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UTILIZATION OF WHEY FOR THE PRODUCTION OF β-GALACTOSIDASE USING YEAST AND FUNGAL
CULTURE
Dr. Parmjit S. PanesarProfessor
Biotechnology Research Laboratory Department of Food Engineering & Technology
Sant Longowal Institute of Engineering & Technology (Deemed University: Estd. by Govt. of India)
Longowal, Punjab, IndiaE. mail: pspbt@yahoo.com
IndiaPunjab
Sant Longowal Institute of Engineering & Technologywww.sliet.ac.in
THE INSTITUTE
Enzyme -galactosidase (-D-galactoside galactohydrolase, E.C. 3.2.1.23), most commonly known as lactase. Technological and nutritional importance Catalyzes the hydrolysis of lactose Has transgalactosyl activity
-GALACTOSIDASE
ACTION OF -GALACTOSIDASE
Hydrolysis of lactose to glucose and galactose
O
OH
HOOH
CH2OH
OO
HOOH
CH2OH
OH
HOO
HOOH
CH2OH
OH
O
OH
HOOH
CH2OH
OH
Lactose-D-Galp-(1 D-Glc
D-GlcD-Gal
-D-Galactosidase
APPLICATIONS OF -GALACTOSIDASE
FOOD, DAIRY AND FERMENTATION SECTOR
Solve the problems of lactose intolerance of the individuals who are deficient in lactase. Reduces set time of yogurt and accelerates the development of flavor in cheese.
Prevents lactose crystallization in frozen
and condensed milk products.
Concentrated hydrolyzed whey or whey
permeates can also be used as a
sweetener in products such as canned
fruit syrups and soft drinks.
Used in the production of different
prebiotics like lactulose, lactosucrose and
galacto-oligosaccharide
Solve the pollution problem caused by
whey and whey permeates.
Prebioti
cs
Animals
Microorganisms Plants
Sources
-GALACTOSIDASE PRODUCTION
Easy handling.
Higher multiplication rate.
High production yields.
Easy genetic manipulation.
Controlled growth.
ADVANTAGES OF USING MICROBES AS SOURCE
MICROBIAL SOURCES
Bacteria: Escherichia coli, Bacillus sp., Lactobacillus sp. etc.
Yeast: Kluyveromyces lactis, Kluyveromyces fragilis, Saccharomyces lactis etc.
Mold: Aspergillus niger, A. oryzae etc.
WIDELY USED MICROBIAL SOURCES
Kluyveromyces sp. and Aspergillus sp. The yeast Kluyveromyces as the most important source for the production of -galactosidase.
As the enzyme from the yeast has an optimum pH suitable for lactose hydrolysis in milk
YEAST-Natural Dairy Environment
-Intracellular location of the enzyme
-Neutral pH required for production
FUNGUS-Extracellular location of the enzyme
-Enzyme stable at low pH
-Stable at high temperature
COMPARISON BETWEEN YEAST AND FUNGUS
Enzymatic processes for lactose hydrolysis are
facing hindrances due to
Intracellular location of enzyme.
Enzyme extraction difficult & expensive.
Enzyme loss.
PROBLEMS WITH YEAST ENZYME
CELL DISRUPTION
Involve disruption of the cell envelope and release of all the intracellular constituents into the surrounding medium
Intact cell Disrupted cell
CELL DISRUPTION TECHNIQUESCELL DISRUPTION
MECHANICAL NON-MECHANICAL
Homogenization in a bead mill
Sonication
French Press
PHYSICAL CHEMICAL ENZYMATIC
Osmotic Shock
Freeze-Thaw
Desiccation
Detergents
Chelating agents
Solvents
Lytic enzymes
APPLICATIONS OF CELL DISRUPTION TECHNIQUES
Extraction of intracellular content from the cells.
Robust method for the downstream process Easy to use Less expensive No adverse affect on the properties of the
enzyme
-Keeping in view of the above, present investigation was carried to study the production of -galactosidase from both yeast as well as fungal cells. Moreover, various disruption techniques were studied for the extraction of intracellular enzyme from yeast cell.
MATERIALS & METHODS
ISOLATION AND PROCUREMENT OF
MICROBIAL CULTURES
-Whey samples were collected from different states of India (Punjab, Haryana, Madhya Pradesh, and Bihar).
-The screening of isolated yeast strains were carried out on the basis of their β-galactosidase activities.
-Further, the novel yeast isolate Kluyveromyces marxianus, WIG2, was used for the further studies.
ISOLATION OF YEAST STRAIN FOR THE PRODUCTION OF β-GALACTOSIDASE
PROCUREMENT OF FUNGAL CULTURES
Microbial Culture Centres
National Chemical Laboratory, Pune (India)
Aureobasidium pullulans
NCIM 1050
Aspergillus niger
NCIM 616
Institute of Microbial Technology, Chandigarh
(India)
Aspergillus flavus MTCC
9349
Aspergillus niger
NCIM 616
PHYSIO-CHEMICAL ANALYSIS OF WHEY
pH Lactose concentration Total protein Fat Solid content
INOCULUM PREPARATION OF YEAST CULTURE
Maintenance medium composition (w/v) Malt extract (0.3%), Yeast extract (0.3%), Peptone (0.5%) and Glucose (1.0%).
Incubated at 30°C for 20 h.
Fungal Spores were grown on PDA.
10 ml of distilled water added to the fully grown spores.
This spore suspension was used as an inoculum.
INOCULUM PREPARATION OF FUNGAL CULTURE
PRODUCTION OF β-GALACTOSIDASE FROM YEAST CELLS
Fermentation media: Whey was supplemented with the following components
Components Concentration Corn Steep Liquor 1.7%
Yeast Extract 0.39%
Magnesium Sulphate 0.05%
Fermentation Parameters
Parameters ValuespH 5.2
Temperature 30°C
Incubation Time 20 h
PRODUCTION OF β-GALACTOSIDASE FROM FUNGAL CELLS
Fermentation media: Whey was supplemented with 0.5% yeast extract.
Fermentation Parameters
Parameters ValuespH 4.5
Temperature 28°C
Incubation Time 7-8 days
Spore Suspension 2%
ENZYME ASSAY FROM YEAST CELLS
1 ml broth containing yeast cells
Centrifuge (5000 rpm x 10 min at 4 oC)
Washed twice with phosphate buffer (0.1M, pH 7.0)
0.1 ml of diluted cell suspension + 0.9 ml of Z- buffer + 50μl of Chloroform + 20μl of sodium dodecyl sulphate
Incubated at 30 °C for 10 min
0.2 ml of O-Nitrophenyl-β-D-galactopyranoside (ONPG)
Incubated at 30°C for 5 min
Reaction stopped by added 1 ml of Na2CO3 (0.5 M)
OD at 420 nmNote Z-buffer: Z-buffer was prepered by dissolved (g/l) Na2HPO4 . 7 H2O: 16.1; NaH2PO4. H2O:
5.5; KCl: 0.75; MgSO4. H2O: 0.246 One unit of enzyme activity is defined as one micromole of o-nitrophenol
liberated per min under standard assay conditionMiller, J. H. (1972) Experiments in Molecular Genetics, p. 352, Cold Spring Harbor, NY.
Cell Broth
Centrifuge (5000 rpm for 10 min)
0.2 ml supernatant +1 ml Ortho-Nitrophenyl β-D-Galactopyranoside (ONPG)
Incubation at 50°C for 5 min
Reaction stopped by addition of 1 ml of Na2CO3 (10%)
O.D. at 420 nm
ENZYME ASSAY FROM FUNGAL CELLS
K. Reczey, H. Stalbrand, B. Hahn-Hegerdal, F.Tijernal, “Mycelia-associated β-galactosidase activity in microbial pellets of Aspergillus and Penicillium strains,” Appl. Microbiol. Biotechnol., vol. 38, pp. 393-397, Dec. 1992.
CELL DISRUPTION TECHNIQUES
HOMOGENIZATION IN A BEAD MILL
Cell Broth
Centrifuge (5000 rpm for 10 min)
Pellet washed twice with phospahte buffer
Pellet suspended in phosphate buffer
Homogenization of cells in a bead beater for 80-90s using silica beads (0.5 mm dia.)
Centrifugation
Supernatant taken for enzyme assay
GRINDING WITH RIVER SAND
Cell Broth
Centrifuge (5000 rpm for 10 min)
Pellet washed twice with phospahte buffer
Pellet suspended in phosphate buffer
Grinding of the cell suspension in pestle and mortar with pre-treated river sand
Centrifugation
Supernatant taken for enzyme assay
SONICATION
Cell Broth
Centrifuge (5000 rpm for 10 min)
Pellet washed twice with phospahte buffer
Pellet suspended in phosphate buffer
Sonication of Cell suspension carried for 160 s with alternate periods of cooling
Centrifugation
Supernatant taken for enzyme assay
FREEZE-THAW
Cell Broth
Centrifuge (5000 rpm for 10 min)
Pellet washed twice with phospahte buffer
Pellet suspended in phosphate buffer
Alternate freezing and thawing of cell suspension at -18°C and 4°C, respectively
Centrifugation
Supernatant taken for enzyme assay
SDS-CHLOROFORM
Cell broth
Centrifuge (5000 rpm x 10 min at 4 oC)
Washed twice with phosphate buffer (0.1M, pH 7.0)
0.1 ml of diluted cell suspension + 0.9 ml of Z- buffer + Chloroform + sodium dodecyl sulphate
Incubated at 30 °C for 10 min
0.2 ml of O-Nitrophenyl-β-D-galactopyranoside (ONPG)
Incubated at 30°C for 5 min
Reaction stopped by added 1 ml of Na2CO3 (0.5 M)
OD at 420 nm
TOLUENE
Cell Broth
Centrifuge (5000 rpm for 10 min)
Pellet washed twice with phospahte buffer
Pellet suspended in phosphate buffer
Aliquot of cell suspension mixed with 1 ml of chilled toluene
Incubated at 30°C for 15 min
Centrifugation
Supernatant taken for enzyme assay
ISO-AMYL ALCOHOL
Cell Broth
Centrifuge (5000 rpm for 10 min)
Pellet washed twice with phospahte buffer
Pellet suspended in phosphate buffer
Aliquot of cell suspension mixed with 0.85 ml iso-amyl alcohol and volume made upto 5 ml with phosphate buffer
Incubated at 30°C for 15 min
Centrifugation
Supernatant taken for enzyme assay
ACETONE
Cell Broth
Centrifuge (5000 rpm for 10 min)
Pellet washed twice with phospahte buffer
Pellet suspended in phosphate buffer
1 ml cell suspension mixed with 5 ml acetone
Incubated at 30°C for 15 min
Centrifugation
Supernatant taken for enzyme assay
RESULTS & DISCUSSION
PHYSIO-CHEMICAL ANALYSIS OF WHEY
Parameters Results
Lactose 4.89±0.11% (w/v)
Total protein content 0.48±0.33% (w/v)
Total fat content 0.18±0.08% (w/v)
Total solid content 7.1±0.13% (w/v)
pH 6.15±0.06
CELL DISRUPTION OF YEAST CELLS
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Physical Disruption Techniques
Enzy
me
Acti
vity
(IU
/gD
W)
Effect of physical disruption techniques on the enzyme activity
0
500
1000
1500
2000
2500
3000
3500
Chemical Disruption Techniques
Enzy
me
Acti
vity
(IU
/gD
W)
Effect of chemical disruption techniques on the enzyme activity
0
500
1000
1500
2000
2500
3000
3500
Concentration of SDS-Chloroform (uL/mL)
En
zym
e A
ctivi
ty (
IU/g
DW
)
Effect of SDS-Chloroform concentration on the enzyme activity
1600
1800
2000
2200
2400
2600
2800
3000
0 5 10 15 20 25
Treatment Time (min)
Enzy
me
Acti
vity
(IU
/gD
W)
Effect of treatment time on the enzyme activity
ENZYME PRODUCTION USING FUNGAL CELLS
0
200
400
600
800
1000
1200
1400
1600
1800
2000
A.pullulans NCIM 1050 A. niger NCIM 616 A. oryzae NCIM 1212 A. flavus MTCC 9349
Fungal Strains
Enzy
me
Acti
vity
(IU
/L)
Screening of different fungal strains for β-galactosidase production
0
200
400
600
800
1000
1200
1400
1600
1800
2000
40 60 80 100 120 140 160 180 200
Incubation Time (h)
Enzy
me
Acti
vity
(IU
/L)
β-galactosidase production by Aureobasidium pullulans NCIM 1050 as a function of incubation time
The novel yeast isolate Kluyveromyces marxianus was found to be efficient β-galactosidase producer culture.
Among the various disruption techniques used for the extraction of the enzyme from the yeast cells, SDS-Chloroform was observed as the best method for extraction with an enzyme activity of 2886 IU/gDW.
The concentration of SDS:Chloroform (200:500 µL/mL) at treatment time of 10 min was found to be the optimum condition for cell disruption.
CONCLUSIONS
Contd.In case of the fungal strains, Aureobasidium pullulans NCIM 1050 showed the maximum β-galactosidase activity (1700 IU/L).
The maximum activity was observed after the incubation period of 168 h.
ACKNOWLEDGEMENTS
Council for Scientific and Industrial Research, New Delhi, India
Sant Longowal Institute of Engineering & Technology, Longowal, India.
World Academy of Science, Engineering and Technology, USA
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