International Journal of Biotechnology. Photon 114 (2015) 444-451 https://sites.google.com/site/photonfoundationorganization/home/international-journal-of-biotechnology Original Research Article. ISJN: 3352-7304: Impact Index: 4.23

International Journal of Biotechnology

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Study of Microbial Beta-Galactosidase Isolated from Fermented Millets
Mixture
Pooja Jagtap, Humera Bhattiwala and Annika Durve*
Department of Biotechnology, Birla College of Arts, Science and Commerce, Kalyan. Dist Thane, 421301, India
Article history:
Received: 31 March, 2015
Accepted: 07 April, 2015
Available online: 28 September, 2015
Keywords:
Beta: galactosidase, ONPG, X: gal, ONPG and X: gal
Corresponding Author:
Durve A.*
Associate Professor
Email: annikadurve ( at ) yahoo ( dot ) com
Bhattiwala H.
Lecturer
Jagtap P.
Scientific Officer

Abstract
In the present study microorganisms producing betagalactosidase enzyme were isolated from fermented millets mixture. Three bacterial isolate C2, C4, Y were found to be Gram positive bacilli, Gram negative cocobacili, Gram positive bacilli respectively while isolate SC2 was found to be yeast. These bacterial

isolates were identified using various biochemical tests
Growth study kinetics of microbial isolates was performed and the optimum pH and temperature was found. Beta-galactosidase enzyme was extracted and the effect of pH, temperature, substrate variation and incubation period on enzyme activity was studied. The activity of beta-galactosidase for all the microbial isolate increased with increase in temperature upto 37°C.
Michaelis-Menten plot and Lineweaver Burk plot were constructed to calculate the Km and Vmax values.
Immobilization of crude enzyme from the isolates was performed and enzyme activity using ONPG and lactose as a substrate was found. SDS PAGE analysis of the dialysate sample of bacterial isolate C2 and yeast isolate
SC2 was carried out. A band having molecular weight of approximately 70-80 kDa was detected.
Citation:
Jagtap P., Bhattiwala H., Durve A., 2015. Study of Microbial
Beta-Galactosidase Isolated from Fermented Millets Mixture.
Photon 114, 444-451
All Rights Reserved with Photon.
Photon Ignitor: ISJN33527304D785828092015

1. Introduction
Lactose is a diasacharide sugar derived from glucose and galactose which is found in milk.
Lactose makes up around 2-8% of milk (by weight) although the amount varies among species and individuals. Lactose intolerance primarily refers to a syndrome having one or more symptoms upon the consumption of food substances containing lactose.
Individuals may be lactose intolerant to varying degrees, depending on the severity of these symptoms. Lactose malabsorption refers to the physiological concomitant of lactase deficiency
(i.e., the body does not have sufficient lactase capacity to digest the amount of lactose ingested).
A medical condition with similar symptoms is fructose malabsorption.
Lactose intolerance is not an allergy because it is not an immune response, but rather a problem with
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digestion caused by lactase deficiency. Milk allergy is a separate condition, with distinct symptoms that occur when the presence of milk proteins trigger an immune reaction.
Lactose intolerance is usually caused by a deficiency of the enzyme called lactase (a protein that causes a chemical reaction to occur). A lactase substitute is available that can be taken to replace the lactase that your body cannot produce. The lactase substitute comes in liquid form (usually as drops) that can be taken before a meal or added to milk. Lactase enzyme, in the form of drops and capsules, are available from most health foods shops (Food Standards Agency, health-library articles, 2011).
The enzyme Beta Galactosidase (β-gal, EC
3.2.1.23) belongs to the group of hydrolases, forming two products from one substrate. The

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enzyme catalyzes the hydrolysis and transgalactosylation reaction of β-D-galactopyranosides.
These enzymes are found widespread in nature: microorganisms, plants, and animals. Microbial
Beta Galactosidases are of major interest due to their easy application in bioprocess technology.
The microbial enzymes have long been used for the hydrolysis of lactose, for increasing the digestibility of milk, or for improving the functional properties of dairy products. During the past decades, the Beta Galactosidase-catalyzed transgalactosylation has proved to be useful for structural and functional modification of food materials, medicines, and other biologically active compounds. Additionally, microbial Beta
Galactosidase are taken as a marker enzyme for coliform bacteria which are indicators of the faecal pollution of water (Nakayama and Amachi, 1999).
In nature, the substrate for Beta Galactosidase is lactose. Many chromogenic, fluorogenic, and luminogenic substrates that are specific for Beta
Galactosidase have been developed both substrate structures together with their respective reaction products are displayed (Nakayama and Amachi,
1999).
The aim of the present work was to isolate microbial sources capable of hydrolyzing a wide range of lactose. Bacteria and yeasts were isolated from fermented mixture of millets. The ability of these microorganisms to grow and produce lactase
(Beta Galactosidase) under different environmental conditions was studied. The microorganisms were capable of producing lactase in the presence of lactose. Various environmental conditions viz. effect of pH, temperature, enzyme concentration and substrate concentration were evaluated and the optimum conditions for the production of lactase were studied.
2. Material and Methods
Millets which included jowar (Sorghum), ragi
(Eleuisine coracana), bajra (Pennisetum glaucum) was purchased from local market. All the chemicals, reagents and media were prepared in distilled water. The prepared media was autoclaved at 15 lbs at 121.7˚C for 20 minutes.
2.1 Isolation, screening and identification of microorganisms producing β-Galactosidase
The millets were soaked in water and incubated at room temperature for 3 days. The millets were crushed with a mortar and pestle. The supernatant was streaked on MRS medium (De Man et al.,
1960) containing 2% lactose. The microorganisms obtained were screened for β-Galactosidase enzyme using Luriya Bertani agar plates infused with 50 µl X-Gal (5-bromo-4-chloro-3-indole-βPh ton

Dgalactopyranoside; 20mg/ml of DMSO) and 10 µl
IPTG (iso-propyl-thio-β-D-galactopyranoside) as an inducer to select the colonies showing lactose fermenting ability. The plates were then incubated at 37ºC for 24 to 48 hrs. The microrganisms were identified using Gram staining and biochemical characteristics (Harley and Prescott, 2002). The effect of pH, temperature and growth kinetics was studied. 2.2. Beta-galactosidase activity measurement using the ONPG standard assay
The enzyme extraction was carried out using toluene/acetone (1:9 v/v) solution and vortexed.
The supernatant was used as a crude enzyme source (Gheytanchi et al., 2010).
The activity assay was carried out by adding 200µL of enzyme extract and 900µL 50mM phosphate buffer (pH 6.5) along with 300µl of 0.132mM
ONPG solution. The reaction mixture was incubated for 15mins at 37°C. The reaction was stopped by adding 1000 µL 0.4 M Na2CO3 solution. The absorption of the yellow colour was measured photo metrically at 420 nm.
The calculation of the enzyme activity was carried out as described by (Nguyen et al., 2006). One unit
(U) of ONPG activity is defined as the amount of enzyme releasing 1 µ mol of ONP per minute under the specific assay conditions.
2.3 Determination of the ONP standard curve
Concentrations of ONP were prepared in sodium phosphate buffer, and the absorption was read at
420 nm, as described by Nguyen (2006). The standard curve was determined.
2.4 Lactose and glucose assay
Beta-galactosidase activity was determined by using lactose as substrate. 400µl of enzyme extract was added to 600µl of 10% and 15% lactose, separately and incubated for 15 minutes. When the mixture was cooled to room temperature, the release of D-glucose was determined photo metrically by using the standard GOD/POD assay
(Nguyen et al., 2006). One unit (U) of betagalactosidase activity is defined as the amount of enzyme releasing 1 µmol of D-glucose per minute under the described conditions.
Glucose concentrations ranging from 0.1- 1g/L were prepared in water and glucose concentration was determined by GOD-POD assay (ERBA GODPOD assay kit). The standard curve was determined. 445

2.5. Protein content
Protein content was found by Folin Lowry method
(Lowry, 1951) using bovine serum albumin as
Standard in range of (100µg-1000µg/ml).
2.6. Enzyme kinetics
The enzyme kinetics was studied for the crude enzymes by determining the optimum pH, temperature, enzyme concentration and substrate concentration. 2.6.1 Determination of pH optimum
To determine the optimum pH for the enzyme, the reaction mixture of enzyme and substrate was incubated in set of buffer of a pH ranging from 4 to
8.5 at 37ºC for 30 minutes. The activity of the betagalactosidase at different pH was determined. The
ONPG standard assay was performed using different pH (Nath et al., 2013; Natrajan et al.,
2012).
2.6.2 Determination of optimum temperature
The optimum temperature for enzyme activity was determined by varying the incubation temperature of the reaction mixture at 4ºC, 28ºC, 37ºC and 55ºC.
The activity of the beta-galactosidase at different temperatures was determined. The ONPG standard assay was performed using different incubation temperatures (Nath et al., 2013; Natrajan et al.,
2012).

2.9 Immobilization
The two isolates which showed maximum enzyme activity using ONPG substrate were immobilized in
Sodium alginate (Ayaz et al., 2011). This involved mixing of extracted enzyme from 100ml of overnight grown culture of the two isolates with
3% alginate. With the help of a syringe this was then dropped drop wise into 6% chilled CaCl2 that resulted in the formation of beads. The beads so formed were cured overnight at 4˚C. The beads were activated by incubating in sterile MRS medium containing 2 % lactose overnight. These beads were then introduced into 10ml of 4mg/ml
ONPG and standard protocol was used to find enzyme activity (Ayaz et al., 2011). Also beads were introduced in 10ml of 10% and 15% lactose, separately and release glucose was found by GODPOD assay (ERBA GOD-POD assay kit).
2.10 Application of Beta Galactosidase- Use of
Lactase in Bread Making
To 80g of flour, 332ml of yeast suspension (7.5g of baker’s yeast suspended in 95 ml of water) and 48 ml of a lactase (beta-galactosidase) enzyme 5ml of milk and sugar was added. The contents were mixed thoroughly with a spatula. After mixing to optimum consistency, the doughs were fermented at 30°C (Husain, 2010).
3. Results and Discussion

2.6.3 Determination of optimum substrate concentration To determine optimum substrate concentration, various concentration of substrate (ONPG) ranging from 0.016, 0.033, 0.049, 0.066, 0.082, 0.099,
0.132 mM, were used and the reactions were carried out at 37ºC for 30minutes. The activity of the beta-galactosidase at different concentration of substrate was determined. The ONPG standard assay was performed using different substrate concentration (Nath et al., 2013; Natrajan et al.,
2012).

3.1 Isolation, screening and identification of microorganisms producing β-Galactosidase
Beta-galctosidase producing colonies were selected by blue white selection method by growing them in the presence of IPTG and X-Gal
(Fig 3.1). Four different beta-galactosidase producing colonies were isolated. The four different colonies which were X-Gal positive were tested for utilization of ONPG substrate. All four colonies were able to break ONPG into ONP, which is a yellow colour compound. Gram staining and colony characters were studied.

2.7. Effect of incubation time on microbial cells to obtain crude enzyme
The effect of incubation period, on betagalactosidase production was investigated by cultivating the organism at different incubation time (24-96 hours). The organism was incubated, the beta-galactosidase activity was determined in supernatant. Figure 3.1: Blue colonies producing Beta-galctosidase

indicating

organisms

2.8 SDS-PAGE analysis
Beta galctosidase enzyme was partially purified by
Ammonium sulphate precipitation followed by dialysis. The enzyme was characterised using SDSPAGE and the molecular weight was determined.
Three bacterial isolate C2, C4, Y were found to be
Gram positive bacilli, Gram negative cocobacili,
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Gram positive bacilli respectively while isolate
SC2 was found to be yeast. The bacterial isolates were subjected to various biochemical tests and were identified according to Bergey’s manual of
Systematic Bacteriology (Volume I and II).
Standard Biochemical were Indole, Methyl Red,
Vogues Proskauer and Citrate Test (IMViC);
Triple Sugar Iron slant test and sugar fermentation containing 1% sugar solutions of sucrose, glucose, lactose, xylose, maltose, mannitol, mannose and fructose containing Andrade’s indicator with an inverted Durham’s tubes. These isolates were identified till species level using biochemical tests.
On the basis of the biochemical results, these isolates were found to belong to Bacilli sp.,
Lactobacillisp. and Vibrio sp.
The optimum pH for growth for bacterial isolate was found to be 7 while for yeast was pH 5. The optimum temperature for all microbial isolates was found to be 37°C. The microbial isolates could grow even in the presence of NaCl. The isolates could tolerate NaCl concentration upto 6.5% for isolate C2, 3 % for isolate C4 and Y and 12.5 % for
SC2.

enzyme activity was found to be 0.0266 enzyme activity /ml. At pH 7.5 for yeast isolate SC2 enzyme activity was found to be 0.0366 enzyme activity /ml (Table 3.1; Fig 3.2).
3.2.2 Determination of Temperature
The optimum temperature required for betagalactosidase activity was checked by incubating the assay reaction at different temperature viz. 4°C,
28°C, 37°C and 55°C. The activity of betagalactosidase for all the microbial bacterial isolate increased with increase in temperature upto 37°C
(Fig 3.3). However, further rise in reaction temperature caused loss of beta-galactosidase activity (Table 3.2).
Temp
(°C)
4
28
37
55

Enzyme activity/ml
C2
C4
0.0033
0.0011
0.0122
0.0044
0.03
0.0111
0.0111
0.0022

Y
0.0033
0.0111
0.0166
0.0044

SC2
0.0077
0.02
0.0355
0.01

Figure 3.3: Effect of temperature on Enzyme activity

3.2 Enzyme kinetics
Beta galactosidase was extracted by treating the culture broth with toluene: acetone (1:9) and the supernatant collected was used as crude enzyme.
The various parameters i.e. effect of pH, temperature, enzyme concentration and substrate concentrations for the crude enzyme were determined. 3.2.1 Determination of pH
The enzyme activity for C2 was found to be 0.029 enzyme activity/ml at pH 5; for C4 bacterial isolate, enzyme activity was found to be
0.0111enzyme activity/ml respectively. The elevation of pH beyond this caused rapid decrease in enzyme activity. At pH 7, for bacterial isolate Y pH 4
5
5.5
6
6.5
7
7.5
8
8.5
9

Enzyme activity/ml
C2
C4
0.027
0.0011
0.029
0.0022
0.0024
0.0066
0.02
0.0077
0.014
0.0111
0.016
0.0099
0.01
0.0088
0.0088
0.0055
0.0033
0.0022
0.0011
0.0022

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Y
0.0044
0.0044
0.0077
0.0111
0.0177
0.0266
0.0088
0.0033
0.0011
0.0011

SC2
0.015
0.019
0.033
0.026
0.019
0.010
0.007
0.005
0.004
0.001

3.2.3 Determination of Substrate variation
The effect of substrate concentration was checked on beta-galactosidase activity. Kinetic constants i.e.
Km and Vmax of the beta-galactosidase enzyme were determined by changing the ONPG substrate concentration. ONPG substrate concentrations in the assay medium were ranging from 0.0016mM to
0.0132mM. Michaelis-Menten plot and Lineweaver
Burk plot were constructed to calculate the Km and
Vmax values. Km and Vmax values for the four microbial isolate were found to be 0.0231, 0.0808,
0.0366, 0.0366, 0.0319 mM and 0.0244, 0.1009,
0.0368, 0.0328 U/mg protein respectively (Fig 3.4).
Figure 3.4: Effect of substrate variation on beta galactosidase enzyme using ONPG as substrate
(Michaelis-Menten graph).

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3.2.4 Effect of incubation time on microbial cells to obtain crude enzyme
According to the results taken at regular intervals, the maximum enzyme activity was obtained at 48th hour of incubation. Beyond this, the enzyme productivity was remains decreased and no further increase in production was observed (Fig. 3.5). It might be due to decrease in the nutrient availabitity in the medium, or catalobic repression of the enzyme. Figure 3.5: Effect of Incubation time on microbial cells to obtain crude enzyme

3.3.1 Lactose assay
Bacterial isolate C2 and yeast isolate SC2 showed highest enzyme activity using ONPG as a substrate so, this two isolate were used to find enzyme activity using lactose as a substrate. Lactose hydrolysis was done using 10% and 15% lactose and released glucose was estimated using god-pod assay (ERBA-God Pod assay kit).
The bacterial isolate C2 which showed highest enzyme activity using ONPG as substrate was used to find enzyme activity of beta-galactosidase using lactose as a substrate (10% and 15%) and comparative study was made using yeast isolate
SC2. Bacterial isolate C2 showed enzyme activity of 0.404µmole/minute and 0.809µmole/minute using 10% and 15% lactose respectively. Similarly, yeast isolate SC2 showed enzyme activity of
2.83µmole/minute and 3.23µmole/minute using
10% and 15% lactose respectively (Table 3.3).
Yeast isolate SC2 showed higher enzyme activity than bacterial isolate at both 10% and 15% lactose as substrate (Fig: 3.6 and Fig 3.7).

The optimum pH for the Lactobacillus acidophilus enzyme was found to be 6.5-7.0 (Akolkar et al.,
2005), pH 6.5 for Thermophile B1.2 (Osiriphun and Jaturapiree, 2010) and pH 7 for Bacillus
Sp.MPTK121 (Mukeshkumar et al., 2012).
The optimum temperature for the Lactobacillus acidophilus enzyme was found to be 50°C (Akolkar et al., 2005), 60°C for Thermophile B1.2
(Osiriphun and Jaturapiree, 2010) and 30°C
Bacillus Sp.MPTK121 (Mukeshkumar et al., 2012).

Table 3.3: Enzyme activity using lactose as a substrate
Isolate
Lactose %

10
15

Enzyme activity (µmole/minute)
C2
SC2
0.404
2.83
0.809
3.23

Figure 3.6: Lactose assay for the isolate C2 and SC2 isolate using 10% lactose

Using ONPG as a substrate Km value for the
Lactobacillus acidophilus enzyme was found to be
0.11mM (Akolkar et al., 2005), 28.85mM for
Thermophile B1.2 (Osiriphun and Jaturapiree,
2010) while Vmax value for the Lactobacillus acidophilus enzyme was found to be 4.94U/mg protein (Akolkar et al., 2005) and 8.38 U/mg protein for Thermophile B1.2 (Osiriphun and
Jaturapiree, 2010).
The maximum enzyme production of beta galactosidase from Bacillus Sp.MPTK 121 was obtained at 48th hour of incubation. Beyond this, the enzyme productivity remained constant and no further increase enzyme production was observed
(Mukeshkumar et al., 2012).

Figure 3.7: Lactose assay for the isolate C2 and SC2 isolate using 15% lactose

3.3 Partial Purification of beta-galactosidase
Enzyme precipitation was carried out by salting out with ammonium sulphate. Further partial purification of beta-galactosidase was carried out by the process of dialysis using 10kDa dialysis bag.

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3.4 Immobilization
Immobilized enzyme was used to find enzyme activity using ONPG (4mg/ml) and lactose (10% and 15%) as substrate. A comparative study was made of free and immobilized enzyme using lactose as substrate over time duration of 1 hour with 15minutes interval (Fig. 3.8).
Figure 3.8: (A) Immobilized beads having crude betagalactosidase from C2
(B) Immobilized beads having crude beta-galactosidase from SC2

A

yeast and bacterial isolate SC2 and C2 in Free State showed increased dissociated glucose as time increased, while in immobilized state the same enzyme showed increased dissociated glucose as time increased (Fig 3.10 and Fig 3.11). It was found that the immobilized enzyme from bacteria and yeast was more effective than compared to its free form.
Figure 3.10: Comparative study of immobilized and free crude beta-galactosidase from isolate C2 using 10% and
15% lactose

B

3.4.1 ONPG assay
Using ONPG as a substrate, immobilized crude beta-galactosidase from both C2 and SC2 isolate was used to find amount of dissociated ONP which is equal to optical density of reaction mixture at
420nm. O.D of reaction mixture was found at interval of 15 minutes till 60 minutes. At 60 minute both immobilized enzyme from C2 and SC2 isolate showed highest O.D (Fig 3.9). The immobilized yeast enzyme SC2 showed higher dissociated ONP than bacterial isolate C2 enzyme.

Figure 3.11: Immobilized and free crude betagalactosidase from isolate SC2 using 10% and 15% lactose Figure 3.9: ONPG assay using immobilised crude betagalactosidase from both C2 and SC2 isolate

3.4.2 Lactose assay
Using lactose (10% and 15%) as a substrate immobilized crude beta-galactosidase from both C2 and SC2 isolate was used to find amount of dissociated glucose which is equal to optical density of reaction mixture at 505nm. Comparative study of free and immobilized enzyme was done. It was found that crude beta-galactosidase from both
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3.5 Electrophoresis and Molecular Weight
Determination by SDS-PAGE
SDS Page analysis of the purified betagalactosidase from C2 and SC2 isolate was carried out. A molecular ladder was run along with the sample. The PAGE analysis showed a band having molecular weight of approximately70-90kDa for bacteria n yeast (Fig 3.12). References have show that the molecular weight of beta-galactosidase enzyme have a molecular weight of 70-75kDa
(Mukeshkumar et al., 2012; Osiriphun and
Jataurapiree, 2010).
3.6 Application- Use of beta-galactosidase in bread making The enzyme from both bacterial isolate C2 and yeast isolate SC2 was use in bread making. The dough containing enzyme and control dough

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having no enzyme was fermented and after 40 mins, the dough containing the enzyme was found to be softer than the control and the texture of the bread was found to be softer compared to the
Figure 3.12: SDS PAGE analysis of crude betagalactosidase enzyme from C2 bacterial isolate and
SC2 yeast isolate

to produce beta-galactosidase enzyme. The enzyme obtained from the bacteria was found to be more effective than the enzyme obtained from yeast. The bacterial isolates were able to grow in acidic environment and showed high enzyme activity at acidic pH which can be used at industrial scale.
These isolates on further strain improvement can be used for producing beta galactosidase commercially.
References
Akolkar S., Sajgure A., Leele S. 2006. β-galactosidase from lactobascillus acidophilus isolated from fermented ragi (Eluisene coracana).Indian journal of biotechnology.5, 184-188.
Ayaz N., Alnahdi H., Danial E. 2011. Immobilization and Properties of β-D-Galactosidase from Bacillus
Licheniformis E66. Journal of Applied Sciences
Research.7(12), 2448-2454

control. The enzyme obtained from the bacteria was found to be more effective than the enzyme obtained Figure 3.13: Dough containing enzyme (C2 and SC2) and no enzyme (control)

Ayaz N., Alnahdi H., Danial E. 2011. Immobilization and Properties of β-D-Galactosidase from Bacillus
Licheniformis E66. Journal of Applied Sciences
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De Man J.C., Rogosa M., Sharpe M.E. 1960. A medium for the cultivation of lactobacilli. J. Applied Bacteriol.
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Figure 3.14: Texture of bread

Husain Q. 2010. β Galactosidases and their potential applications: a review. Critical Reviews in
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Lowry O. H., Rosebrough J., Farr A. L., Randall R. J.
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Conclusion
In the present study microorganisms producing beta-galactosidase enzyme were isolated from fermented millets mixture. Three bacterial isolates and yeast were selected on the basis of their ability
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Nath A., Sarkar S., Maitra M., Bhattacharjee C.,
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Characterization of β-galactosidase Synthesized by

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