Production and estimation of alkaline protease by immobilized Bacillus licheniformis isolated from poultry farm soil of 24 Parganas and its reusability

Abstract

Microbial alkaline protease has become an important industrial and commercial biotech product in the recent years and exerts major applications in food, textile, detergent, and pharmaceutical industries. By immobilization of microbes in different entrapment matrices, the enzyme produced can be more stable, pure, continuous, and can be reused which in turn modulates the enzyme production in an economical manner. There have been reports in support of calcium alginate and corn cab as excellent matrices for immobilization of Bacillus subtilis and Bacillus licheniformis, respectively. This study has been carried out using calcium alginate, κ-carrageenan, agar-agar, polyacrylamide gel, and gelatin which emphasizes not only on enzyme activity of immobilized whole cells by different entrapment matrices but also on their efficiency with respect to their reusability as first attempt. Gelatin was found to be the best matrix among all with highest enzyme activity (517 U/ml) at 24 h incubation point and also showed efficiency when reused.

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2Journal of Advanced Pharmaceutical Technology & Research | Jan-Mar 2015 | Vol 6 | Issue 1
Production and estimation of alkaline protease by
immobilized Bacillus licheniformis isolated from
poultry farm soil of 24 Parganas and its reusability
abstract
Microbial alkaline protease has become an important industrial and commercial biotech
product in the recent years and exerts major applications in food, textile, detergent,
and pharmaceutical industries. By immobilization of microbes in different entrapment
matrices, the enzyme produced can be more stable, pure, continuous, and can be
reused which in turn modulates the enzyme production in an economical manner. There
have been reports in support of calcium alginate and corn cab as excellent matrices for
immobilization of Bacillus subtilis and Bacillus licheniformis, respectively. This study has
been carried out using calcium alginate, κ‑carrageenan, agar‑agar, polyacrylamide gel,
and gelatin which emphasizes not only on enzyme activity of immobilized whole cells by
different entrapment matrices but also on their efficiency with respect to their reusability
as first attempt. Gelatin was found to be the best matrix among all with highest enzyme
activity (517 U/ml) at 24 h incubation point and also showed efficiency when reused.
Key words: Bacillus licheniformis, calcium alginate, cell immobilization, entrapment
matrices, gelatin, microbial alkaline protease
Shamba Chatterjee
Department of Biotechnology,
Haldia Institute of Technology,
West Bengal University of Technology,
West Bengal, India
J. Adv. Pharm. Technol. Res.
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DOI:
10.4103/2231-4040.150361
INTRODUCTION
Protease enzyme plays the pivotal role in the hydrolysis
of the peptide bonds that link amino acids to one another
while forming a polypeptide chain. Amidst all the dierent
types of proteases, alkaline protease is active from neutral
to alkaline pH and is of maximal industrial interest. From
the viewpoint of biotechnology, alkaline protease has its
importance in pharmaceutical and medicinal industry,
food industry, peptide synthesis in brewing and baking
industry, detergent industry, leather and textile industry,
and wastewater treatment.[1-3]
Micro-organisms were found to be an excellent source
for production of alkaline protease than plants and
animals as they exhibit beer advantages such as broad
biochemical diversity, simplicity in genetic manipulation,
and feasibility in mass culture. Among many alkaline
protease producing microbes, bacteria of Bacillus genus
are active producers of extracellular alkaline proteases.
They are industrially very useful specically as commercial
alkaline protease producers because of their high pH and
thermal stability.[2,3]
Due to the wide range of industrial application of alkaline
protease, dierent measures have been taken to reduce
the production cost and increase its industrial utility.
Immobilization of enzymes and whole cells has been
an efficient tool for this purpose for years as it offers
numerous advantages over free enzyme like increase in
production rate, operational stability, possibility in reuse
of enzyme, and recovery of nal product free of enzyme
contamination.[1,2,4] Depending on the materials used for
entrapment, whole cells show modications in increased
mechanical, chemical, thermal resistance, and stability in
changes in pH.[2,5] There have been reports where dierent
Bacillus species like alkalophylic and thermophylic Bacillus
have been immobilized in various substances like calcium
alginate, polyacrylamide gel, gelatin, chitosan, corn cab,
Address for correspondence:
Dr. Shamba Chatterjee,
Department of Biotechnology, Haldia Institute of Technology,
Haldia ‑ 721 657, West Bengal, India.
E‑mail: shambac@gmail.com
original articlE
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Chatterjee: Production and estimation of protease by B. licheniformis
3
Journal of Advanced Pharmaceutical Technology & Research | Jan-Mar 2015 | Vol 6 | Issue 1
κ-carrageenan, etc.[2-4] Calcium alginate has been reported
as a beer matrix for immobilization of Bacillus subtilis
by Mahto and Bose[2] and corn cab for B. licheniformis as
reported by Maghsoodi et al.[3]
In this present study, we have reported the use of
dierent entrapment matrices such as calcium alginate,
κ-carrageenan, agar-agar, polyacrylamide gel, and gelatin
to immobilize B. licheniformis isolated from poultry farm
soil of 24 Parganas (West Bengal) for beer production of
alkaline protease and its reusability as rst comparative
approach. B. licheniformis is a mesophylic soil bacterium,
mainly found in the poultry farm soil,[6] and also in feathers
and breast of birds mainly hen, sparrow, and ducks.
Besides, the prospect of reusability of the enzyme has
also been investigated. Calcium alginate has been earlier
successfully used to immobilize rifamycin oxidase in the
form of Chryseobacterium species whole cells[5] and Candida
antarctica Lipase B.[7] κ-carrageenan is a naturally occurring
polysaccharide isolated from marine red algae.[8] It has been
in extensive use for immobilization worldwide. It has been
eectively employed in immobilization of Halobacterium
sp. JS1 cells for the production of haloalkaliphilic protease[9]
and lipase enzyme from Burkholderia cepacia.[8] Agar-agar
is a polysaccharide having strong gel forming ability and
shows no reactivity with protein.[10] It has been eectively
used for immobilization of thermostable α-amylase
extracted from soybean seeds[10] and pectinase isolated
from B. licheniformis KIBGE-IG21.[11] Polyacrylamide gels
have been in use for immobilization and entrapment
for decades. It has a special characteristic of having
all its charges embedded in the interior providing a
negatively charged three dimensional network for enzyme
entrapment.[12] It has also been used for immobilization
of Halobacterium sp. JS1 whole cells[9] as well as alkaline
phosphatase obtained from the Escherichia coli.[12]
Recent studies has shown that gelatin cross linked with
glutaraldehyde has been a good prospect for immobilizing
enzymes like hyperthermostable β-amylase isolated
from B. subtilis DJ5.[13] Earlier gelatin was used along
with polyacrylamide for entrapment and pure invertase
enzyme.[14]
MATERIALS AND METHODS
Materials
Media required for bacterial growth, Luria-Bertani (LB)
broth and LB agar as well as Casein were procured from
HiMedia. All the rest of the reagents and chemicals were
brought from SRL.
Sample collection and bacterial culture
Soil samples were collected from the poultry farm of
24 Parganas region (West Bengal), crushed into powder
and sieved to get rid of large pieces and sticks. 1% (w/v)
soil sample was prepared by mixing it properly in 9 ml of
double distilled autoclaved water. The soil solution was
then serially diluted. The last dilution (1 × 10 − 8) was chosen
for culture. 100 µl of the solution was used for preparing a
spread plate in LB agar. The plates were incubated at 37°C
for 24 h.
Screening and identication of isolates
For successful screening of the microbes, the colonies grown
on LB agar were then grown in two selective media, viz.
Brilliance™ Bacillus cereus agar and HiCrome Bacillus agar.[15]
Subsequently microscopic and biochemical analysis as par
Bergey’s manual[16] was done for further identication of
the isolated microbe.
Protease production and enzyme assay
Aer screening and identication of the microbes, a loop
full of colonies were transferred into 100 ml of LB broth in
aseptic condition and grown for 24 h. 10 ml of the 24 h old
LB broth culture was then transferred aseptically to a 250 ml
ask containing a complex media having 6% glucose, 2%
soybean meal, 0.04% CaCl2 and 0.02% MgCl2.[3] The culture
was incubated for 48 h at 37°C under shaking condition at
150 rpm. Incubation was followed by centrifugation of the
culture at 10,000 g for 10 min at 4°C. The cell pellet obtained
was washed thoroughly with sterile 2% KCl solution
followed by double distilled water. The cell mass was
then suspended in sterile 0.85% saline solution.[5] This cell
suspension was used later as inoculum for immobilization.
The supernatant was further used for protease assay by
following modied Folin and Ciocalteu method.[17] 0.65%
casein solution in 800 µl of 50 mm phosphate buer (pH 9)
was added as substrate to 200 µl of supernatant. The reaction
mixture was incubated at 75°C for 10 min followed by
addition of 5% trichloroacetic acid to stop the enzymatic
reaction. The reaction mixture was centrifuged at 10,000 g
for 15 min.[2] Absorbance was measured at 660 nm.[3] One
unit enzyme activity was dened as the amount of enzyme
that liberated 1 µg tyrosine/min under assay condition. The
enzyme activity was measured using tyrosine solutions as
standard.[18]
Immobilization of whole cells
Calcium alginate
The cell suspension was added to 2% sodium alginate
solution in 1:1 ratio and vortexed for thorough mixing.
The obtained solution was then added drop-wise to
30 ml of 0.2 M CaCl2 solution from a fair height. Colorless
transparent gel beads of 2–5 mm were formed. They were
allowed to stand for 30–60 min in the CaCl2 solution,
followed by thorough washing in double distilled water
and stored at −11°C for a maximum of 1-week.[5,7]
κ‑carrageenan
Ten milliliter of 4% (w/v) κ-carrageenan solution was
prepared by heating at around 80°C.[8] It was then cooled
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Chatterjee: Production and estimation of protease by B. licheniformis
4Journal of Advanced Pharmaceutical Technology & Research | Jan-Mar 2015 | Vol 6 | Issue 1
to around 45°C; 5 ml of cell suspension was added to it
and mixed thoroughly by vortexing. This mixture was then
added drop-wise to 2% of cold KCl solution for formations
of beads.[19] The beads were then washed thoroughly in
double distilled water stored in the refrigerator.
Agar‑agar
A 4% (w/v) agar-agar solution was prepared in 25 mM
sodium acetate buer (pH 5.5) by heating at a temperature
of 50°C.[10] Aer cooling to room temperature, 5 ml of
cell suspension was mixed thoroughly with 10 ml of
the agar-agar solution.[19] The solution was then poured
on a at boom petridish and allowed to solidify. Aer
solidification, the agar-agar block containing the cell
suspension immobilized in it was cut into small equal
sized cubes and washed properly with double distilled
water and stored in fresh sterile double distilled water at
4°C for further studies.
Polyacrylamide gel
2.85 g of acrylamide, 0.15 g of bisacrylamide 10 mg ammonium
phosphate and 1 ml of tetramethylethylenediamine was
added to 10 ml of 0.2M sterile phosphate buer (pH 7.0).
To this solution, prechilled cell suspension was mixed at the
ratio of 1:1. The solution was mixed thoroughly and poured
onto a petridish and allowed to polymerize. The rm gel
was then cut into equal sized small cubes and transferred
to 0.2 M sterile phosphate buer (pH 7.0) and kept in the
refrigerator for 1 h for curing. The cubes were then washed
properly and stored in double distilled water at 4°C.[20]
Gelatin
Ten milliliter of the solution was prepared by heating
10% (w/v) gelatin in 0.1 M phosphate buer (pH 6.9) at
50°C.[13] Cell suspension was added to the gelatin solution
at the ratio of 1:2, mixed and poured onto petridish
followed by overlaying it with 10 ml of 5% glutaraldehyde
and le for solidication at room temperature.[19] The gel
was then cut into small equal sized cubes and washed
thoroughly with double distilled water to remove the excess
glutaraldehyde.[20] The cubes were then stored in the buer
at 4°C for future use.
Enzyme production and assay by immobilized whole cells
Instead of LB broth immobilized whole cells were
added to 50 ml of complex media having 6% glucose,
2% soybean meal, 0.04% CaCl2 and 0.02% MgCl2 and
incubated in shaking condition at 150 rpm for 48 h at
room temperature. Rest of the extraction and enzyme
assay procedure has already been discussed above. To
study the reusability of immobilized whole cells, aer
incubation, the cell beads were washed and added to
fresh complex media.
Statistical analysis
All the data obtained regarding the assay of enzyme
production, and the reusability of immobilized whole cells
are conrmed by carrying out independent experiments
in triplicate and presented as mean ± standard error of the
mean.[21]
RESULTS AND DISCUSSION
Screening and identication of the soil isolates
The soil isolates grown on LB agar were grown on
Brilliance™ B. cereus agar and HiCrome Bacillus agar. Gram
staining [Figure 1] and other biochemical assays [Figure 2]
according to Bergey’s manual[16] was used in identifying
the soil isolate, namely B. licheniformis [Table 1] which was
at par with Němečková et al.[15] and Vigneshwaran et al.,[6]
respectively.
Production of alkaline protease by the immobilized
whole cells
Immobilization of B. licheniformis was done with various
entrapment matrices like calcium alginate, κ-carrageenan,
agar-agar, polyacrylamide gel and gelatin for production
of alkaline protease [Figure 3] with the sole interest to nd
out the best entrapment matrices with respect to enzyme
activity as well as its reusability. Experimental studies reveal
that there was a gradual increase in enzyme production with
the immobilized whole cells which was at its maximum at
24 h incubation point. Comparing the enzyme production
with the free cells, the yield of enzyme was more in case
of immobilized cells of all the matrices. Excess incubation
showed a decline in enzyme production [Figure 4]. When
the same cells entrapped in matrices were washed and
replenished with fresh media, a rise in enzyme production
was observed. However, when the same approach was
taken for free cells reverse results were obtained. This
dierence in results unveils the fact that immobilized whole
cells are highly suitable for reuse [Figure 5]. Nevertheless,
Figure 1: Isolation of Bacillus licheniformis from soil and its Gram staining
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Chatterjee: Production and estimation of protease by B. licheniformis
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Journal of Advanced Pharmaceutical Technology & Research | Jan-Mar 2015 | Vol 6 | Issue 1
Table 1: Microscopic and biochemical assays
for identication of soil isolates
Identifying tests and
biochemical assays
Observations
Growth on Brilliance™
Bacillus cereus agar
Negative
Growth on HiCrome
Bacillus agar
Positive-middle irregular or creeping
yellow colonies with surroundings
slightly changed to yellow
Gram stain Positive
Endospore stain Positive
Litmus milk reaction Peptonization
Carbohydrate fermentation
with lactose, sucrose, and
dextrose
All shows acid production with gas
Nitrate reduction Positive
Indole production Positive
Methyl red test Negative
Voges proskauer test Positive
Citrate utilization Positive
Catalase activity Positive
Starch Hydrolysis Positive
Casein hydrolysis Positive
Urease hydrolysis Positive
Figure 3: Immobilization of Bacillus licheniformiss whole cells. (a) Cell Suspension. (b) Formation of calcium alginate cell beads. (c) Cell
immobilizedinκ-carrageenan.(d)Cellimmobilizedinagar-agar.(e)Immobilizationofcellsinpolyacrylamidegel.(f)Formationofgelatin
cell beads
d
c
b
f
a
e
Figure 2:Biochemicalassays.(a)Nitratereductiontest.(b)Indole
productiontest.(c)Methylredtest.(d)Citrateutilizationtest
d
c
b
a
the production of the enzyme by reused immobilized
whole cells aer 24 h incubation was less than the fresh
immobilized whole cells. With respect to the production
of alkaline protease and reusability of immobilized whole
cells, gelatin manifested beer immobilizing ability than
calcium alginate which has already been established as
a beer entrapment matrix for Bacillus sp. by dierent
research groups.[1,2,4,5]
CONCLUSION
The present study emphasizes on the production of alkaline
protease by immobilized cells using various entrapment
matrices like calcium alginate, κ-carrageenan, agar-agar,
polyacrylamide gel, and gelatin. It also focuses on the degree
of reusability of the immobilized cells compared to the free
cells. The experimental data obtained indicate gelatin as a
promising matrix for immobilization of B. licheniformis and
optimum production of alkaline protease and its reusability
when compared to calcium alginate. κ-carrageenan
exhibited moderate ability, and agar-agar showed least
ability towards immobilization of B. licheniformis and
production of alkaline protease.
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Chatterjee: Production and estimation of protease by B. licheniformis
6Journal of Advanced Pharmaceutical Technology & Research | Jan-Mar 2015 | Vol 6 | Issue 1
Figure 4:Timecourseproleofproductionofalkalineproteaseby
Bacillus licheniformis immobilized in different entrapment matrices
Figure 5: Decrease in production of alkaline protease of reused
immobilized Bacillus licheniformis whole cells
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How to cite this article: Chaerjee S. Production and estimation
of alkaline protease by immobilized Bacillus licheniformis isolated
from poultry farm soil of 24 Parganas and its reusability. J Adv
Pharm Technol Res 2015;6:2-6.
Source of Support: The authors are grateful to the home institute
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Conict of Interest: Nil.
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