β-galactosidase (β-D-galactoside galacto hydrolase, E.C.3.2.1.23, trivially lactate) known primarily as a hydrolase enzyme. β-galactosidase generally hydrolysis of lactose into glucose and galactose. β-galactosidase Lactose → Galactose + Glucose

1. PRODUCTION, PURIFICATION AND
CHARACTERIZATION OF β-GALACTOSIDASE
ENZYME FROM SOIL MICROBES
ANKIT KUMAR PATI
1660027
Dr. Mahesh M &
PURUSHOTHAMA D

2. Introduction
β-galactosidase enzyme is used in dairy
industries for improvisation of technological
and sensory characteristics of foods via
increasing solubility of sugars, creating
specially designed foods for lactose
intolerant people(reduction of lactose
content) because of its lactose-hydrolyzing
capabilities
β-galactosidase (β-D-galactoside
galacto hydrolase, E.C.3.2.1.23, trivially
lactate) known primarily as a hydrolase
enzyme. β-galactosidase generally
hydrolysis of lactose into glucose and
galactose.
β-galactosidase
Lactose → Galactose + Glucose
Based on the review β-galactosidase has a wide application & there has a lot of work been done in the production of β-
galactosidase from microbes. It is also available commercially but increasing the production to reach the present needs
always will be challenging, for retrieving we made an small attempt by production of the enzyme from soil microbes.
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3. β-galactosidase enzyme is generally extracted from various different sources like microbes yeast (intracellular
enzyme), bacteria , Fungi (extracellular enzyme), Plant sources like almonds, apples and apricots. Industrial &
commercial applicable beta β-galactosidase enzymes is preferably extracted from Aspergillus and Kluyveromyces
.Various bacterial sources are preferred for extraction of beta β-galactosidase such as Aspergillus, E.coli, Lactic
acid bacteria (LAB) and many new bacterial sources come up every now and then and accepted as the best
source both economically and industrially as it can comparatively produce more yield in the same required time
frame than that of other available sources.
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4. Why this & why from soil ?
It’s pretty commom for you to ask that...

5. What’s the scope of this work ??
The ease in production and increased yield of beta β-
galactosidase from any soil microbe can help the dairy
industries to economically produce lactose free milk and by-
products with cutting down the cost of the products. The
comparison of various research done, in progress to this will
also open up new observations, limitations, and scope for
further studies on beta β-galactosidase.
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6. The 4 phase process
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Confirmatory phase
Production and
Purification phase
Experimental phase
The Isolation phase

7. Roadmap
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1 3 5
4
2
Isolation & identification of a
microbe producing the β-
galactosidase enz yme from soil
samples.
Optimiz e the chemical parameters
i.e. carbon & nitrogen sources &
concentration to enhance the
enz yme activity.
Purification of B-gal
from cell free crude
extract.
Optimiz ation of pH and
Temperature.
Production of the β-
galactosidase enz yme
under optimal
conditions.

8. Now results of each phase is
systematically described and the reasons &
possible errors are also highlighted.
RESULTS
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9. Selection of Media and Strain:
Various soil samples are
experimented
in different media to select the
one - Lactose Media & the B1
Strain from the soil sample
collected from cubbon park,
Banglore.
Isolation Phase
O-NPG assay
The ON fits right into the active-site of β-galactosidase enzyme
and cleaves it producing galactose and ONP.The ONP
produces a yellow color after cleavage that can be measured
using the Spectrophotometer.
ONPG
β-galactosidase → Galactose + ONP
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10. Isolation Phase
10
Figure: Sub-culturing Plate
Figure: Gram staining slide
Figure: B1 strain in Lactose media

11. Experimental phase
Biochemical Tests: Various tests are
conducted to characterize the bacteria &
standardise the strain.
Incubation Time: The Strain B1 confirmed
Bacillus Sp. showed promising observable
values of enzyme activity standing at peak in
72 h incubation time, hence selected for
further optimisation.
pH & Temperature
Optimization:The various physical
parameters like pH and temperature
were experimented and optimized to
check for enzyme activity peak and
the best suitable pH 10 and
temperature 35℃ for the production
of β-galactosidase enzyme from the
selected stain B1 was optimised.
Chemical Parameters:The
chemical parameters plays an
important role in the enzyme
production.Different sources and its
concentrations optimization of this
can give the best yield during the
production of β-galactosidase
enzyme & fro the B1 the best
optimisation was set for
Carbon: Maltose (1.55%)
Nitrogen: Gelatin (1.5%)
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12. A Check stop before next phase
The Check post stop is just to confirm the byfar procedure
and the possible errors and move to the production phase
with an minimum error percentage by comparison of pre
existing datas and our objectives and calculations as
expected beforehand.
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13. 13

14. Production and Purification
After successfully isolation of a microbe that can produce β-galactosidase enzyme from the soil sample collected
(Cubbon Park Wasteland), screening and confirming through regression biochemical tests, optimizing the
different physical, chemical parameters & incubation period and calculating the enzyme activity of the strain at
each step just to be sure its the suitable organism to move forward to produce and purify the β-galactosidase
enzyme.

15. The purification step is systematically proceeded
• SALT PRECIPITATION,
• DIALYSIS,
• ION EXCHANGE CHROMATOGRAPHY,
• GEL FILTERATION
The SDS PAGE was perform to check for the Mw of the purified
protein and the band was formed somewhere between 115-118kDa,
which is approx. the molecular weight of the protein.
At each Step O-NPG essay was performed to check for presence of the enzyme.
The enzyme activity was also calculated and noted after each succesive steps.

16. Tablular data
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The above tabulation shows the enzyme activity of the each step from isolation to purification, which is the peak
compared to the previous researches. Hence, we can conclude that the thesis procceded as expected with a minimum
error percentage giving us promising results and future research scopes.
Samples Enzyme activity
B1 Strain (Bacillus Sp.) 0.633
pH optimization (10) 0.925
Temperature optimization
(35℃)
0.936
Carbon source (Maltose) 1.00
Carbon Concentration (1.5%) 1.00
Nitrogen Source (Gelatin) 1.04
Nitrogen Concentration (1.5%) 1.046
Crude 1.057
Salt 0.987
Dialysis 0.75
IE 5 0.626
GF 4 0.420
**NOTE: The samples were screened time after
time and the strains selected went through
rigorous biochemical and other tests. Standard
protein estimation, protein concentration,
enzyme activity, specific activity was noted after
each step. The observed results were cross
checked and double confirmed before
processing further. The SDS PAGE run shows
the band in the required ran

17. Conclusion
Finally, summarizing the thesis B1 Strain of Bacillus Sp. Isolated from Cubbon Park wasteland soil, Bangalore was
tested and optimized with pH10, 1.5% of Maltose & Gelatin as carbon and nitrogen source yielded the highest
enzyme activity of 1.057 after the growth period with the incubation period of 72 h at 35℃ in a shaking
incubator in the production phase.
In purification phase the enzyme activity tends to decrease from crude to the gel filtration observations. And each step
the selection was narrowed down to the best and maximized yield. With the enzyme activity as of the following :
Crude 1.057
Salt 0.987
Dialysis 0.75
IE 5 0.626
GF 4 0.420
The SDS PAGE run, after proper staining and destaining procedure the
run showed the bands at around in a range of 115-118kDa, which is the
appropriate Molecular Weight of the protein according to the thesis and
the pre mentioned data.

18. Future Work
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The work was done to its best as per my knowledge but there might be some limitations and errors which
might have occurred. There are some some new values findings which can be worked upon and explored a
little more. Other strains isolated apart from the selected can be experimented with optimization of
different parameters and it might give a result close to the observed data(s). The effects of different metal
ions is what the next work can be optimized to check for more increase in the yield of the β-galactosidase
enzyme production.
Several Cell disruption techniques, Effects of different Growth media, Optimization of mixed buffers, etc..
are few topic now open to work on after the successful completion of this thesis work.
Time limitations bounded us to wind up the work early as possible else few more tests and areas of work
could have been possible to compile in the thesis.
This thesis is still all the hard work that have been done, wide knowledge application, efforts of myself, my
supervisors & the people involved.

19. Credits
Special thanks to all including my supervisior and co-supervisior for
their guidance throughout.
Besides my supervisor, my special thanks goes to my fellow research students
for their help regarding these project aspects. I am very much thankful to Dr.
SRINIVAS PATNAIK, DEAN and Dr. VAISHAKHA RAINA,
ASSOCIATE DEAN Academics, of School of Biotechnology and authorities
of KSBT, KIIT, BHUBANESWAR for their co-operation. My generic
ample thanks also goes to my mentor Dr. RAHUL MODAK for his support
and timely help during my small to large requirements through out this
academic year.
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20. THANK YOU