Recorded

results showed that maximum polygalacturonase

production by Penicillium citrinum was obtained in the

presence of yeast extract this result is in agreement with

that reported by Bai et al (2004) who found that high

monosodium glutamate water Yeast extract served as the

best inducer of exopectinase by Aspergillus sp (Mrudula

and Anitharaj 2011) Also Thakur et al (2010)

reported that the best PGase production was obtained when

casein hydrolysate and yeast extract were used together It

has been reported that nitrogen limitation decreases the

polygalacturonase production Also Aguilar et al (1991)

Discussion

showed that yeast extract (organic nitrogen source) was the

best inducer of exopectinases by Aspergillus sp Moreover

Kashyap et al (2003) found that yeast extract peptone

and ammonium chloride were found to enhance pectinase

production up to 24 and addition of ammonium nitrate

inhibited pectinase production In this context yeast extract

proved to be the best nitrogen source likely because it

provided other stimulatory components such as vitamins

(Qureshi 2012)Yeast extract has previously proved

superior to other nitrogen sources in the production of

pectinases by the thermophilic fungus Sporotrichum

thermophile (Kaur et al 2004) Bacillus shaericus

produced maximum polygalactouronase when grown on

mineral medium containing yeast extract as sole nitrogen

source (Ranveer et al 2010) Ammonium sulphate was

also effective in the induction of polygalacturonase

production Galiotou-Panayotou and Kapantai (1993)

observed that ammonium phosphate and ammonium

sulphate did influence production of pectinase positively

but also recorded an inhibitory effects of ammonium nitrate

and potassium nitrate on pectinase production Moreover

Patil and Dayanand (2006) revealed that both ammonium

phosphate and ammonium sulphate did influence

production of pectinase positively in both submerged and

Discussion

solid-state conditions In addition Sapunova (1990) found

that ammonium salts stimulated the pectinolytic enzyme

production in Aspergillus alliaceus Moreover Sapunova

et al (1997) has also observed that (NH4)2SO4 stimulated

pectinase synthesis as in its absence fungus did not

produce extracellular pectinases In addition Fawole and

Odunfa (2003) found ammonium sulphate and ammonium

nitrate were good nitrogen sources for pectic enzyme

production from Aspergillus niger Also Phutela et al

(2005) found that presence of yeast extract + (NH4)2 SO4 in

growth medium supported maximal production of pectinase

followed by malt sprouts+ (NH4)2 SO4 which also

supported maximal polygalacturonase activity In addition

Rasheedha et al (2010) found that ammonium sulphate

has enhanced the production of Penicillium chrysogenum

pectinase On the contrary Alcacircntara et al( 2010)

reported that the concentration of ammonium sulphate had

a negative effect on enzyme activities The observations of

Hours et al (1998) who suggested that lower levels of

(NH4)2SO4 or K2HPO4 added to the growth medium as

inorganic nitrogen sources did not influence pectinase

yield In addition Vivek et al (2010) found that organic

nitrogen sources showed higher endo exo pectinases

activities than inorganic nitrogen source The nitrogen

Discussion

source can play an important role in affecting the pH

changes in the substrate during the fermentation The

ammonium ion was taken up as ammonia thereby releasing

a proton into the medium and causing a decrease in pH

(Qureshi et al 2012)

The size of inoculum added to the fermentation

medium has significant effect on growth and enzyme

production Maximum polygalacturonase production took

place at the inoculum size of (18 times105

sporesml) for SSF

but decrease subsequently with the increase in the inoculum

size Low inoculum density than the optimum may not be

sufficient to initiate growth and to produce the required

biomass whereas highe inoculum can cause competition

for nutrients (Jacob and Prema 2008) Mrudula and

Anitharaj (2011) reported that the optimum inoculum

density is an important consideration for SSF process

since over crowding of spores can inhibit growth and

development Higher inoculum levels besides increasing

spores density increase water content of the medium as

well The inoculum size of 1times105ml

-1 resulted the

maximum production of endo- and exo-pectinases by

Penicillium sp in submerged conditions and 1times107ml

-1 had

given maximum amount in solid-state condition (Patil and

Dayanand

2006)Similar observations were made by

Discussion

Aguilar and Huitron(1987) for submerged condition and

Pereira et al( 1994) for solid-state condition

pH stongly affects many enzymatic processes and

transport of various components across the cell membrane

(Moon amp Parulekar 1991) The effect of hydrogen ion

concentration on the enzyme activity may be explained in

part in terms of the relative molecular stability of the

enzyme itself and in part on the ionizable groups (COO-

OH-) of the tertiary protein structure of the enzyme

complex (Lehninger 1973)In this study the maximum

production of polygalacturonase was recorded at a pH

range of 5-6 with optimum production at pH 55 Boccas et

al (1994) also reported similar observations The pH of the

medium will also limit the growth of the culture or exert

influence upon catalytic activity of the enzyme (Adeleke et

al 2012) Maximum polygalacturonase production was

observed in the medium with acidic pH values within a

range of 4 to 6 (Aminzadeh et al 2007)Also

Ramanujam and Subramani (2008) reported that the

optimum pH for Aspergillus niger was 60 using citrus peel

and sugarcane bagasse respectively for the production of

pectinase in SSF Observation in the study by Adeleke et

al (2012) showed optimum pH for enzymes production

within 5 to 55 Banu et al (2010) presented similar

Discussion

observations for polygalacturonase production by

Penicillium viridicatum Trichoderma longibrachiatum

showed high production of glucose on the day 7at pH 5

and 450C Wide range of initial pH of the medium during

the upstream bioprocess make the end product either acidic

or alkaline which tend to have varied applications

(Hoondal et al 2002) The pH regulates the growth and

the synthesis of extracellular enzyme by several

microorganisms particularly fungal strains (Suresh and

Chandrasekaran 1999) Fungi and yeasts produce mainly

acidic PGases whilst alkaline pectinases are mainly

produced by bacteriaThe highest titres of acidic PGase

have been obtained with strains of Aspergillus Penicillium

and Candida (Torres et al 2006) revealed that pH is the

most significant factor that influence the enzyme

production and that the optimal value of 5 resulted in an

increase in PGase production up to 667 fold

Temperature is another critical parameter and must

be controlled to get the optimum enzyme production It has

been found that temperature is a significant controlling

factor for enzyme production (Kitpreechavanich et al

1984) Temperature in solid state fermentation is

maintained at 30-320C as it cannot be precisely controlled

due to the reason that solid-state fermentation has solid

Discussion

substances which limited heat transfer capacity In the

current study the obtained results revealed that the highest

polygalacturonase production has been achieved at 25degC

during optimization using the classical methods

(1271Ugdfs) and at 30degC using the full factorial design

(132Ugdfs) Most microorganisms are mesophiles which

grow over a range of 25degC -300C while others are

psychrophiles or thermophiles in nature Akintobi et al

(2012) reported that the temperature of the medium also

affected both growth and enzyme production by

Penicillium variabile Growth of the organism and

production of pectinolytic enzymes were optimum at 30degC

According to Bailey and Pessa (1990) lower temperature

slows down the hydrolysis of pectin At low temperature

(40C) there was no growth and at high temperature

generation of metabolic heat in solid state fermentation

might be a reason for growth inhibition in microorganisms

Release of proteins into the medium was also optimum at

30degC Growth and enzymes production were least

supported at 20degC and 35degC In general temperature is

believed to be the most important physical factor affecting

enzyme activity (Dixon and Webbs 1971) In contrast

Freitas et al (2006) reported that the fungal species

Discussion

investigated for pectinase production showed optimum

growth in the range of 45 to 600C

Patil and Dayanand (2006) stated that the period of

fermentation depends upon the nature of the medium

fermenting organisms concentration of nutrients and

physiological conditions Penicillium citrinum started

polygalacturonase production from the second day of

incubation period with low enzyme activity (78Ugds)

which increased gradually as the incubation period was

increased reaching its maximum activity on the seventh

day of incubation (1292Ugds)which decreased thereafter

showing moderate increase on the ninth day of the

incubation period and the activity reached (1002Ugds)

These results are in agreement with that of Akhter et al

(2011) who demonstrated that the maximum pectinase

production by Aniger was peaked on the seventh day of

incubation In contrast Silva et al (2002) reported that

Polygalacturonase production by Penicillium viridicatum

peaked between the 4th

and the 6th

days Another study

(Gupta et al 1996) showed that the maximum production

of polygalacturonase in SSF by Penicillium citrinum was at

the 120th

hour (ie the fifth day) Many results showed that

PG activity increased during the primary metabolism and

decreased when the secondary metabolism started In

Discussion

Botrytis cinerea (Martinez et al 1988) and Fusarium

oxysporum (Martinez et al 1991) the highest PG

activities were obtained during the primary growth phase

In Trametes trogii (Ramos et al 2010) the highest PGase

activity was obtained when the biomass was at its highest

level The incubation period for maximum enzyme

production was found to vary with different strains

Alternaria alternata (Kunte and Shastri 1980) showed

maximum polygalacturonase activity on the 4th day The

decrease in the activity can be due to the depletion of

nutrients in the medium The incubation period is generally

dictated by the composition of the substrate and properities

of the strain such as its growth rate enzyme production

profile initial inoculum and others (Lonsane and Ramesh

Considering surfactants application high level of

polygalacturonase production was obtained upon addition

of Tween 40 (01) to the culture medium (1401 Ugdfs)

Also Tween 20 and 60 1261Ugdfs128Ugdfs

respectively slightly increased PGase activities than the

enzyme produced in the surfactant free medium These

results are in agreement with Kapoor et al 2000 and Zu-

ming et al 2008 who reported stimulation of pectinases

when Tween-20 was supplemented to the medium The

Discussion

reason is probably is due to the possibility that the

surfactants might improve the turnover number of PGs by

increasing the contact frequency between the active site of

the enzyme and the substrate by lowering the surface

tension of the aqueous medium(Kapoor et al 2000)

Moreover Surfactants have been reported to affect the

growth rate and enzyme production of many fungi Similar

finding have been recorded with respect to the action of

surfactant on different microbial enzymes (Sukan et al

1989) The mechanisms by which detergents enhance

extracellular enzyme production were reported to be due to

increased cell membrane permeability change in lipid

metabolism and stimulation of the release of enzymes are

among the possible modes of the action (Omar et al

1988) Mrudula and Anitharaj (2011) reported that

production of pectinase is highest when Triton-X-100 was

supplemented to the orange peel in SSF

Full Factorial Statistical Design

Full factorial design was used in order to identify

important parameters in the screening analysis The factors

were yeast extract incubation period inoculums size pH

and temperature Selection of the best combination has

been done using factorial design of 32 runs Activities were

Discussion

measured after using sugar beet pulp as the best carbon

source The carbon substrate was determined for the

screening study based on the results of the preliminary

experiments A significant model was obtained in which

yeast extract Inoculum size and Temperature had

significant effects on the exo-PG activity while incubation

period and pH factors did not show significant variations

All interaction effects were also insignificant Small p-

values (p lt00250) show that the parameters (yeast extract

inoculum size and temperature) are significant on the

response The P-values used as a tool to check the

significance of each of the coefficients in turn indicate the

pattern of interactions between the variables Smaller value

of P was more significant to the corresponding coefficient

According to the model the highest exo-PG activity

(132Ugds) has been obtained using 12 yeast extract as

the best nitrogen source inoculated with 18times105sporesml

incubated for 8 days at pH 55 and temperature 30degC

According to the results the model predicts the

experimental results well and estimated factors effects were

real as indicated by R2 value (o74) R

2 value being the

measure of the goodness to fit the model indicated that

74 of the total variation was explained by the model ie

the good correlation between the experimental and

Discussion

predicted results verified the goodness of fit of the model

(R2 = 0 74) It is a known fact that the value of R

2 varies

from 0 to plusmn1 When R2

=0 there is no correlation between

experimental and predicted activities For R2= plusmn1 perfect

straight line relationship exists between the experimental

and predicted activities (Naidu and Panda 1998) On the

other hand the conventional method (ie change-one-

factor-at-a-time) traditionally used for optimization of

multifactor experimental design had limitations because (i)

it generates large quantities of data which are often difficult

to interpret (ii) it is time consuming and expensive (iii)

ignores the effect of interactions among factors which have

a great bearing on the response To overcome these

problems a full factorial design was applied to determine

the optimal levels of process variables on pectinase enzyme

production The results indicated that (Full factorial design

FFD) not only helps us locate the optimum conditions of

the process variables in order to enhance the maximum

pectinase enzyme production but also proves to be well

suited to evaluating the main and interaction effects of the

process variables on pectinase production from waste

agricultural residues There are few works in literature that

report the effects of culture media on the optimization of

PG activityTari et al (2007) who evaluated the biomass

Discussion

pellet size and polygalacturonase (PG) production by

Aspergillus sojae using response surface methodology

showing that concentrations of malt dextrin corn steep

liquor and stirring rate were significant (plt005) on both

PG and biomass production

Effect of gamma radiation on polygalacturonase

production

Radiation effect on enzymes or on the energy

metabolism was postulated

Gamma irradiation potentiates the productivity of

the enzyme to its maximum value (1522Ugdfs) post

exposure to 07 kGy This enhancement of enzyme

production might have been due to either an increase in the

gene copy number or the improvement in gene expression

or both (Meyrath et al 1971 Rajoka et al 1998 El-

Batal et al 2000 and El-Batal and Abdel-Karim 2001)

Also induction of gene transcriptions or proteins has been

found after low dose irradiation (Wolff 1998 and Saint-

Georges 2004) indicating that the induction of gene

transcription through the activation of signal transduction

may be involved in the low dose effects A gradual

decrease in the enzyme activity after exposure to the

different doses of 1 15kGy was observed The complete

Discussion

inhibition of growth and consequently on enzyme

production has been obtained at a level of 2kGy dose This

could be explained by damage or deterioration in the

vitality of the microorganism as radiation causes damage to

the cell membrane This major injury to the cell allows the

extracellular fluids to enter into the cell Inversely it also

allows leakage out of essential ions and nutrients which the

cell brought inside El-Batal and Khalaf (2002)

evidenced that production of pectinases increased by

gamma irradiated interspecific hybrids of Aspergillussp

using agroindustrial wastes

Enzyme purification

Pectinase enzyme was purified from crude sample by

ammonium sulfate fractionation and further dialysis was

carried out The 75 ammonium-dialysate fractionated

sample showed 12 purification fold and a yield of 91

Elution profile of the crude enzyme subjected to gel

filtration on sephadex G-100 column chromatography

showed 16 purification fold and 87 yield Enzyme

activity at 540 nm and protein content at 280 nm were

determined for each fraction The enzyme activity has been

detected between the fractions No16 to the fraction No20

while fraction No10 to the fraction No13 had no enzyme

Discussion

activity suggesting a number of isoforms of PGase

According to Viniegra-Gonzalez and Favela-Torres

(2006) and Torres et al ( 2006) variation in the isoforms

of extracellular enzymes obtained by SSF can be attributed

to alteration of the water activity (aw) that results in changes

in the permeability of fungal membranes limitation of

sugar transport and presence or absence of inducer It is

even reported that pectinases produced by the same

microorganism have exhibited different molecular weights

degrees of glycosylation and specificities These variations

may be due to the post transitional modification of a protein

from a single gene or may be the products of different

genes (Cotton et al 2003 and Serrat et al 2002)

Enzyme characterization

Effect of pH on polygalacturonase activity and stability

The enzyme of Pcitrinum was active over a broad pH

range displaying over 60 of its activity within the pH

range of 40 to70 with an optimum pH at 60 Optimum pH

for different pectinases has been reported to vary from 38

to 95 depending upon the type of enzyme and the source

(Joshi et al 2011) Meanwhile Pviridicatum showed an

optimum pH at 60 as mentioned by Silva et al (2007)

Moniliella sp showed its maximum activity at pH 45 and at

Discussion

pH 45-50 for Penicillium sp (Martin et al 2004) The

maximum activity of Monascus sp and Aspergillus sp for

exo-PGase was obtained at pH 55 (Freitas et al 2006)

Also Silva et al( 2002) and Zhang et al (2009 ) reported

that optimum pH for pectinase activity was 50 for both

Penicillium viridicatum and Penicillium oxalicum

respectivielySimilarily PGases of Aspergillis niger were

shown to possess maximum catalytic activity at pH 50

(Shubakov and Elkina 2002) However the optimal pH

of polymethylploygalacturonase was found to be 40

(Kollar 1966 and Kollar and Neukom 1967) Dixon and

Webbs (1971) amp Conn and Stump (1989) separately

reported that the changes in pH have an effect on the

affinity of the enzyme for the substrate The effect of pH on

the structure and activity of polygalacturonase from Aniger

was described by Jyothi et al (2005) They reported that

the active conformation of PGase was favored at pH

between 35 and 45 alterations in the secondary and

tertiary structures resulted at pH (from 50 to 70) This

could be attributed to Histidine residues that have ionizable

side-chains increasing the net negative charge on the

molecule in the neutral-alkaline pH range and leading to

repulsion between the strands resulting in a destabilization

Discussion

of the hydrogen-bond structure of the enzyme (Jyothi et al

Stability of the enzyme when incubated at pH in suitable

buffer systems for 2hs at 30degC was also investigated during

this work The results revealed that the polygalacturonase

enzyme of Pcitrinum was stable at a broad pH range 4 -7

retaining more than 66 of its activity PGase activity was

more stable at pH 60 However the stability was

significantly reduced to 58 at pH 8 It was reported that

the inactivation process was found to be faster at high

alkaline pHs due to disulfide exchange which usually

occur at alkaline condition (Dogan and Tari 2008) In this

sense Gadre et al (2003) reported that PGase activity

show higher stability in the range from 25 to 60 however

at pH 70 the stability was 60 lower On the other hand

Hoondal et al (2002) evaluated a PGase from Aspergillus

fumigates that kept their activity in a range of pH from 3 to

Effect of temperature on polygalacturonase activity and

stability

The results showed that the activity of Pcitrinum

polygalacturonase increased gradually within temperature

range from 200C up to 60

0C Moreover the optimum

Discussion

temperature was achieved at 40oC and a relative activity of

49 was attained at 700C This is supported by results of

Juwon et al (2012) who reported a decline in the enzyme

activity at temperatures more than 400C Similar

observation had been reported by Palaniyappan et al

(2009) by Aspergillus niger Also PGase produced by

Aspergillus flavus Aspergillus fumigatus and Aspergillus

repens exhibited maximum activity at 350C 40

0C and 45

respectively (Arotupin 2007) Similarly Barthe et al

(1981) and Yoon et al (1994) documented temperature of

400C for the maximum PGase activity from Colletotrichum

lindemuthianum and Ganoderma lucidum The same

optimum temperature was implicated for the PGase

obtained from Aspergillus niger Botryodiplodia

theobromae and Penicillium variabile and Aspergillus

alliaceus(Juwon et al 2012) On the other hand other

studies conducted by several authors using different strains

revealed that optimum temperature of an

exopolygalacturonase from Aspergillus niger was 60degC

(Sakamoto et al 2002)Furthermore the partially purified

polygalacturonase from Sporotrichum thermophile apinis

was optimally active at 55degC (Jayani et al 2005

Kashyap et al 2001)These variations in the optimum

temperature of fungal PGase suggested a broad range of

Discussion

temperature tolerable by the enzyme In addition nature

source and differences in the physiological activities of

fungi may be responsible for these variable observations

(Arotupin 1991)

Thermostability is the ability of the enzyme to

tolerate against thermal changes in the absence of

substrates (Bhatti et al 2006) The thermostability of the

purified polygalacturonase was determined by measuring

the residual activity of the enzyme after incubation at

different ranges of temperatures (20degC - 70degC) after 30

minutes The increase in temperature caused an overall

increase in the stability up to 600C of PGase from

Pcitrinum rising temperature above 60degC caused a decline

in thermostability It is worth mentioned that the maximum

stability of 100 was observed at 500C Similarly the

optimum temperatures for PGase of Aspergillus niger and

Penicillium dierckii were shown to be 500

C and 600C

respectively (Shubakov and Elkina 2002) However the

residual activity declined up to 58 at 700C Also Exo-PG

of Monascus sp and Aspergillus sp showed stability at

temperature up to 500C (Freitas et al 2006)

A loss in PGase activity percentage obtained at 700

C from

Aspergillus nigerBotryodiplodia theobromae and

Discussion

Penicillium variabile was reported by Oyede (1998) and

Ajayi et al( 2003) Daniel et al 1996 who also reported

the thermal inactivation of the enzymes at high

temperature It was reported that extremely high

temperature lead to deamination hydrolysis of the peptide

bonds interchange and destruction of disulphide bonds

and oxidation of the amino acids side chains of the enzyme

protein molecules (Creighton 1990 and Daniel et al

The study conducted by Maciel et al (2011) is not in

agreement with our study they recorded that exo-PGase

was stable at 80degC and showed 60 residual activity

remaining after 1 h at this temperature

Effect of metal ions on polygalacturonase activity

Results in the present study revealed that the enzyme

activity was enhanced in the presence of Mg+2

and Zn+2

12 and 5 respectively whereas Ca+2

resulted in a

reduction in the enzyme activity by 12 The cations may

affect protein stability by electrostatic interaction with a

negatively charged protein surface by induction of dipoles

changes in the inter-strand dispersion forces and by their

ability to modify the water structure in the vicinity of the

protein and thus influence its hydration environment (Zarei

Discussion

et al 2011) Salts such as Ba (NO3) CoCl26H2O

CuSO45H2O and EDTA inhibited enzyme activity up to

50 Jurick et al (2009) reported that there was an

increase in PG enzyme activity by adding magnesium and

iron whereas a decrease in activity occurred when calcium

and manganese were included in the PGase assay Also

Banu et al (2010) reported that HgCl2 CoCl2 and CuSO4

caused inhibition of pectinase activity by Pchrysogenum

up to 60 Thus Hg+2

and Cu+2

block thiol groups on the

protein (Skrebsky et al 2008 and Tabaldi et al 2007)

Besides this effectCu+2

induces protein polymerization by

forming Histidine-Cu-Histidine bridges between adjacent

peptide chains(Follmer and Carlini 2005) and can

interfere in the structure of some proteins through its

coordination geometry (Pauza et al 2005) Similarly

BaCl2 and EDTA resulted in the maximum inhibition of

pectinases activity up to 40 (Banu et al 2010) Also

Oyede (1998) reported the stimulatory role of K+2

and Mg+2

on PGase activity from Penicillium sp while

concentrations of Ca+2

beyond 15mM inhibited the enzyme

activity This variation in degrees of stimulation and

inhibition could be a function of the sources of enzyme

from different mould genera Also Murray et al (1990)

showed that the formation of a chelate compound between

Discussion

the substrate and metal ions could form a more stable

metal-enzyme-substrate complex and stabilizing the

catalytically active protein conformation Also Brown and

Kelly (1993) affirmed the ability of metal ions often acting

as salt or ion bridges between two adjacent amino acids

Famurewa et al (1993) and Sakamoto et al (1994)

confirmed the inhibitory activity of EDTA on enzyme The

metal building reagent like EDTA can inactivate enzyme

either by removing the metal ions from the enzyme forming

coordination complex or by building inside enzyme as a

ligand ( Schmid 1979)

Concluding Remarks

5-Concluding remarks

Pectinases are among the first enzymes to be used at

homes Their commercial application was first observed in

1930 for the preparation of wines and fruit juices As a

result pectinases are today one of the upcoming enzymes

of the commercial sector It has been reported that

microbial pectinases account for 25 of the global food

enzymes sales (Jayani et al 2005)

Higher cost of the production is the major problem in

commercialization of new sources of enzymes Though

using high yielding strains optimal fermentation conditions

and cheap raw materials as a carbon source can reduce the

cost of enzyme production for subsequent applications in

industrial processes So the production of pectinases from

agro-wastes is promising and required further

investigations

In the coming times it should increase attention

toward the study of the molecular aspects of pectinases the

impact effect of radiation exposure on pectinase as well as

developing the mutant of the superior pectinase producing

strains Also further studies should be devoted to the

understanding of the regulatory mechanism of the enzyme

secretion at the molecular level

References

Adeleke AJ SA Odunfa A Olanbiwonninu MC

Owoseni(2012) Production of Cellulase and

Pectinase from Orange Peels by Fungi Nature and

Science10 (5)107-112

Aguilar G and C Huitron (1987) Stimulation of the

production of extracellular pectinolytic activities of

Aspergillus sp by galactouronic acid and glucose

addition Enzyme Microb Technol 9 690-696

Aguilar G B Trejo J Garcia and G Huitron(1991)

Influence of pH on endo and exopectinase

production by Aspergillus species CH-Y-1043 Can

J Microbiol 37 912-917

Aidoo KE Hendry R and Wood BJB (1982)Solid

state fermentation Adv Appl Microbiol 28-201-

Ajayi A A Olutiola P O and Fakunle J B

(2003)Studies on Polygalacturonase associated with

the deterioration of tomato fruits (Lycopersicon

esculentum Mill) infected by Botryodiplodia

theobromae Pat Science Focus 5 68 ndash 77

Akhter N Morshed1 M A Uddin A Begum F Tipu

Sultan and Azad A K (2011) Production of

Pectinase by Aspergillus niger Cultured in Solid

State Media International Journal of Biosciences

Vol 1 No 1 p 33-42

References

Akintobi AO Oluitiola PO Olawale AK Odu NN Okonko

IO(2012) Production of Pectinase Enzymes system

in culture filtrates of Penicillium variabile

SoppNature and Science 10 (7)

Albershein P (1966) Pectin lyase from fungi Method

Enzymology 8 628-631

Alcacircntara S R Almeida F A C Silva F L H(2010)

Pectinases production by solid state fermentation

with apple bagasse water activity and influence of

nitrogen source Chem Eng Trans 20 121-126

Alkorta I Garbisu C Liama J Sera J(1998)

ldquoIndustrial applications of pectic enzymes A

reviewrdquo Process Biochemistry33 pp21-28

Aminzadeh S Naderi-Manesh H and Khadesh K(2007)

Isolation and characterization of polygalacturonase

produced by Tetracoccosporium spIran J Chem

Eng 26(1) 47 ndash 54

Arotupin D J (1991) Studies on the microorganisms

associated with the degradation of sawdust M

ScThesis University of Ilorin Ilorin Nigeria

Arotupin D J (2007) Effect of different carbon sources

on the growth and polygalacturonase activity of

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Research Journal of Microbiology 2(4) 362-368

Ashford M Fell JT Attwood D Sharma H Wood-head P

(1993)An evaluation of pectin as a carrier for drug

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eliciting plant disease resistance

Bailey MJ Pessa E(1990) Strain and process for

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Banu AR Devi MK Gnanaprabhal GR Pradeep

BVand Palaniswamy M (2010) Production and

characterization of pectinase enzyme from

Penicillium chysogenum Indian Journal of Science

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Baracet MC Vanetti M CD Araujo EF and Silva

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Aspergillus fumigates for degumming of natural

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BartheJP Canhenys D and Tauze A

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Beltman H and Plinik W(1971)Die Krameersche

Scherpresse als Laboratoriums-Pressvorrichtung

und Ergebnisse von Versucher mit

AepfelnConfructa16(1) 4-9

Berovič M and Ostroveršnik H( 1997) ldquoProduction of

Aspergillus niger pectolytic enzymes by solid state

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Bhatti HN M Asgher A Abbas R Nawaz MA

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Boccas F Roussos S Gutierrez M Serrano L and

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Technol 31(1) 22ndash26

Boudart G Lafitte C Barthe JP Frasez D and

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Brown SH and Kelly RM (1993)Characterization of

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hydrolytic activity from the thermophilic

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59 26122621

Cavalitto SF Arcas JA Hours RA (1996) Pectinase

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18 (3) 251-256

Cervone F Hahn MG Lorenzo GD Darvill A and

Albersheim P (1989) Host-pathogen interactions

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Charley VLS (1969)Some advances in Food processing

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Chun-hui Z Zu-ming LI Xia-wei P Yue J Hong-xun

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and Characterization of Three Endo-

polygalacturonases from a Newly Isolated

Penicillum oxalicum The Chinese Journal of Process

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Conn E E and Stump K P (1989) Outline of

Biochemistry 4th edition Wiley Eastern Limited

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Cook PE(1994) Fermented foods as biotechnological

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Cotton P Kasza Z Bruel C Rascle C Fevre M(

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endopolygalacturonse genes in the nectrotrophic

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Lett227163-9

Creighton T E (1990) Protein Function A practical

Approach Oxford University Press Oxford 306 pp

Daniel R M Dines M and Petach H H (1996) The

denaturation and degradation of stable enzymes at

high temperatures Biochemical Journal 317 1 -11

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Dixon M and Webbs E C (1971) Enzymes Williams

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Dogan N Tari C( 2008)Characterization of Three-phase

Partitioned Exo-polygalacturonase from Aspergillus

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43minus50

Dunaif G and Schneeman BO (1981) The effect of

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1034-1035

El-BatalAI and Abdel-KarimH(2001)Phytase

production and phytic acid reduction in rapeseed

meal by Aspergillus niger during solid state

fermentationFood ResInternatinal 34715-720

El-Batal A I and SA Khalaf (2002) Production of

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of Aspergillus sp using agro-industrial wastes

EgyptJBiotechnol1292-106

El-Batal A I Abo-State M M and Shihab A(2000)

Phenylalanine ammonia lyase production by gamma

irradiated and analog resistant mutants of

Rhodotorula glutinisActa MicrobialPolonica 4951-

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Englyst HN et al (1987) Polysaccharide breakdown by

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Microbiology and Ecology 95pp 163-171

Famurewa O Oyede MA Olutiola PO(1993)Pectin

transeliminase complex in culture filtrates of

Aspergillus flavus Folia Microbiol 38 459466

Fawole OB and SA Odunfa (2003) Some factors

affecting production of pectic enzymes by

Aspergillus niger Int Biodeterioration

Biodegradation 52 223-227

Fawole OB and Odunfa SA(1992) Pectolytic moulds in

Nigeria Letters in Applied Microbiology 15 266 ndash

Flourie B Vidon N Florent CH Bernier JJ (1984) Effects

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layer thickness in normal man Gut 25(9) pp 936-

Follmer C and Carlini C R (2005) Effect of chemical

modification of histidines on the copper-induced

oligomerization of jack bean urease (EC 3515)

Arch Biochem Biophys 435 15-20

Freedman DA (2005) Statistical Models Theory and

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Freitas PMN Martin D Silva R and Gomes E(2006)

Production and partial characterization of

polygalacturonase production by thermophilic

Monascus sp N8 and by thermotolerant Aspergillus

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Fujian Xu Chen Hong Zhang Li Zuohu(2001) Solid

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Gadre R et al (2003) Purification characterization and

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Microb Technol New York v32p321-333

Galiotou-Panayotou MPR Kapantai M (1993)

Enhanced polygalacturonase production by

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17 145ndash148

Ghanem NB HH Yusef HK Mahrouse

(2000)Production of Aspergullus terrus xylanase in

solid state cultures application of the plachett

Burman experimental design to evaluate nutritional

requirements Biores Technol 73113-121

Ginter E Kubec F J Vozar J and Bobek P (1979)

Natural hypocholesterolemic agentpectin plus

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Gummadi SN and T Panda( 2003) Purification and

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review Process Biochem 38 987-996

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Gupta MN RKaul DGuoqiangCDissing and

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Hang Y and Woodams E (1994) Production of fungal

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SciTechnol27 pp194-96

Hoondal GS Tiwari RP Tewari R Dahiya N Beg Q

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Biotechnol 59409-418

Harholt J Suttangkakul A Vibe Scheller H (2010)

Biosynthesis of pectinPlant Physiology 153 384-

Hours R Voget C Ertola R (1988) ldquoApple pomace as

raw material for pectinases production in solid state

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HoursRA CEVoget and RJErtola(1998)Some factors

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Hulme MA Stranks DW (1970) Induction and the

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226 469ndash470

Ishii S and Yokotsuka T(1972)Clarification of fruit juice

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Pp 787 791

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Jacob N and Prema P Novel process for the simultaneous

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Jayani RS Saxena S Gupta R (2005) Microbial

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(9) Pp 2931-2944

Joseph GH (1956) Pectin Bibliography of

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Growers)

Joshi V Mukesh P Rana N( 2006) ldquoPectin esterase

production from apple pomace in solid-state and

submerged fermentations (Special issue Food

enzymes and additives Part 1 Enzymes and organic

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Biotechnology44(2) pp253-56

JoshiVK ParmarM and Rana N(2011) Purification

and Characterization of Pectinase produced from

Applr Pomace and Evaluation of its Efficacy in Fruit

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Jurick WM Vico I Mcevoy JL Whitaker BD Janisiewicz

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characterization of a polygalacturonase produced in

Penicillium solitum-decayed bdquoGolden Delicious‟

apple fruit Phytopathology 99(6)636ndash641

Juwon A D Akinyosoye F A and Kayode OA(2012)

Purification Characterization and Application of

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Jyothi TCSingh SARao AGA(2005)The contribution of

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Biol Macromol36310-7

Kabli SA and Al-Garni SM (2006) Bioextraction of

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Research Journal of Biotechnology 1 (1) 10-16

Kapoor M Beg QK Bhushan B Dadhich KS and

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purification and characterization of a thermo-

alkalistable polygalacturoanse from Bacillus sp

MGcp-2 Proc Biochem 36 467ndash473

Karthik JL Kumar KV G and Rao B (2011)

Screening of Pectinase Producing Microorganisms

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Biochemical and pharmaceutical research 1(2)

2231-2560

Kashyap DR Soni KS and Tewari R( 2003)

Enhanced production of pectinase by Bacillus sp

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Technol 88 251-254

Kashyap DR Voha PK Chopra S Tewari R (2001)

Application of pectinases in the commercial sector

A Review Bioresour Technol 77216-285

Kaur G Kumar S Satyarnarayana T (2004) Production

characterization and application of a thermostable

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Kitpreechavanich V Hayashi M Nagai S (1984)

Productionof xylan-degrading enzymes by

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Kollar A (1966) Fractionierrung und charakterizerung der

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Kumar CG and Takagi H (1999) Microbial alkaline

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Biotechnol Adv 17 561-594

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Lehninger AL (1973) A short Course in Biochemistry

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Leuchtenberger A Friese E Ruttloff H (1989)

Variation of polygalacturonase and pectinesterase

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Lonsane BK Ramesh MV (1990) Production of

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Lowry O H Rosebrough N J Farr A L and Randall

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Maciel MHC Herculano PN Porto TS Teixeira

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Production and partial characterization of pectinases

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Afr J Biotechnol 10 2469ndash2475

Maldonado M Navarro A Calleri D (1986)

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Martin NSouza SRSilva RGomes E (2004)Pectinase

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Martiacutenez MJ Martiacutenez R Reyes F( 1988) Effect of pectin

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Mycopathologia 10237-43

Martinez MJ Alconda MT Guillrn F Vazquez C amp

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Martins E S Silva R and Gomes E (2000) Solid state

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MeyrathJBahnMHanHE and Altmann H (1971)

Induction of amylase producing mutants in

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Miller GH (1959) Use of dinitrosalicylic acid reagent for

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Chemistry and Functions of Pectins ACS

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Moon SH and Parulekar SJ (1991) A parametric study

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Mrudula M and Anithaj R (2011) Pectinase production

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Natalia M Simone RDS Roberto DS Aleni G (2004)

Pectinase production by fungal strains in solid state

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Brazilian Archives of biology and Technology

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ObiSK and Moneke NA(1985) Pectin Lyase and

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Oyede M A (1998) Studies on cell wall degrading

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Nigeria

Palaniyappan M Vijayagopal V Renuka V Viruthagiri T

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Pandey A Selvakumar P Soccoi CR and Nigam

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httptejasserciiscernetin~currscijuly10articles2

Patil N P and Chaudhari B L(2010) Production and

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Recent Research in Science and Technology 2(7)

Patil S R and Dayanand A (2006)Production of

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Pauza NL Cotti MJP Godar L Sancovich AMF and

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Characterization and Industrial Application of

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Pereira SS Torres ET Gonzalez GV Rojas MG (1992)

Effect of different carbon sources on the synthesis of

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Pereira BMC JLC Coelho and DO Silva

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ApplMicrobiol 18127-129

Peričin D Jarak M Antov M Vujičič B Kevrešan

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PhutelaU Dhuna V Sandhu S and BSChadha

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3663-69

Pilnik W and Voragen A G J (1993) Pectic enzymes in

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Pushpa S and Madhava MN (2010) Protease production

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Utilizing Coffee By-Products World Applied

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QureshiAS Muhammad Aqeel Bhutto Yusuf Chisti

Imrana Khushk Muhammad Umar Dahot and Safia

Bano(2012) Production of pectinase by Bacillus

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RajokaMIBashirAHussainSRS and Malik

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Ramanujam N and subramani SP (2008)Production of

pectiniyase by solid-state fermentation of sugarcane

bagasse using Aspergillus niger Advanced Biotech

Ramos Araceli Marcela Marcela Gally Maria CGarcia

and Laura Levin (2010)rdquo Pectinolytic enzyme

production by Colletotrichumtruncatumcausal

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Ranveer SJ Surendra KS Reena G (2010) Screening of

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Production by Bacillus sphaericus (MTCC 7542)

Enzyme Res Article ID 306785 5 pages

Rasheedha AB MD Kalpana GR Gnanaprabhal BV

Pradeep and M Palaniswamy (2010) Production

and characterization of pectinase enzyme from

Penicillium chrysogenum Indian J Sci Technol 3

377-381

Reese E T amp McGuire A (1969) Applied Microbiology 17 242ndash245

Ricker AJ and RSRicker( 1936)Introduction to

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Rosenbaum P R (2002) Observational Studies (2nd ed)

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Rubinstein A Radai R Ezra M Pathak J S and

Rokem S (1993) In vitro evaluation of calcium

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Pharmaceutical Research 10 pp 258-263

Said S Fonseca MJV Siessere V(1991) Pectinase

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Saint-Georges dL (2004) Low-dose ionizing radiation

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Sakamoto T Hours R A Sakai T (1994) Purification

characterization and production of two pectic

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Sakamoto T E Bonnin B Quemener JF

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Sandberg AS Ahderinne R Andersson H Hallgren B

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Sanzo AV Hasan SDM Costa JAV and Bertolin

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Sapunova LI (1990) Pectinohydrolases from Aspergillus

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Sapunova LI G Lobanok and RV Mickhailova( 1997)

Conditions of synthesis of pectinases and proteases

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Schmid RD (1979) Protein Function A practical

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Serrat MBermudez RCVilla TG

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Biotechnol97193-208

Shevchik V Evtushenkov A Babitskaya H and

Fomichev Y( 1992) ldquoProduction of pectolytic

enzymes from Erwinia grown on different carbon

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Biotechnology Vol (8) Pp115-20

Shubakov AA and Elkina EA (2002) Production of

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Silva D Martins E S Silva R and Gomes E (2002)

Pectinase production from Penicillium viridicatum

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SilvaRFerreiraVGomesE(2007) Purifiaction and

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Singh SA M Ramakrishna and AGA Rao (1999)

Optimization of downstream processing parameters

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Biochem 35 411-417

Skrebsky E C Tabaldi L A Pereira L B Rauber R

Maldaner J Cargnelutti D Gonccedilalves J F

Castro G Y Shetinger M RC Nicoloso F T

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Smith JE and Aidoo KE (1988) Growth of fungi on

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Soares M M C N Silva R Carmona E C and Gomes

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Bacillus species and their potential application on

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Soliacutes-Pereira S EF Torres GV Gonzaacutelez and M

Gutieacuterrez Rojas (1993) Effects of different carbon

sources on the synthesis of pectinase by Aspergillus

niger in submerged and solid state fermentations

Appl Microbiol Biotechnol 3936-41

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Solis-Pereyra S Favela-Torres E Gutierrez Rojas M

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fermentation at high initial glucose concentrations

World J Microbiol Biotechnol12 257ndash260

Spalding DH and Abdul-Baki AA (1973) In Vitro and In

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Sriamornsak P (2001) Pectin The role in health Journal

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Sukan SS Guray A and Vardar-Sukan F (1989)

Effects of natural oils and surfactants on cellulase

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Suresh PV and MChandrasekaran(1999)Impact of

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Tabaldi LA Ruppenthal R Cargnelutti D Morsh VM

Pereira LB Schetinger MRC (2007) Effects of metal

elements on acid phosphatase activity in cucumber

(Cucumis sativus L) seedlings EnvironExp Bot

5943-48

Taragano V Sanchez VE Pilosof AMR (1997)

Combined effect of water activity depression and

glucose addition on pectinase and protease

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production by Aspergillus niger Biotechnol Lett 19

(3) 233ndash236

Tari C Gogus N Tokatli F (2007) Optimization of

biomass pellet size and polygalacturonase

production by Aspergillus sojae ATCC 20235 using

response surface methodology Enzyme Microb

Technol 40 1108-16

Taflove A and Hagness SC (2005) Computational

Electrodynamics The Finite-Difference Time-

Domain Method 3rd ed Artech House Publishers

Tipler and Paul (2004) Physics for Scientists and

Engineers Electricity Magnetism Light and

Elementary Modern Physics (5th ed) W H

Freeman

TorresEF Sepulved TV and Gonzalez V (2006)

Production of hydrolytic depolymerizing pectinase

Food TechnolBiotechnol 44221-227

Tsereteli A Daushvili L Buachidze T Kvesitadze E

Butskhrikidze N(2009) ldquoProduction of pectolytic

enzymes by microscopic fungi Mucor sp 7 and

Monilia sp 10rdquo Bull Georg Natl Acad Sci 3(2)

Pp126-29

Thakur Akhilesh Roma Pahwa and Smarika

Singh(2010)rdquo Production Purification and

Characterization of Polygalacturonase from Mucor

circinelloidesrdquo Enzyme research

References

TuckerGA and WoodsL FJ(1991) Enzymes in

production of Beverages and Fruit juices Enzymes

in Food Processing Blackie New York 201-203

Uenojo M Pastore GM (2006) Isolamento e seleccedilatildeo de

microrganismos pectinoliacuteticos a partir de resiacuteduos

provenientes de agroinduacutestrias para produccedilatildeo de

aromas frutais Ciecircnc Tecnol Aliment 26 509-515

Venugopal C Jayachandra T Appaiah KA (2007) Effect

of aeration on the production of Endo-pectinase from

coffee pulp by a novel thermophilic fungi Mycotypha

sp Strain No AKM1801 6(2) 245-250

Viniegra-Gonzalez G and Favela-Torres E (2006) Why

solid state fermentation seems to be resisitant to

catabolite repression Food Technol Biotechnol

44397-406

Vivek R M Rajasekharan R Ravichandran K

Sriganesh and V Vaitheeswaran( 2010) Pectinase

production from orange peel extract and dried orange

peel solid as substrates using Aspergillus niger Int

J Biotechnol Biochem 6 445-453

Wilson F and Dietschy J (1974) The intestinal unstirred

water layer its WilsonK and WaikerJ(1995)

Practical biochemistry Principles and

techniquesfourth

editionCambridge University

Presspp182-191

Wilson K Waiker J (1995) Practical biochemistry

Principles and techniques 4th EditionCambridge

University Press 182-91

References

Wolff S (1998)The adaptive response in radiobiology

evolving insights and implications Environ Health

Perspect 106277-283

Xue M Lui D Zhang H Qi H and Lei Z (1992)

Pilot process of Solid State fermentation from Sugar

Beet Pulp for production of Microbial Protein J

Ferment Bioeng 73 203-205

Yoon S Kim M K Hong J S and Kim M S (1994)

Purification and properties of polygalacturonase

from Genoderma incidum Korean Journal of

Mycology 22 298 ndash 304

YoungM M Moriera A R and Tengerdy R P(1983)

Principles of Solid state Fermentation in Smith JE

Berry D Rand Kristiansen B eds Filamentous

fungi Fungal Technology Arnold E London

Pp117-144

Zarei M Aminzadeh S Zolgharnein H Safahieh

Daliri M Noghabi K A Ghoroghi A Motallebi

A (2011)Characterization of a chitinase with

antifungal activity from a native Serratia marcescens

B4A Braz J Microbiol vol42 (3) Satildeo Paulo

Zhang C Z Li X Peng Y Jia H Zhang and Z Z Bai

(2009) Separation Purification and Characterization

of Three Endo-polygalacturonases from a Newly

Isolated Penicillum oxalicumThe Chinese Journal

of Process Engineering 9242-250

Zheng Zuo-Xing and Kalidas S (2000) ldquoSolid state

production of polygalacturonase by Lentinus edodes

References

using fruit processing wastesrdquo Process

Biochemistry35 (8) Pp825-30

Zhong-Tao S Lin-Mao T Cheng L Jin-Hua D

(2009)ldquoBioconversion of apple pomace into a

multienzyme bio-feed by two mixed strains of

Aspergillus niger in solid state fermentationrdquo

Electronic Journal of Biotechnology12(1) pp1-13

Zu-ming LI Hong-xun Z Zhi-hui B Wen-tong X

and Hong-yu LI(2008) Purification and

Characterization of Three Alkaline Endo-

polygalacturonases from a Newly Isolated Bacillus

gibsonii The Chinese Journal of Process

Engineering 8(4) Pp 769-773

جحسيي الاحاج الفطري للازيوات الوحللة للبكحيي باسحخدام اشعة جاها جحث

ظروف الحخور شبه الجافة

شيواء عبد الوحسي ابراهين((

جاهعة حلواى-كلية العلوم-قسن البات والويكروبيولوجي

الوسحخلص العربي

رؼطي اػهي ازبط يرى في ذ انذراصخ فحص نغػخ ي انفطزيبد انز

ي ازيبد انجكزييز قذ عذ ا فطز انجضهيو صيززيى يؼطي اػهي

قذ رى دراصخ ربصيز انؼايم انزي انجني عبلاكزرييزازبط ي ازيى

رؤصز ػهي ازبط الازيى حيش عذ ا يبدح نت انجغز رؼطي اػهي ازبط

انصبدر انخزهفخ نهيززعي ثي ينهكزث حيذ نلازيى كصذر

عذ ا خلاصخ انخيزح رؼطي اػهي قيخ ي ازبط الازيى ي

انهقبػ ػهي ازبط الازيى كيخ خ ربصيزبانزي رى دراص الاخزي انؼايم

81times81عذ ا رزكيز حيش5

فززح انزحضي كبذيؼطي اػهي ازبط

ازبط نلازيى يحذس في انيو ي اى انؼايم انؤصزح حيش عذ ا اػهي

رجي ا ربصيزانزقى انيذرعيي دراصخ ذانضبثغ ي انزحضي ر

يؼطي اػهي ازبط نلازيى ا درعخ حزارح 55الاس انيذرعيي

رذدرعخ يئيخ رؼطي اػهي ازبط نلازيى اخيزا (55انزحضي )

رؼطي 01بدح ريرجي ا ي ربصيز يخزصبد انزرز انضطحيدراصخ

انذعخ الاحصبئي نذراصخ ربصيز اصهة رى اصزخذاواػهي ضجخ ازبط قذ

فززح انزحضي انزقى انيذرعييخش يزغيزاد )خلاصخ انخيزح

( ػهي ازبط ازيى انجني انهقبػدرعخ حزارح انزحضي كيخ

ػهي اػهي ازبط رى انحصل قذ اصفزد انزبئظ ػهي الاريعبلاكزرييز

الاس Cdeg30لازيى انجني عبلاكزرييزثؼذ صبي ايبو في درعخ حزارح

يغ خلاصخ انخيزح كبفضم يصذر نهيززعي ثززكيز 55انيذرعيي

ثبصزخذاو ذ انظزف انجيئيخ انضهي يحزي يززعيي15

اي رى كيهعز10ثبلاضبفخ اني اصزخذاو الاشؼبع انغبيي ثغزػخ

قذ انجني عبلاكزرييز يزرفغ ضجيب ي ازيى انحصل ػهي ازبط

ػهيبد رقيخ عزئيخ لازيى انجني عبلاكزرييز ثؼذ رزصيج اعزيذ

انفصم صى انذيهز صى ي كجزيزبد الاييو 05ثاصطخ اصزخذاو

قذ عذ ا انظزف انضهي 811انكزيبرعزافي ثاصطخ صيفبدكش

1-0اس يذرعيي Cdeg40ػذ درعخ انحزارح يكنشبط الازيى

درعخ يئيخػذ دراصخ ربصيز ايبد 01-51 انضجبد انيذرعيي ثي

انؼبد ػهي انشبط الازيي عذ ا انغضيو انزك يحفزح نهشبط

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

Review20of20literatures[1]pdf

MATERIALampMETHODpdf

RESULTSpdf

DISCUSSION CORRECTED[1]pdf

Concluding remarks 6pdf

REFERENCES20FINAL207[1]pdf

المستخلص العربيpdf

Faculty of Science

fermentation

Thesis

Presented BY

Ain Shams University

Faculty of Science

Botany and Microbiology Department

ث إشرافجح

Approval Sheet

fermentation

Submitted to

Department of

ACKNOWLEDGMENT

Helwan University

Fermentation

University)

Summary

citrinum

20degC -60degC Mg+2

and Zn+2

Contents

No Title Page

1 Introduction 1

42 Wine industry 25

57- Surfactants 32

7 Gamma Rays 35

32Culture media 40

pulp medium

fermentation

filtrate

83 Dialysis 52

enzyme

Pcitrinum

4- Results 58

enzyme production

citrinum

state fermentation

design

5- Discussion 98

7- References 127 7

List of tables

No Title page

state fermentation

fermentation

Pcitrinum

List of Figures

No Title page

by Pcitrinum

production

Pcitrinum

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

sp species

Aim of the study

state fermentation

sources

Introduction

by-products

Introduction

inorganic chemicals

Introduction

ammonium ions

11Protopectin

12Pectic acids

called pectates

13Pectinic acids

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Orange pulp

Dry matter

124ndash280

They include

esterified pectin

the pectin chain

cleavage They are

33221 Endo-PGL (Ec 4222)

pectic acid

33 Protopectinase

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

aseptic control

Vapor treatment non

sterile conditions

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

451 Protopectinases

respectively

453 Pectinesterase

47Disadvantages

monitoring

Yokotsuka 1972)

513 Liquefaction

514 Maceration

52 Wine industry

53 Textile industry

62 Carbon Sources

63-Nitrogen sources

64ndashTemperature

was found at 7th

and 4th

66- Inoculum size

1times10 7

67- Surfactants

cell membrane

7 Factorial Design

factors

conclusions derived

Factorial Design

8 Gamma Rays

neutral (neutrons)

al 2009)

31-Microorganisms

32Culture media

Dextrose 20 g

Agar 17 -20 g

production

4 Culture condition

solution pH 59)

of distilled water

beet pulp medium

spores

61 Pectinases assay

6111Reagents

distilled water

6112 Procedure

) of the

version 11

spores

ml and 9times104

incubation period

activity

state fermentation

cell-free filtrate

(59) to be dialyzed

small portions

appropriate buffer

83 Dialysis

Walker 1995)-

fridge

bubbles)

enzyme activity

70degC for 30 min

Pcitrinum

and Co+2

and Al-Garni 2006)

Results

4-Results

producer

Results

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

tested fungal

species

Results

fermentation

1122plusmn 19 b

Results

Urea 831plusmn 18 c

Results

fermentation

Results

fermentation

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

citrinum

Results

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Results

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

between each other

Results

citrinum

Results

by Pcitrinum

state fermentation

Results

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Results

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Yeast extractpH 58589844 3822781 153 01462

Inoculm sizepH -3608984 3822781 -094 03601

pHTemp -2652734 3822781 -069 04983

Results

of factorial design

Results

citrinum

Radiation dose

Enzyme activity

(Ugds)

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

ND not determined

Results

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Results

Pcitrinum

Results

Pcitrinum

40 100

Results

respectively

Results

100 50

Results

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

at concentration of

presence of Mg+2

and Zn+2

whereas Ca+2

Results

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

with Pcitrinum

pectin(gm)

Discussion

other substrate

and less expensive

Discussion

Aspergillus niger

Discussion

sporesml) for SSF

-1 resulted the

-1 had

Dayanand

Discussion

and the 6th

days Another study

the 120th

Discussion

2 value being the

Discussion

2 varies

Discussion

production

Discussion

Enzyme purification

Discussion

stability

Discussion

0C and 45

Discussion

(Arotupin 1991)

C and 600C

C from

Discussion

and Zn+2

resulted in a

Discussion

up to 60 Thus Hg+2

and Cu+2

and Mg+2

Discussion

Concluding Remarks

investigations

References

Science10 (5)107-112

Vol 1 No 1 p 33-42

References

Eng 26(1) 47 ndash 54

References

Technol 12 266-271

106Pp162-171

References

59 26122621

18 (3) 251-256

References

Lett227163-9

References

43minus50

1034-1035

References

17 145ndash148

References

226 469ndash470

Pp 787 791

References

39(1) 115-121

(9) Pp 2931-2944

Growers)

References

2231-2560

Technol 88 251-254

References

Technol 94239-234

Technology 62 63-69

TH Zurich (3374)

References

Letters10 pp 825-28

References

7B 159 - 209

References

polymers 32283-292

31426-429

Washington DC

37467-483

DC 66-83

References

47(5) 813-819

Nigeria

(4)682-686

References

87680-06-7 pp 221

activity by Pb2+

and Li+

References

3663-69

References

363-399

1 3-45

References

27(4)186ndash190

377-381

Yorkpp117

References

Nutr Clin Nutr 1983 37(3)171-83

Engenharia 10 59-62

References

Biotechnol97193-208

References

Biochem 35 411-417

Londrina

References

5943-48

References

(3) 233ndash236

Technol 40 1108-16

Freeman

Pp126-29

References

sp Strain No AKM1801 6(2) 245-250

44397-406

techniquesfourth

Presspp182-191

References

Perspect 106277-283

Pp117-144

References

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

RESULTSpdf

ث إشرافجح

Approval Sheet

fermentation

Submitted to

Department of

ACKNOWLEDGMENT

Helwan University

Fermentation

University)

Summary

citrinum

20degC -60degC Mg+2

and Zn+2

Contents

No Title Page

1 Introduction 1

42 Wine industry 25

57- Surfactants 32

7 Gamma Rays 35

32Culture media 40

pulp medium

fermentation

filtrate

83 Dialysis 52

enzyme

Pcitrinum

4- Results 58

enzyme production

citrinum

state fermentation

design

5- Discussion 98

7- References 127 7

List of tables

No Title page

state fermentation

fermentation

Pcitrinum

List of Figures

No Title page

by Pcitrinum

production

Pcitrinum

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

sp species

Aim of the study

state fermentation

sources

Introduction

by-products

Introduction

inorganic chemicals

Introduction

ammonium ions

11Protopectin

12Pectic acids

called pectates

13Pectinic acids

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Orange pulp

Dry matter

124ndash280

They include

esterified pectin

the pectin chain

cleavage They are

33221 Endo-PGL (Ec 4222)

pectic acid

33 Protopectinase

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

aseptic control

Vapor treatment non

sterile conditions

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

451 Protopectinases

respectively

453 Pectinesterase

47Disadvantages

monitoring

Yokotsuka 1972)

513 Liquefaction

514 Maceration

52 Wine industry

53 Textile industry

62 Carbon Sources

63-Nitrogen sources

64ndashTemperature

was found at 7th

and 4th

66- Inoculum size

1times10 7

67- Surfactants

cell membrane

7 Factorial Design

factors

conclusions derived

Factorial Design

8 Gamma Rays

neutral (neutrons)

al 2009)

31-Microorganisms

32Culture media

Dextrose 20 g

Agar 17 -20 g

production

4 Culture condition

solution pH 59)

of distilled water

beet pulp medium

spores

61 Pectinases assay

6111Reagents

distilled water

6112 Procedure

) of the

version 11

spores

ml and 9times104

incubation period

activity

state fermentation

cell-free filtrate

(59) to be dialyzed

small portions

appropriate buffer

83 Dialysis

Walker 1995)-

fridge

bubbles)

enzyme activity

70degC for 30 min

Pcitrinum

and Co+2

and Al-Garni 2006)

Results

4-Results

producer

Results

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

tested fungal

species

Results

fermentation

1122plusmn 19 b

Results

Urea 831plusmn 18 c

Results

fermentation

Results

fermentation

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

citrinum

Results

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Results

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

between each other

Results

citrinum

Results

by Pcitrinum

state fermentation

Results

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Results

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Yeast extractpH 58589844 3822781 153 01462

Inoculm sizepH -3608984 3822781 -094 03601

pHTemp -2652734 3822781 -069 04983

Results

of factorial design

Results

citrinum

Radiation dose

Enzyme activity

(Ugds)

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

ND not determined

Results

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Results

Pcitrinum

Results

Pcitrinum

40 100

Results

respectively

Results

100 50

Results

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

at concentration of

presence of Mg+2

and Zn+2

whereas Ca+2

Results

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

with Pcitrinum

pectin(gm)

Discussion

other substrate

and less expensive

Discussion

Aspergillus niger

Discussion

sporesml) for SSF

-1 resulted the

-1 had

Dayanand

Discussion

and the 6th

days Another study

the 120th

Discussion

2 value being the

Discussion

2 varies

Discussion

production

Discussion

Enzyme purification

Discussion

stability

Discussion

0C and 45

Discussion

(Arotupin 1991)

C and 600C

C from

Discussion

and Zn+2

resulted in a

Discussion

up to 60 Thus Hg+2

and Cu+2

and Mg+2

Discussion

Concluding Remarks

investigations

References

Science10 (5)107-112

Vol 1 No 1 p 33-42

References

Eng 26(1) 47 ndash 54

References

Technol 12 266-271

106Pp162-171

References

59 26122621

18 (3) 251-256

References

Lett227163-9

References

43minus50

1034-1035

References

17 145ndash148

References

226 469ndash470

Pp 787 791

References

39(1) 115-121

(9) Pp 2931-2944

Growers)

References

2231-2560

Technol 88 251-254

References

Technol 94239-234

Technology 62 63-69

TH Zurich (3374)

References

Letters10 pp 825-28

References

7B 159 - 209

References

polymers 32283-292

31426-429

Washington DC

37467-483

DC 66-83

References

47(5) 813-819

Nigeria

(4)682-686

References

87680-06-7 pp 221

activity by Pb2+

and Li+

References

3663-69

References

363-399

1 3-45

References

27(4)186ndash190

377-381

Yorkpp117

References

Nutr Clin Nutr 1983 37(3)171-83

Engenharia 10 59-62

References

Biotechnol97193-208

References

Biochem 35 411-417

Londrina

References

5943-48

References

(3) 233ndash236

Technol 40 1108-16

Freeman

Pp126-29

References

sp Strain No AKM1801 6(2) 245-250

44397-406

techniquesfourth

Presspp182-191

References

Perspect 106277-283

Pp117-144

References

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

RESULTSpdf

Approval Sheet

fermentation

Submitted to

Department of

ACKNOWLEDGMENT

Helwan University

Fermentation

University)

Summary

citrinum

20degC -60degC Mg+2

and Zn+2

Contents

No Title Page

1 Introduction 1

42 Wine industry 25

57- Surfactants 32

7 Gamma Rays 35

32Culture media 40

pulp medium

fermentation

filtrate

83 Dialysis 52

enzyme

Pcitrinum

4- Results 58

enzyme production

citrinum

state fermentation

design

5- Discussion 98

7- References 127 7

List of tables

No Title page

state fermentation

fermentation

Pcitrinum

List of Figures

No Title page

by Pcitrinum

production

Pcitrinum

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

sp species

Aim of the study

state fermentation

sources

Introduction

by-products

Introduction

inorganic chemicals

Introduction

ammonium ions

11Protopectin

12Pectic acids

called pectates

13Pectinic acids

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Orange pulp

Dry matter

124ndash280

They include

esterified pectin

the pectin chain

cleavage They are

33221 Endo-PGL (Ec 4222)

pectic acid

33 Protopectinase

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

aseptic control

Vapor treatment non

sterile conditions

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

451 Protopectinases

respectively

453 Pectinesterase

47Disadvantages

monitoring

Yokotsuka 1972)

513 Liquefaction

514 Maceration

52 Wine industry

53 Textile industry

62 Carbon Sources

63-Nitrogen sources

64ndashTemperature

was found at 7th

and 4th

66- Inoculum size

1times10 7

67- Surfactants

cell membrane

7 Factorial Design

factors

conclusions derived

Factorial Design

8 Gamma Rays

neutral (neutrons)

al 2009)

31-Microorganisms

32Culture media

Dextrose 20 g

Agar 17 -20 g

production

4 Culture condition

solution pH 59)

of distilled water

beet pulp medium

spores

61 Pectinases assay

6111Reagents

distilled water

6112 Procedure

) of the

version 11

spores

ml and 9times104

incubation period

activity

state fermentation

cell-free filtrate

(59) to be dialyzed

small portions

appropriate buffer

83 Dialysis

Walker 1995)-

fridge

bubbles)

enzyme activity

70degC for 30 min

Pcitrinum

and Co+2

and Al-Garni 2006)

Results

4-Results

producer

Results

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

tested fungal

species

Results

fermentation

1122plusmn 19 b

Results

Urea 831plusmn 18 c

Results

fermentation

Results

fermentation

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

citrinum

Results

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Results

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

between each other

Results

citrinum

Results

by Pcitrinum

state fermentation

Results

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Results

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Yeast extractpH 58589844 3822781 153 01462

Inoculm sizepH -3608984 3822781 -094 03601

pHTemp -2652734 3822781 -069 04983

Results

of factorial design

Results

citrinum

Radiation dose

Enzyme activity

(Ugds)

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

ND not determined

Results

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Results

Pcitrinum

Results

Pcitrinum

40 100

Results

respectively

Results

100 50

Results

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

at concentration of

presence of Mg+2

and Zn+2

whereas Ca+2

Results

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

with Pcitrinum

pectin(gm)

Discussion

other substrate

and less expensive

Discussion

Aspergillus niger

Discussion

sporesml) for SSF

-1 resulted the

-1 had

Dayanand

Discussion

and the 6th

days Another study

the 120th

Discussion

2 value being the

Discussion

2 varies

Discussion

production

Discussion

Enzyme purification

Discussion

stability

Discussion

0C and 45

Discussion

(Arotupin 1991)

C and 600C

C from

Discussion

and Zn+2

resulted in a

Discussion

up to 60 Thus Hg+2

and Cu+2

and Mg+2

Discussion

Concluding Remarks

investigations

References

Science10 (5)107-112

Vol 1 No 1 p 33-42

References

Eng 26(1) 47 ndash 54

References

Technol 12 266-271

106Pp162-171

References

59 26122621

18 (3) 251-256

References

Lett227163-9

References

43minus50

1034-1035

References

17 145ndash148

References

226 469ndash470

Pp 787 791

References

39(1) 115-121

(9) Pp 2931-2944

Growers)

References

2231-2560

Technol 88 251-254

References

Technol 94239-234

Technology 62 63-69

TH Zurich (3374)

References

Letters10 pp 825-28

References

7B 159 - 209

References

polymers 32283-292

31426-429

Washington DC

37467-483

DC 66-83

References

47(5) 813-819

Nigeria

(4)682-686

References

87680-06-7 pp 221

activity by Pb2+

and Li+

References

3663-69

References

363-399

1 3-45

References

27(4)186ndash190

377-381

Yorkpp117

References

Nutr Clin Nutr 1983 37(3)171-83

Engenharia 10 59-62

References

Biotechnol97193-208

References

Biochem 35 411-417

Londrina

References

5943-48

References

(3) 233ndash236

Technol 40 1108-16

Freeman

Pp126-29

References

sp Strain No AKM1801 6(2) 245-250

44397-406

techniquesfourth

Presspp182-191

References

Perspect 106277-283

Pp117-144

References

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

RESULTSpdf

Approval Sheet

fermentation

Submitted to

Department of

ACKNOWLEDGMENT

Helwan University

Fermentation

University)

Summary

citrinum

20degC -60degC Mg+2

and Zn+2

Contents

No Title Page

1 Introduction 1

42 Wine industry 25

57- Surfactants 32

7 Gamma Rays 35

32Culture media 40

pulp medium

fermentation

filtrate

83 Dialysis 52

enzyme

Pcitrinum

4- Results 58

enzyme production

citrinum

state fermentation

design

5- Discussion 98

7- References 127 7

List of tables

No Title page

state fermentation

fermentation

Pcitrinum

List of Figures

No Title page

by Pcitrinum

production

Pcitrinum

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

sp species

Aim of the study

state fermentation

sources

Introduction

by-products

Introduction

inorganic chemicals

Introduction

ammonium ions

11Protopectin

12Pectic acids

called pectates

13Pectinic acids

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Orange pulp

Dry matter

124ndash280

They include

esterified pectin

the pectin chain

cleavage They are

33221 Endo-PGL (Ec 4222)

pectic acid

33 Protopectinase

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

aseptic control

Vapor treatment non

sterile conditions

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

451 Protopectinases

respectively

453 Pectinesterase

47Disadvantages

monitoring

Yokotsuka 1972)

513 Liquefaction

514 Maceration

52 Wine industry

53 Textile industry

62 Carbon Sources

63-Nitrogen sources

64ndashTemperature

was found at 7th

and 4th

66- Inoculum size

1times10 7

67- Surfactants

cell membrane

7 Factorial Design

factors

conclusions derived

Factorial Design

8 Gamma Rays

neutral (neutrons)

al 2009)

31-Microorganisms

32Culture media

Dextrose 20 g

Agar 17 -20 g

production

4 Culture condition

solution pH 59)

of distilled water

beet pulp medium

spores

61 Pectinases assay

6111Reagents

distilled water

6112 Procedure

) of the

version 11

spores

ml and 9times104

incubation period

activity

state fermentation

cell-free filtrate

(59) to be dialyzed

small portions

appropriate buffer

83 Dialysis

Walker 1995)-

fridge

bubbles)

enzyme activity

70degC for 30 min

Pcitrinum

and Co+2

and Al-Garni 2006)

Results

4-Results

producer

Results

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

tested fungal

species

Results

fermentation

1122plusmn 19 b

Results

Urea 831plusmn 18 c

Results

fermentation

Results

fermentation

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

citrinum

Results

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Results

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

between each other

Results

citrinum

Results

by Pcitrinum

state fermentation

Results

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Results

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Yeast extractpH 58589844 3822781 153 01462

Inoculm sizepH -3608984 3822781 -094 03601

pHTemp -2652734 3822781 -069 04983

Results

of factorial design

Results

citrinum

Radiation dose

Enzyme activity

(Ugds)

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

ND not determined

Results

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Results

Pcitrinum

Results

Pcitrinum

40 100

Results

respectively

Results

100 50

Results

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

at concentration of

presence of Mg+2

and Zn+2

whereas Ca+2

Results

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

with Pcitrinum

pectin(gm)

Discussion

other substrate

and less expensive

Discussion

Aspergillus niger

Discussion

sporesml) for SSF

-1 resulted the

-1 had

Dayanand

Discussion

and the 6th

days Another study

the 120th

Discussion

2 value being the

Discussion

2 varies

Discussion

production

Discussion

Enzyme purification

Discussion

stability

Discussion

0C and 45

Discussion

(Arotupin 1991)

C and 600C

C from

Discussion

and Zn+2

resulted in a

Discussion

up to 60 Thus Hg+2

and Cu+2

and Mg+2

Discussion

Concluding Remarks

investigations

References

Science10 (5)107-112

Vol 1 No 1 p 33-42

References

Eng 26(1) 47 ndash 54

References

Technol 12 266-271

106Pp162-171

References

59 26122621

18 (3) 251-256

References

Lett227163-9

References

43minus50

1034-1035

References

17 145ndash148

References

226 469ndash470

Pp 787 791

References

39(1) 115-121

(9) Pp 2931-2944

Growers)

References

2231-2560

Technol 88 251-254

References

Technol 94239-234

Technology 62 63-69

TH Zurich (3374)

References

Letters10 pp 825-28

References

7B 159 - 209

References

polymers 32283-292

31426-429

Washington DC

37467-483

DC 66-83

References

47(5) 813-819

Nigeria

(4)682-686

References

87680-06-7 pp 221

activity by Pb2+

and Li+

References

3663-69

References

363-399

1 3-45

References

27(4)186ndash190

377-381

Yorkpp117

References

Nutr Clin Nutr 1983 37(3)171-83

Engenharia 10 59-62

References

Biotechnol97193-208

References

Biochem 35 411-417

Londrina

References

5943-48

References

(3) 233ndash236

Technol 40 1108-16

Freeman

Pp126-29

References

sp Strain No AKM1801 6(2) 245-250

44397-406

techniquesfourth

Presspp182-191

References

Perspect 106277-283

Pp117-144

References

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

RESULTSpdf

ACKNOWLEDGMENT

Helwan University

Fermentation

University)

Summary

citrinum

20degC -60degC Mg+2

and Zn+2

Contents

No Title Page

1 Introduction 1

42 Wine industry 25

57- Surfactants 32

7 Gamma Rays 35

32Culture media 40

pulp medium

fermentation

filtrate

83 Dialysis 52

enzyme

Pcitrinum

4- Results 58

enzyme production

citrinum

state fermentation

design

5- Discussion 98

7- References 127 7

List of tables

No Title page

state fermentation

fermentation

Pcitrinum

List of Figures

No Title page

by Pcitrinum

production

Pcitrinum

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

sp species

Aim of the study

state fermentation

sources

Introduction

by-products

Introduction

inorganic chemicals

Introduction

ammonium ions

11Protopectin

12Pectic acids

called pectates

13Pectinic acids

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Orange pulp

Dry matter

124ndash280

They include

esterified pectin

the pectin chain

cleavage They are

33221 Endo-PGL (Ec 4222)

pectic acid

33 Protopectinase

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

aseptic control

Vapor treatment non

sterile conditions

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

451 Protopectinases

respectively

453 Pectinesterase

47Disadvantages

monitoring

Yokotsuka 1972)

513 Liquefaction

514 Maceration

52 Wine industry

53 Textile industry

62 Carbon Sources

63-Nitrogen sources

64ndashTemperature

was found at 7th

and 4th

66- Inoculum size

1times10 7

67- Surfactants

cell membrane

7 Factorial Design

factors

conclusions derived

Factorial Design

8 Gamma Rays

neutral (neutrons)

al 2009)

31-Microorganisms

32Culture media

Dextrose 20 g

Agar 17 -20 g

production

4 Culture condition

solution pH 59)

of distilled water

beet pulp medium

spores

61 Pectinases assay

6111Reagents

distilled water

6112 Procedure

) of the

version 11

spores

ml and 9times104

incubation period

activity

state fermentation

cell-free filtrate

(59) to be dialyzed

small portions

appropriate buffer

83 Dialysis

Walker 1995)-

fridge

bubbles)

enzyme activity

70degC for 30 min

Pcitrinum

and Co+2

and Al-Garni 2006)

Results

4-Results

producer

Results

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

tested fungal

species

Results

fermentation

1122plusmn 19 b

Results

Urea 831plusmn 18 c

Results

fermentation

Results

fermentation

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

citrinum

Results

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Results

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

between each other

Results

citrinum

Results

by Pcitrinum

state fermentation

Results

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Results

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Yeast extractpH 58589844 3822781 153 01462

Inoculm sizepH -3608984 3822781 -094 03601

pHTemp -2652734 3822781 -069 04983

Results

of factorial design

Results

citrinum

Radiation dose

Enzyme activity

(Ugds)

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

ND not determined

Results

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Results

Pcitrinum

Results

Pcitrinum

40 100

Results

respectively

Results

100 50

Results

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

at concentration of

presence of Mg+2

and Zn+2

whereas Ca+2

Results

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

with Pcitrinum

pectin(gm)

Discussion

other substrate

and less expensive

Discussion

Aspergillus niger

Discussion

sporesml) for SSF

-1 resulted the

-1 had

Dayanand

Discussion

and the 6th

days Another study

the 120th

Discussion

2 value being the

Discussion

2 varies

Discussion

production

Discussion

Enzyme purification

Discussion

stability

Discussion

0C and 45

Discussion

(Arotupin 1991)

C and 600C

C from

Discussion

and Zn+2

resulted in a

Discussion

up to 60 Thus Hg+2

and Cu+2

and Mg+2

Discussion

Concluding Remarks

investigations

References

Science10 (5)107-112

Vol 1 No 1 p 33-42

References

Eng 26(1) 47 ndash 54

References

Technol 12 266-271

106Pp162-171

References

59 26122621

18 (3) 251-256

References

Lett227163-9

References

43minus50

1034-1035

References

17 145ndash148

References

226 469ndash470

Pp 787 791

References

39(1) 115-121

(9) Pp 2931-2944

Growers)

References

2231-2560

Technol 88 251-254

References

Technol 94239-234

Technology 62 63-69

TH Zurich (3374)

References

Letters10 pp 825-28

References

7B 159 - 209

References

polymers 32283-292

31426-429

Washington DC

37467-483

DC 66-83

References

47(5) 813-819

Nigeria

(4)682-686

References

87680-06-7 pp 221

activity by Pb2+

and Li+

References

3663-69

References

363-399

1 3-45

References

27(4)186ndash190

377-381

Yorkpp117

References

Nutr Clin Nutr 1983 37(3)171-83

Engenharia 10 59-62

References

Biotechnol97193-208

References

Biochem 35 411-417

Londrina

References

5943-48

References

(3) 233ndash236

Technol 40 1108-16

Freeman

Pp126-29

References

sp Strain No AKM1801 6(2) 245-250

44397-406

techniquesfourth

Presspp182-191

References

Perspect 106277-283

Pp117-144

References

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

RESULTSpdf

Fermentation

University)

Summary

citrinum

20degC -60degC Mg+2

and Zn+2

Contents

No Title Page

1 Introduction 1

42 Wine industry 25

57- Surfactants 32

7 Gamma Rays 35

32Culture media 40

pulp medium

fermentation

filtrate

83 Dialysis 52

enzyme

Pcitrinum

4- Results 58

enzyme production

citrinum

state fermentation

design

5- Discussion 98

7- References 127 7

List of tables

No Title page

state fermentation

fermentation

Pcitrinum

List of Figures

No Title page

by Pcitrinum

production

Pcitrinum

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

sp species

Aim of the study

state fermentation

sources

Introduction

by-products

Introduction

inorganic chemicals

Introduction

ammonium ions

11Protopectin

12Pectic acids

called pectates

13Pectinic acids

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Orange pulp

Dry matter

124ndash280

They include

esterified pectin

the pectin chain

cleavage They are

33221 Endo-PGL (Ec 4222)

pectic acid

33 Protopectinase

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

aseptic control

Vapor treatment non

sterile conditions

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

451 Protopectinases

respectively

453 Pectinesterase

47Disadvantages

monitoring

Yokotsuka 1972)

513 Liquefaction

514 Maceration

52 Wine industry

53 Textile industry

62 Carbon Sources

63-Nitrogen sources

64ndashTemperature

was found at 7th

and 4th

66- Inoculum size

1times10 7

67- Surfactants

cell membrane

7 Factorial Design

factors

conclusions derived

Factorial Design

8 Gamma Rays

neutral (neutrons)

al 2009)

31-Microorganisms

32Culture media

Dextrose 20 g

Agar 17 -20 g

production

4 Culture condition

solution pH 59)

of distilled water

beet pulp medium

spores

61 Pectinases assay

6111Reagents

distilled water

6112 Procedure

) of the

version 11

spores

ml and 9times104

incubation period

activity

state fermentation

cell-free filtrate

(59) to be dialyzed

small portions

appropriate buffer

83 Dialysis

Walker 1995)-

fridge

bubbles)

enzyme activity

70degC for 30 min

Pcitrinum

and Co+2

and Al-Garni 2006)

Results

4-Results

producer

Results

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

tested fungal

species

Results

fermentation

1122plusmn 19 b

Results

Urea 831plusmn 18 c

Results

fermentation

Results

fermentation

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

citrinum

Results

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Results

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

between each other

Results

citrinum

Results

by Pcitrinum

state fermentation

Results

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Results

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Yeast extractpH 58589844 3822781 153 01462

Inoculm sizepH -3608984 3822781 -094 03601

pHTemp -2652734 3822781 -069 04983

Results

of factorial design

Results

citrinum

Radiation dose

Enzyme activity

(Ugds)

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

ND not determined

Results

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Results

Pcitrinum

Results

Pcitrinum

40 100

Results

respectively

Results

100 50

Results

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

at concentration of

presence of Mg+2

and Zn+2

whereas Ca+2

Results

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

with Pcitrinum

pectin(gm)

Discussion

other substrate

and less expensive

Discussion

Aspergillus niger

Discussion

sporesml) for SSF

-1 resulted the

-1 had

Dayanand

Discussion

and the 6th

days Another study

the 120th

Discussion

2 value being the

Discussion

2 varies

Discussion

production

Discussion

Enzyme purification

Discussion

stability

Discussion

0C and 45

Discussion

(Arotupin 1991)

C and 600C

C from

Discussion

and Zn+2

resulted in a

Discussion

up to 60 Thus Hg+2

and Cu+2

and Mg+2

Discussion

Concluding Remarks

investigations

References

Science10 (5)107-112

Vol 1 No 1 p 33-42

References

Eng 26(1) 47 ndash 54

References

Technol 12 266-271

106Pp162-171

References

59 26122621

18 (3) 251-256

References

Lett227163-9

References

43minus50

1034-1035

References

17 145ndash148

References

226 469ndash470

Pp 787 791

References

39(1) 115-121

(9) Pp 2931-2944

Growers)

References

2231-2560

Technol 88 251-254

References

Technol 94239-234

Technology 62 63-69

TH Zurich (3374)

References

Letters10 pp 825-28

References

7B 159 - 209

References

polymers 32283-292

31426-429

Washington DC

37467-483

DC 66-83

References

47(5) 813-819

Nigeria

(4)682-686

References

87680-06-7 pp 221

activity by Pb2+

and Li+

References

3663-69

References

363-399

1 3-45

References

27(4)186ndash190

377-381

Yorkpp117

References

Nutr Clin Nutr 1983 37(3)171-83

Engenharia 10 59-62

References

Biotechnol97193-208

References

Biochem 35 411-417

Londrina

References

5943-48

References

(3) 233ndash236

Technol 40 1108-16

Freeman

Pp126-29

References

sp Strain No AKM1801 6(2) 245-250

44397-406

techniquesfourth

Presspp182-191

References

Perspect 106277-283

Pp117-144

References

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

RESULTSpdf

20degC -60degC Mg+2

and Zn+2

Contents

No Title Page

1 Introduction 1

42 Wine industry 25

57- Surfactants 32

7 Gamma Rays 35

32Culture media 40

pulp medium

fermentation

filtrate

83 Dialysis 52

enzyme

Pcitrinum

4- Results 58

enzyme production

citrinum

state fermentation

design

5- Discussion 98

7- References 127 7

List of tables

No Title page

state fermentation

fermentation

Pcitrinum

List of Figures

No Title page

by Pcitrinum

production

Pcitrinum

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

sp species

Aim of the study

state fermentation

sources

Introduction

by-products

Introduction

inorganic chemicals

Introduction

ammonium ions

11Protopectin

12Pectic acids

called pectates

13Pectinic acids

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Orange pulp

Dry matter

124ndash280

They include

esterified pectin

the pectin chain

cleavage They are

33221 Endo-PGL (Ec 4222)

pectic acid

33 Protopectinase

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

aseptic control

Vapor treatment non

sterile conditions

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

451 Protopectinases

respectively

453 Pectinesterase

47Disadvantages

monitoring

Yokotsuka 1972)

513 Liquefaction

514 Maceration

52 Wine industry

53 Textile industry

62 Carbon Sources

63-Nitrogen sources

64ndashTemperature

was found at 7th

and 4th

66- Inoculum size

1times10 7

67- Surfactants

cell membrane

7 Factorial Design

factors

conclusions derived

Factorial Design

8 Gamma Rays

neutral (neutrons)

al 2009)

31-Microorganisms

32Culture media

Dextrose 20 g

Agar 17 -20 g

production

4 Culture condition

solution pH 59)

of distilled water

beet pulp medium

spores

61 Pectinases assay

6111Reagents

distilled water

6112 Procedure

) of the

version 11

spores

ml and 9times104

incubation period

activity

state fermentation

cell-free filtrate

(59) to be dialyzed

small portions

appropriate buffer

83 Dialysis

Walker 1995)-

fridge

bubbles)

enzyme activity

70degC for 30 min

Pcitrinum

and Co+2

and Al-Garni 2006)

Results

4-Results

producer

Results

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

tested fungal

species

Results

fermentation

1122plusmn 19 b

Results

Urea 831plusmn 18 c

Results

fermentation

Results

fermentation

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

citrinum

Results

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Results

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

between each other

Results

citrinum

Results

by Pcitrinum

state fermentation

Results

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Results

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Yeast extractpH 58589844 3822781 153 01462

Inoculm sizepH -3608984 3822781 -094 03601

pHTemp -2652734 3822781 -069 04983

Results

of factorial design

Results

citrinum

Radiation dose

Enzyme activity

(Ugds)

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

ND not determined

Results

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Results

Pcitrinum

Results

Pcitrinum

40 100

Results

respectively

Results

100 50

Results

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

at concentration of

presence of Mg+2

and Zn+2

whereas Ca+2

Results

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

with Pcitrinum

pectin(gm)

Discussion

other substrate

and less expensive

Discussion

Aspergillus niger

Discussion

sporesml) for SSF

-1 resulted the

-1 had

Dayanand

Discussion

and the 6th

days Another study

the 120th

Discussion

2 value being the

Discussion

2 varies

Discussion

production

Discussion

Enzyme purification

Discussion

stability

Discussion

0C and 45

Discussion

(Arotupin 1991)

C and 600C

C from

Discussion

and Zn+2

resulted in a

Discussion

up to 60 Thus Hg+2

and Cu+2

and Mg+2

Discussion

Concluding Remarks

investigations

References

Science10 (5)107-112

Vol 1 No 1 p 33-42

References

Eng 26(1) 47 ndash 54

References

Technol 12 266-271

106Pp162-171

References

59 26122621

18 (3) 251-256

References

Lett227163-9

References

43minus50

1034-1035

References

17 145ndash148

References

226 469ndash470

Pp 787 791

References

39(1) 115-121

(9) Pp 2931-2944

Growers)

References

2231-2560

Technol 88 251-254

References

Technol 94239-234

Technology 62 63-69

TH Zurich (3374)

References

Letters10 pp 825-28

References

7B 159 - 209

References

polymers 32283-292

31426-429

Washington DC

37467-483

DC 66-83

References

47(5) 813-819

Nigeria

(4)682-686

References

87680-06-7 pp 221

activity by Pb2+

and Li+

References

3663-69

References

363-399

1 3-45

References

27(4)186ndash190

377-381

Yorkpp117

References

Nutr Clin Nutr 1983 37(3)171-83

Engenharia 10 59-62

References

Biotechnol97193-208

References

Biochem 35 411-417

Londrina

References

5943-48

References

(3) 233ndash236

Technol 40 1108-16

Freeman

Pp126-29

References

sp Strain No AKM1801 6(2) 245-250

44397-406

techniquesfourth

Presspp182-191

References

Perspect 106277-283

Pp117-144

References

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

RESULTSpdf

Contents

No Title Page

1 Introduction 1

42 Wine industry 25

57- Surfactants 32

7 Gamma Rays 35

32Culture media 40

pulp medium

fermentation

filtrate

83 Dialysis 52

enzyme

Pcitrinum

4- Results 58

enzyme production

citrinum

state fermentation

design

5- Discussion 98

7- References 127 7

List of tables

No Title page

state fermentation

fermentation

Pcitrinum

List of Figures

No Title page

by Pcitrinum

production

Pcitrinum

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

sp species

Aim of the study

state fermentation

sources

Introduction

by-products

Introduction

inorganic chemicals

Introduction

ammonium ions

11Protopectin

12Pectic acids

called pectates

13Pectinic acids

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Orange pulp

Dry matter

124ndash280

They include

esterified pectin

the pectin chain

cleavage They are

33221 Endo-PGL (Ec 4222)

pectic acid

33 Protopectinase

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

aseptic control

Vapor treatment non

sterile conditions

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

451 Protopectinases

respectively

453 Pectinesterase

47Disadvantages

monitoring

Yokotsuka 1972)

513 Liquefaction

514 Maceration

52 Wine industry

53 Textile industry

62 Carbon Sources

63-Nitrogen sources

64ndashTemperature

was found at 7th

and 4th

66- Inoculum size

1times10 7

67- Surfactants

cell membrane

7 Factorial Design

factors

conclusions derived

Factorial Design

8 Gamma Rays

neutral (neutrons)

al 2009)

31-Microorganisms

32Culture media

Dextrose 20 g

Agar 17 -20 g

production

4 Culture condition

solution pH 59)

of distilled water

beet pulp medium

spores

61 Pectinases assay

6111Reagents

distilled water

6112 Procedure

) of the

version 11

spores

ml and 9times104

incubation period

activity

state fermentation

cell-free filtrate

(59) to be dialyzed

small portions

appropriate buffer

83 Dialysis

Walker 1995)-

fridge

bubbles)

enzyme activity

70degC for 30 min

Pcitrinum

and Co+2

and Al-Garni 2006)

Results

4-Results

producer

Results

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

tested fungal

species

Results

fermentation

1122plusmn 19 b

Results

Urea 831plusmn 18 c

Results

fermentation

Results

fermentation

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

citrinum

Results

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Results

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

between each other

Results

citrinum

Results

by Pcitrinum

state fermentation

Results

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Results

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Yeast extractpH 58589844 3822781 153 01462

Inoculm sizepH -3608984 3822781 -094 03601

pHTemp -2652734 3822781 -069 04983

Results

of factorial design

Results

citrinum

Radiation dose

Enzyme activity

(Ugds)

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

ND not determined

Results

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Results

Pcitrinum

Results

Pcitrinum

40 100

Results

respectively

Results

100 50

Results

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

at concentration of

presence of Mg+2

and Zn+2

whereas Ca+2

Results

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

with Pcitrinum

pectin(gm)

Discussion

other substrate

and less expensive

Discussion

Aspergillus niger

Discussion

sporesml) for SSF

-1 resulted the

-1 had

Dayanand

Discussion

and the 6th

days Another study

the 120th

Discussion

2 value being the

Discussion

2 varies

Discussion

production

Discussion

Enzyme purification

Discussion

stability

Discussion

0C and 45

Discussion

(Arotupin 1991)

C and 600C

C from

Discussion

and Zn+2

resulted in a

Discussion

up to 60 Thus Hg+2

and Cu+2

and Mg+2

Discussion

Concluding Remarks

investigations

References

Science10 (5)107-112

Vol 1 No 1 p 33-42

References

Eng 26(1) 47 ndash 54

References

Technol 12 266-271

106Pp162-171

References

59 26122621

18 (3) 251-256

References

Lett227163-9

References

43minus50

1034-1035

References

17 145ndash148

References

226 469ndash470

Pp 787 791

References

39(1) 115-121

(9) Pp 2931-2944

Growers)

References

2231-2560

Technol 88 251-254

References

Technol 94239-234

Technology 62 63-69

TH Zurich (3374)

References

Letters10 pp 825-28

References

7B 159 - 209

References

polymers 32283-292

31426-429

Washington DC

37467-483

DC 66-83

References

47(5) 813-819

Nigeria

(4)682-686

References

87680-06-7 pp 221

activity by Pb2+

and Li+

References

3663-69

References

363-399

1 3-45

References

27(4)186ndash190

377-381

Yorkpp117

References

Nutr Clin Nutr 1983 37(3)171-83

Engenharia 10 59-62

References

Biotechnol97193-208

References

Biochem 35 411-417

Londrina

References

5943-48

References

(3) 233ndash236

Technol 40 1108-16

Freeman

Pp126-29

References

sp Strain No AKM1801 6(2) 245-250

44397-406

techniquesfourth

Presspp182-191

References

Perspect 106277-283

Pp117-144

References

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

RESULTSpdf

32Culture media 40

pulp medium

fermentation

filtrate

83 Dialysis 52

enzyme

Pcitrinum

4- Results 58

enzyme production

citrinum

state fermentation

design

5- Discussion 98

7- References 127 7

List of tables

No Title page

state fermentation

fermentation

Pcitrinum

List of Figures

No Title page

by Pcitrinum

production

Pcitrinum

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

sp species

Aim of the study

state fermentation

sources

Introduction

by-products

Introduction

inorganic chemicals

Introduction

ammonium ions

11Protopectin

12Pectic acids

called pectates

13Pectinic acids

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Orange pulp

Dry matter

124ndash280

They include

esterified pectin

the pectin chain

cleavage They are

33221 Endo-PGL (Ec 4222)

pectic acid

33 Protopectinase

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

aseptic control

Vapor treatment non

sterile conditions

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

451 Protopectinases

respectively

453 Pectinesterase

47Disadvantages

monitoring

Yokotsuka 1972)

513 Liquefaction

514 Maceration

52 Wine industry

53 Textile industry

62 Carbon Sources

63-Nitrogen sources

64ndashTemperature

was found at 7th

and 4th

66- Inoculum size

1times10 7

67- Surfactants

cell membrane

7 Factorial Design

factors

conclusions derived

Factorial Design

8 Gamma Rays

neutral (neutrons)

al 2009)

31-Microorganisms

32Culture media

Dextrose 20 g

Agar 17 -20 g

production

4 Culture condition

solution pH 59)

of distilled water

beet pulp medium

spores

61 Pectinases assay

6111Reagents

distilled water

6112 Procedure

) of the

version 11

spores

ml and 9times104

incubation period

activity

state fermentation

cell-free filtrate

(59) to be dialyzed

small portions

appropriate buffer

83 Dialysis

Walker 1995)-

fridge

bubbles)

enzyme activity

70degC for 30 min

Pcitrinum

and Co+2

and Al-Garni 2006)

Results

4-Results

producer

Results

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

tested fungal

species

Results

fermentation

1122plusmn 19 b

Results

Urea 831plusmn 18 c

Results

fermentation

Results

fermentation

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

citrinum

Results

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Results

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

between each other

Results

citrinum

Results

by Pcitrinum

state fermentation

Results

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Results

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Yeast extractpH 58589844 3822781 153 01462

Inoculm sizepH -3608984 3822781 -094 03601

pHTemp -2652734 3822781 -069 04983

Results

of factorial design

Results

citrinum

Radiation dose

Enzyme activity

(Ugds)

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

ND not determined

Results

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Results

Pcitrinum

Results

Pcitrinum

40 100

Results

respectively

Results

100 50

Results

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

at concentration of

presence of Mg+2

and Zn+2

whereas Ca+2

Results

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

with Pcitrinum

pectin(gm)

Discussion

other substrate

and less expensive

Discussion

Aspergillus niger

Discussion

sporesml) for SSF

-1 resulted the

-1 had

Dayanand

Discussion

and the 6th

days Another study

the 120th

Discussion

2 value being the

Discussion

2 varies

Discussion

production

Discussion

Enzyme purification

Discussion

stability

Discussion

0C and 45

Discussion

(Arotupin 1991)

C and 600C

C from

Discussion

and Zn+2

resulted in a

Discussion

up to 60 Thus Hg+2

and Cu+2

and Mg+2

Discussion

Concluding Remarks

investigations

References

Science10 (5)107-112

Vol 1 No 1 p 33-42

References

Eng 26(1) 47 ndash 54

References

Technol 12 266-271

106Pp162-171

References

59 26122621

18 (3) 251-256

References

Lett227163-9

References

43minus50

1034-1035

References

17 145ndash148

References

226 469ndash470

Pp 787 791

References

39(1) 115-121

(9) Pp 2931-2944

Growers)

References

2231-2560

Technol 88 251-254

References

Technol 94239-234

Technology 62 63-69

TH Zurich (3374)

References

Letters10 pp 825-28

References

7B 159 - 209

References

polymers 32283-292

31426-429

Washington DC

37467-483

DC 66-83

References

47(5) 813-819

Nigeria

(4)682-686

References

87680-06-7 pp 221

activity by Pb2+

and Li+

References

3663-69

References

363-399

1 3-45

References

27(4)186ndash190

377-381

Yorkpp117

References

Nutr Clin Nutr 1983 37(3)171-83

Engenharia 10 59-62

References

Biotechnol97193-208

References

Biochem 35 411-417

Londrina

References

5943-48

References

(3) 233ndash236

Technol 40 1108-16

Freeman

Pp126-29

References

sp Strain No AKM1801 6(2) 245-250

44397-406

techniquesfourth

Presspp182-191

References

Perspect 106277-283

Pp117-144

References

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

RESULTSpdf

Pcitrinum

4- Results 58

enzyme production

citrinum

state fermentation

design

5- Discussion 98

7- References 127 7

List of tables

No Title page

state fermentation

fermentation

Pcitrinum

List of Figures

No Title page

by Pcitrinum

production

Pcitrinum

Conc Concentration

g gram

microg microgram

hr hour

L Liter

M Molar

mg milligram

min minute

ml milliliter

mM millimolar

microM Micromolar

sp species

Aim of the study

state fermentation

sources

Introduction

by-products

Introduction

inorganic chemicals

Introduction

ammonium ions

11Protopectin

12Pectic acids

called pectates

13Pectinic acids

Fruit vegetable

Tissue

Pectic

Substance ()

Apple peel

05ndash16

Banana peel

Fresh 07ndash12

Peaches pulp

01ndash09

Strawberries pulp

06ndash07

Cherries pulp

02ndash05

Peas pulp

09ndash14

Carrots peel

Orange pulp

Dry matter

124ndash280

They include

esterified pectin

the pectin chain

cleavage They are

33221 Endo-PGL (Ec 4222)

pectic acid

33 Protopectinase

Factor

Liquid Substrate

fermentation

Solid Substrate

Fermentation

Substrates

Soluble

Substrates(sugars)

Polymer Insoluble

Substrates Starch

Cellulose Pectins

Lignin

Aseptic conditions

aseptic control

Vapor treatment non

sterile conditions

discarded

Limited Consumption

of water low Aw No

effluent

Metabolic Heating

Easy control of

temperature

Low heat transfer

capacity

451 Protopectinases

respectively

453 Pectinesterase

47Disadvantages

monitoring

Yokotsuka 1972)

513 Liquefaction

514 Maceration

52 Wine industry

53 Textile industry

62 Carbon Sources

63-Nitrogen sources

64ndashTemperature

was found at 7th

and 4th

66- Inoculum size

1times10 7

67- Surfactants

cell membrane

7 Factorial Design

factors

conclusions derived

Factorial Design

8 Gamma Rays

neutral (neutrons)

al 2009)

31-Microorganisms

32Culture media

Dextrose 20 g

Agar 17 -20 g

production

4 Culture condition

solution pH 59)

of distilled water

beet pulp medium

spores

61 Pectinases assay

6111Reagents

distilled water

6112 Procedure

) of the

version 11

spores

ml and 9times104

incubation period

activity

state fermentation

cell-free filtrate

(59) to be dialyzed

small portions

appropriate buffer

83 Dialysis

Walker 1995)-

fridge

bubbles)

enzyme activity

70degC for 30 min

Pcitrinum

and Co+2

and Al-Garni 2006)

Results

4-Results

producer

Results

Pectin lyase

activity(Ugdfs)

Polygalacturonase

activity(Ugdfs)

Fungal strains

tested fungal

species

Results

fermentation

1122plusmn 19 b

Results

Urea 831plusmn 18 c

Results

fermentation

Results

fermentation

Enzyme activity

(Ugdfs)

Inoculum size

(Sporesml)

812 plusmn 19 d

9times104

951 plusmn 18 c

54times105

1151plusmn19b

36times105

1272plusmn2a

18times105

Results

citrinum

Results

2 783plusmn23a

3 952plusmn18b

4 98plusmn22 b

5 1082plusmn19c

6 1141plusmn23d

7 1292plusmn22e

8 12801plusmn18 e

9 1002plusmn2c

10 942plusmn2 b

Results

3 802plusmn2a

35 87plusmn19b

4 981plusmn18c

45 1009plusmn22c

5 1142plusmn21 d

55 1261plusmn18e

6 114plusmn18 d

65 1123plusmn21 d

7 952plusmn11f

75 905plusmn20g

between each other

Results

citrinum

Results

by Pcitrinum

state fermentation

Results

the 25

Factors (Enzyme

production(

Ugdfs)

Trials

Temperat

(ordmC)

pH Inoculum

size(sporesml)

Incubation

period(day)

content

+ - + + - 866 1

+ - + + + 1037 2

+ - + - - 1136 3

- + 703 4

+ - 1008 5

+ - - + + 1115 6

+ - - - - 659 7

+ - - - + 1194 8

+ + + + - 609 9

+ + + + + 735 10

+ + + - - 556 11

+ + + - + 1224 12

+ + - + - 889 13

+ + - + + 1320 14

+ + - - - 819 15

Results

+ + - - + 948 16

- - + + - 582 17

- + + + + 447 18

- - + - - 405 19

- - + - + 501 20

- - - + - 621 21

- - - + + 784 22

- - - - - 845 23

- - - - + 919 24

- + + + - 640 25

- + + + + 387 26

- + + - - 304 27

- + + - + 331 28

- + - + - 488 29

- + - + + 1272 30

- + - - - 686 31

- - - - + 978 32

Results

Intercept 78552734 3822781 2055 lt0001

Yeast extract(041) 81972656 3822781 214 00488

Inoculm size(1836) -1225977 3822781 -321 00059

pH(555) -2108984 3822781 -055 05893

Temp(2530) 14958984 3822781 391 00014

Results

Yeast extractpH 58589844 3822781 153 01462

Inoculm sizepH -3608984 3822781 -094 03601

pHTemp -2652734 3822781 -069 04983

Results

of factorial design

Results

citrinum

Radiation dose

Enzyme activity

(Ugds)

01 1378plusmn21b

02 1422plusmn13c

05 1455plusmn21d

07 1522plusmn22e

1 1002plusmn23f

15 955plusmn2 g

ND not determined

Results

Purification

Protein

activity

Specific

activity

Purification

exract

1300 2500 19 1 100

(NH4)SO4 1000 2275 23 12 91

G-100 720 2192 30 16 87

Results

1 6 11 16 21 26 31 36

Fraction Number

Results

Pcitrinum

Results

Pcitrinum

40 100

Results

respectively

Results

100 50

Results

chlorides of Ca+2

and Mg+2

sulphates of Cu+2

at concentration of

presence of Mg+2

and Zn+2

whereas Ca+2

Results

Cacl2 88

CuSO45H2O 690

ZnSO4 105

CoCl26H2O 590

MgCl2 1120

EDTA 500

CaSO4 881

CONTROL 100

Results

with Pcitrinum

pectin(gm)

Discussion

other substrate

and less expensive

Discussion

Aspergillus niger

Discussion

sporesml) for SSF

-1 resulted the

-1 had

Dayanand

Discussion

and the 6th

days Another study

the 120th

Discussion

2 value being the

Discussion

2 varies

Discussion

production

Discussion

Enzyme purification

Discussion

stability

Discussion

0C and 45

Discussion

(Arotupin 1991)

C and 600C

C from

Discussion

and Zn+2

resulted in a

Discussion

up to 60 Thus Hg+2

and Cu+2

and Mg+2

Discussion

Concluding Remarks

investigations

References

Science10 (5)107-112

Vol 1 No 1 p 33-42

References

Eng 26(1) 47 ndash 54

References

Technol 12 266-271

106Pp162-171

References

59 26122621

18 (3) 251-256

References

Lett227163-9

References

43minus50

1034-1035

References

17 145ndash148

References

226 469ndash470

Pp 787 791

References

39(1) 115-121

(9) Pp 2931-2944

Growers)

References

2231-2560

Technol 88 251-254

References

Technol 94239-234

Technology 62 63-69

TH Zurich (3374)

References

Letters10 pp 825-28

References

7B 159 - 209

References

polymers 32283-292

31426-429

Washington DC

37467-483

DC 66-83

References

47(5) 813-819

Nigeria

(4)682-686

References

87680-06-7 pp 221

activity by Pb2+

and Li+

References

3663-69

References

363-399

1 3-45

References

27(4)186ndash190

377-381

Yorkpp117

References

Nutr Clin Nutr 1983 37(3)171-83

Engenharia 10 59-62

References

Biotechnol97193-208

References

Biochem 35 411-417

Londrina

References

5943-48

References

(3) 233ndash236

Technol 40 1108-16

Freeman

Pp126-29

References

sp Strain No AKM1801 6(2) 245-250

44397-406

techniquesfourth

Presspp182-191

References

Perspect 106277-283

Pp117-144

References

الازيي

Coverpdf

AKNOWLEDGMENTpdf

Abstractpdf

Contents[1]pdf

List of tablespdf

List of Figurespdf

abbreviationspdf

Aim of workpdf

introduction[1]pdf

RESULTSpdf

Documents

Enhancement of fungal pectinolytic enzymes production