Extraction and Purification of β-galactosidase from yeast isolated from

curd sample
Vinaya Sara Jacob1, Jovis Jacob1, Saira Varghese1 and Dr. V.Mohanasrinivasan2,
Nivetha. A3
1M.Sc Student of Department of Applied Microbiology, School of Biosciences and
Technology, VIT University, Vellore, Tamil Nadu, India.
2Senior Assistant Professor, Department of Biomedical Sciences, School of Biosciences and
Technology, VIT University, Vellore, Tamil Nadu, India.
3Research Associate, Department of Biomedical Sciences, School of Biosciences and
Technology, VIT University, Vellore, Tamil Nadu, India.

ABSTRACT
Lactose intolerance is a condition in which individuals have symptoms (like abdominal
pain, bloating, diarrhoea, gas, and nausea) due to the decreased ability to digest the lactose
present in dairy products. Lactose intolerance is due to the lack of enzyme lactase (beta
galactosidase) in the small intestine, to break lactose into simpler sugars (monosaccharides like
glucose and galactose) through the breaking of a glycosidic bond. The main objective of this
study is to isolate and select some yeast strains from curd sample which produce β-
galactosidase and for the large scale production of the same and use it for the treatment of
lactose intolerant individuals. The samples were serially diluted and isolated on MRS agar
followed by the screening of the organisms using X-gal and IPTG. The green colour colonies
were primarily screened for β-galactosidase production by ONPG assay. Synthesis of β-
galactosidase by yeasts can either be intracellular or extracellular. Hence by centrifugation, the
culture was separated into pellet and supernatant and ONPG assay was done to see which forms
the yellow colour. The purification of the crude extract was done by ammonium sulphate
precipitation followed by gel permeation chromatography and SDS-PAGE. The protein
estimation was done by Bradford method.
INTRODUCTION
β-D-galactosidase otherwise called lactase is an enzyme or protein which catalyzes the
hydrolysis of lactose which is a fundamental and most prominent carbohydrate present in
majority of the dairy items. The lactose gets converted to monosaccharides glucose and
galactose to get ingested over the intestinal epithelium and has a potential centrality in the dairy
business. β-galactosidase additionally hydrolyzes the D-galactosyl buildups in polymers,
oligosaccharide and secondary metabolites. β-Galactosidases which are found in plants
(especially in almonds, peaches, apricots, apples), microorganisms (bacteria, fungi, yeasts),
and animal organs has been widely used for the hydrolysis of lactose because of the ease of
fermentation, good stability and high activity of the enzyme.
Lactose is a disaccharide made up of two monosaccharides: glucose and galactose which is the
primary and essential carbohydrate present in milk. Absorption of lactose involves lactase,
present in the brush border of small intestinal tract. Lactose intolerance is a state in which
individuals are unable to hydrolyze lactose, sugar present in dairy products such as milk, curd,
butter, cheese, yoghurt. Less production of β-galactosidase results in higher concentrations of
indigested lactose. Lactose intolerance or lactose malabsorption was found to be a major health
problem for more than 70% of the health problems in the world. The β-galactosidase enzyme
is located on the tips of the villi. The activity of this enzyme originates from the lactase
phlorizin hydrolase, within the intestinal mucosa. Lactose absorption takes place by lactose
hydrolysis to glucose and galactose. In the intestinal enterocytes, monosaccharides of glucose
and galactose are assimilated into the blood stream. Glucose absorbed initially is used as the
most important source of energy, while galactose acts as a glycolipid and glycoprotein
component. However with low β-galactosidase activity, lactose remains unabsorbed which
leads to intestinal discomfort.
Treating milk products with β-galactosidase offers a solution to this problem through pre
hydrolyzation of milk and milk-related products that reduce lactose. On a commercial scale,
bacterial and fungal lactase enzymes are being used to treat milk products. Medicines
containing β-galactosidase are also available, which are taken before consuming milk products.
These medicines possess fungal derived β-galactosidases, usually Aspergillus, that is stable at
low pH allowing for the proper functioning in stomach.
In food industries, lactose hydrolysis is not only preferred to produce lactose-free products but
also used to reduce the crystallization in ice creams and condensed milk, which occurs due to
high lactose concentration. Use of β-galactosidase in treating lactose not only improves the
texture but also makes the products more easily digestible. The end-products of lactose
hydrolysis (i.e., glucose and galactose) ferment more easily, thus reducing the overall time
required to achieve the preferred pH in various food items such as yogurts and cottage cheese.
Furthermore, it also reduces the need to add additional sweeteners, thus lowering the amount
of calories in the final product.

MATERIALS AND METHODS

Collection of Sample
The curd sample for the isolation of microorganism was bought from the local market in
Vellore and maintained in the laboratory of School of Biosciences and Technology, VIT which
was used throughout the work.
Isolation and Screening of β-galactosidase producing Organisms
For the isolation of organism from curd sample, 1ml of the sample was added to 100ml distilled
water and kept for shaking for 3hours, after which the serial dilution technique was performed
followed by plating on MRS agar (De Man, Rogosa and Sharpe agar) which consists of
1.0 % peptone, 1.0 % beef extract, 0.4 % yeast extract, 2.0 % glucose, 0.5 % sodium
acetate trihydrate, 0.1 % polysorbate 80 (also known as Tween 80), 0.2 % dipotassium
hydrogen phosphate, 0.2 % triammonium citrate, 0.02 % magnesium sulfate heptahydrate,
0.005 % manganese sulfate tetrahydrate, 1.0 % agar and pH adjusted to 6.2) using spread plate
technique and incubated at 37°C for 24hours. The screening of the organisms producing β-
galactosidase was done by streaking the isolated colonies on MRS agar with 50µl X-gal
(20mg/ml in DMSO) and 30µl IPTG (Isopropyl β-D-1-thiogalactopyranoside). Incubation was done
at 37°C for 24-72hours.

Characterization of the Isolated Organism

The isolated organisms with β-galactosidase activity was identified based on macroscopic
characteristics, microscopic examination and biochemical tests. Gram staining and LPCB
staining was done for microscopic identification. Biochemical tests (IMViC, Catalase and
Oxidase) was also performed.

Inoculum preparations for Enzyme Production

One loop full of master culture was transferred to the MRS broth. For effective production it
was kept in a shaker incubator at 37°C for 48hours.

Estimation of Enzyme activity (ONPG assay)

Presence of β-galactosidase has been detected using ONPG as a substrate. For this 5ml of the
culture was transferred aseptically to falcon tubes and centrifuged at 5000rpm for 20minutes
at -4°C. After centrifugation supernatant was transferred to a fresh tube without disturbing the
pellet. To both the pellet and the supernatant add 1ml Z buffer (10 mM KCl, 1 mM MgSO4,
60 mM Na2HPO4⋅ 7H2O, 40 mM NaH2PO4⋅ H2O, 50 mM β-mercaptoethanol), 100µl
chloroform and 50µl of TA (toluene: acetone in 1:9 ratio) and vortex for 5minutes. To both the
tubes add 500µl ONPG (4 mg/mL o-nitrophenyl-β-d-galactopyranoside in 0.1 M phosphate
buffer pH 7.0). The tubes were incubated at room temperature for 30 minutes. Yellow colour
was observed. To stop the reaction after sufficient yellow colour formation, Na2CO3 was added
(200µl). Then the optical density was recorded at 420 nm and 550nm using UV
spectrophotometer and Miller units were calculated (Units= 1000 x OD420 / volume(1ml)x
time(1min)x OD550)

Protein Precipitation using Ammonium Sulphate

Crude enzyme with β-galactosidase activity was subjected to precipitation by ammonium
sulfate with 70 % saturation under ice cooled conditions. The broth culture was centrifuged at
5000rpm at -4°C for 20minutes and the supernatant was transferred to a beaker and stirred on
mechanical shaker under ice cooled conditions. 3g of ammonium sulphate was added in
pinches to the culture. The culture was then centrifuged and the supernatant was taken. Again
3g of ammonium sulphate was added and centrifugation was performed. The precipitate
obtained after the addition (in pinches) of 12g of ammonium sulphate was centrifuged at 5000
rpm for 20 min at -4°C and was redissolved in a small volume of Z buffer.
Chromatographic Fractionation by Gel Permeation Chromatography
Gel permeation chromatography (GPC), sometimes referred to as gel filtration
chromatography (GFC) or size exclusion chromatography (SEC), entails the
chromatographic fractionation of macromolecules according to molecular size. In the early
development of this technique, cross-linked polydextran gels having varying pore sizes
(Sephadex) was used as the stationary phase. When swollen in water, sephadex gels form a
three-dimensional network that acts as a molecular sieve. When an aqueous solution of
macromolecules is allowed to move through a column containing the gel, a chromatographic
separation takes place; molecules of low molecular weight (size) (i.e., molecules having a small
hydrodynamic radius) are able to penetrate into the gel particle pores but large molecules are
excluded from the pores and pass directly through the column. Consequently, the largest
molecules elute first and the smallest last.
Estimation of molecular weight of protein by SDS-PAGE Electrophoresis
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a widely used
electrophoretic technique. In SDS-PAGE, proteins are separated in a polyacrylamide gel
based on their molecular weight. Proteins are amphoteric molecules, i.e. they have both
positive and negative charges. To make them move in a single direction, a uniform negative
charge is created on them. When the proteins are mixed with SDS, they acquire a net negative
charge.
SDS is a detergent having a negative charge, therefore it is an anionic detergent. SDS
denatures the native proteins by disturbing the non-covalent forces. The non-covalent forces
include hydrogen-bonding, hydrophobic and ionic interactions which are responsible for the
three-dimensional structure of a native protein. SDS also gives a uniform net negative charge
to the protein molecules. The denatured linear protein molecules are loaded onto the
polyacrylamide gel (PAGE) which is made by polymerizing the acrylamide monomers.
PAGE is prepared using acrylamide, bisacrylamide, TEMED, ammonium persulfate and Tris-
HCl buffer. PAGE has two phases: a stacking gel and a separating gel. Under an applied
electric field, the stacking gel concentrates the SDS-loaded linear protein molecules while the
separating gel separates the proteins on the basis of molecular weight. After the run, PAGE
gel is placed in a Coomassie Brilliant Blue R250 dye solution for staining for a few hours and
is de-stained to visualize the separated protein molecules as bands.
An intact SDS PAGE electrophoresis system should include: a tank, lid with power cables,
electrode assembly, cell buffer dam, casting stands, casting frames, combs(usually 10-well or
15-well), and glass plates (thickness 0.75mm or 1.0mm or 1.5mm). Stacking gel (acrylamide
7%) is poured on top of the separating gel (after solidification) and a gel comb is inserted in
the stacking gel. The acrylamide percentage in SDS PAGE gel depends on the size of the target
protein in the sample.
 For a 5 ml stacking gel:
H2O 2.975 ml
0.5 M Tris-HCl,
pH 6.8
1.25 ml 10% (w/v) SDS
0.05 ml Acrylamide/Bis-acrylamide (30%/0.8% w/v)
0.67 ml 10% (w/v) ammonium persulfate (AP)
0.05 ml TEMED 0.005 ml
 For a 10ml separating gel:
H2O
Acrylamide/Bis-acrylamide (30%/0.8% w/v)
1.5M Tris(pH=8.8)
10% (w/v)SDS
10% (w/v) ammonium persulfate (AP)
TEMED
 5X Sample buffer (loading buffer):
10% w/v SDS
10 mM Dithiothreitol, or beta-mercapto-ethanol
20 % v/v Glycerol
0.2 M Tris-HCl,
pH 6.8
0.05% w/v Bromophenolblue
 1x Running Buffer:
25 mM Tris-HCl
200 mM Glycine
0.1% (w/v) SDS

Protein Estimation by Bradford Protein Assay

The Bradford protein assay is used to measure the concentration of total protein in a sample.
The principle of this assay is that the binding of protein molecules to Coomassie dye under
acidic conditions results in a colour change from brown to blue. This method actually measures
the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes
to formation of the protein-dye complex. The Bradford reagent is prepared by dissolving 100
mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol. To this 100 ml 85% (w/v)
phosphoric acid was added. The solution was diluted to 1 litre when the dye had completely
dissolved, and filtered through Whatman paper just before use.

RESULTS AND DISCUSSION

Isolation and identification of β-galactosidase producing organisms

Organisms were isolated from curd sample. An isolated colony with cream-colored or off white
to grey, dull, smooth, soft and creamy or wrinkled or rough appearance was selected . The
isolated colony when streaked on MRS agar with X-gal and IPTG produced green colour
colonies which indicates the β-galactosidase activity. The green colour producing colonies
were further identified by microscopy. In gram staining, slightly oval or round colonies were
observed. In LPCB staining also round blue coloured colonies were observed. The biochemical
tests IMViC was negative for this organism.

Isolated colonies on MRS agar

 Gram Staining LPCB Staining

Biochemical Tests (IMViC)

ONPG Assay
When the ONPG assay was done for both the supernatant and the pellet, only the supernatant
showed yellow colour. The absorbance at 420nm and at 550nm was checked using a UV-
spectrophotometer. The OD at 420nm and 550nm was found as 0.463 and 0.095 respectively.
The miller units was calculated using the formula [1000 x OD420 / volume(1ml)x time(1min)x
OD550] and was found to be 4873.68
Ammonium Sulphate Precipitation
100ml of the overnight grown broth culture (at 37°C in a shaker incubator) was centrifuged at
5000rpm at -4°C for 20minutes and the supernatant was transferred to a sterile beaker. The
culture was kept for stirring on a mechanical stirrer for 1hour under ice cold conditions and
12g of ammonium sulphate was added in pinches (centrifugation was also done after the
addition of every 3g of ammonium sulphate and only the supernatant was taken). After stirring
for 1hour the culture was stored in the refrigerator and precipitates was formed after 24hours.
This precipitate was taken and 1ml of Z-buffer was added to it.

Falcon tubes with separated Mechanical stirring of the

supernatant and pellet supernatant

Gel permeation chromatography

After packing the sephadex beads in the column, freshly prepared Z buffer was eluted through
the column. The sample which was diluted with Z buffer was also added slowly to the column
and the eluent was collected in different eppendorf tubes (1.5ml in each tube). The absorbance
of the eluent collected (16 eluents) was recorded at 250nm .

Fractions OD at 250nm
1 0.716
2 0.793
3 0.695
4 0.616
5 0.560
6 0.637
7 0.585
8 0.681
9 0.928
10 0.677
11 0.988
12 1.193
Gel permeation
13 0.901
chromatography column
14 0.960 packed with sephadex beads
15 0.931
16 0.984
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