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