International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
Fermentative Production of Bacillus Protease using Vegetable
Waste as Substrate
1
Jayant P.Parpalliwar, Dr. I.D.Patil2, Aaisha Parin Sayyad3
Assistant Professor1, Professor2, Assistant Professor3
Department of Biotechnology,
Shram Sadhana Bombay Trust College of Engineering & Technology Bambhori, Jalgaon - 425-001,
Maharashtra, India.
Abstract: Protease is one of the most important
industrial enzymes due to having some
biotechnological interests It account for about 60%
of the total worldwide sale of enzymes. Microbial
proteases were produced from high yielding strain
Bacillus subtilis2724 by using vegetable waste
especially vegetable waste as a substrate in
submerged fermentation. Protein and carbohydrate
estimation techniques were used for measuring their
quantity present in the media. Thefermentation
profile like pH (7),temperature (37°C),inoculum size
(3mL/100mL media),incubation time (48 hrs).Glucose
as a carbon source, peptone as a nitrogen source and
casein as a protein source were used for the
optimization of media. The recovery of crude enzyme
done by centrifugation at 10,000 rpm for 10 min.
Qualitative analysis done by HPLC. Ammonium
sulphate precipitation method was used for the
recovery of protease from supernatant. The main
purpose of the project was to use the agro-waste
efficiently for the production of protease. Purified
protease has the potential application of distaining
and degradation of gelatinous coating of x-ray films.
Keywords: Bacillus subtilis2724, vegetable waste,
protease, submerged fermentation, HPLC.
INTRODUCTION:
Any enzyme that catalyses the splitting of proteins
into smaller peptide fractions and amino acids by a
process known as proteolysis. An enzyme that
hydrolyses the peptide bond is called protease. It
works without consuming energy because peptide
bond hydrolysis is exothermic. This reaction releases
~ 2 kcal/mol when the bond breaks. The resonance
structure of protease gives it to partial double bond
characteristics makes the bond kinetically stable
although thermodynamically unstable. Proteases
possess some characteristics of biotechnological
interest due to which these have become the most
important industrial enzymes. Alkaline proteases
possess considerable industrial potential due to their
biochemical diversity and wide applications in
tannery and food industries, medicinal formulations,
detergents and processes like waste treatment, silver
recovery and resolution of amino acid mixtures. In
leather industries alkaline proteases are exhibiting a
ISSN: 2231-5381
prominent role in unharing and bating processes.
Protease catalysis of peptide bonds (proteolysis).
Proteases (peptidyl-peptide hydrolases) are a group of
enzymes (also known as peptidases, proteinases or
proteolytic enzymes) that hydrolyse (break down) a
variety of proteins via the addition of water across
peptide bonds (i.e., bonds that join two adjacent
amino acids to form a polypeptide) and catalyse
peptide synthesis in organic solvents and in solvents
with low water content. Proteases are one of the most
important classes of enzymes, occupying a major
share of 60% of total enzyme market. This biocatalyst
hydrolyses peptide bonds in proteins. All proteases
are heat resistant Proteases play a crucial role in
many physiological and patho physiological
processes. Microbial proteases account for
approximately 40% of the total worldwide enzyme
sales. Microbial proteases are preferred to the
enzymes from plant and animal sources, since they
possess almost all the characteristics desired for
biotechnological applications. On the basis of their
acid-base behaviour, proteases are classified into
three categories i.e. acid, neutral and alkaline
proteases. The acid proteases are those which have
pH optimum in the range of 2.0-5.0 and these are
mainly fungal in origin. Acidic proteases have
application in meat tenderization, in the production of
fermented foods and also in acidic cleaning
compositions. Proteases have optimum pH in the
range of 7.0 or around are neutral and they are mainly
originated from plants however some bacteria and
fungi are also able to produce neutral proteases.
While those which work in the pH range of 8.0-11.0
areal kaline proteases. Some of the important alkaline
proteases are Solanain, Hurain and Proteolytic
enzymes of Bacillus and Streptomyces species.
Neutral and alkaline protease showed great potential
for application in the detergent and leather tanning
industries due to the increasing trend in developing
environment friendly technologies.
MATERIALS AND METHODS:
Collection of substrate:
Vegetable waste was used as a substrate/media for
carrying out the fermentation. Bulk amount of
vegetable waste was collected from the vegetable
market of Jalgaon city. The vegetables that consist of
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International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
high protein content and sugar content was selected
as substrate.
Washing and mixing of vegetable:
The collected vegetables was thoroughly washed with
distilled water 3-4 times and also cleaned with 70%
alcohol in order to remove the microbial load. These
washed vegetables were crushed in mixer grinder.
The juice filtered and sterilized in autoclave for
removing the contamination.
PROTEIN ESTIMATION:
Protein content was estimated by Lowry method with
bovine serum albumin as the Standard as per the
following protocol.
Sr.
Working
Distille
No.
stock(ml)
d water
1
Blank
(ml)
Alkaline
Cu
Solution
(ml)
02
05
Mix
thoroug
hly
And
allow
to stand
at room
tempera
ture.
for
10min.
(00)
F.C.
Reag
ent
(ml)
O.D.at
650nm
0.5
0.0050
0.5
0.0091
0.5
0.0492
0.5
0.1126
2
0.2
1.8
05
3
0.4
1.6
05
4
0.6
1.4
05
5
0.8
1.2
05
0.5
0.2159
6
1.0
1
05
0.5
0.3548
7
Test
05
0.7276
TABLE I
FERMENTATIVE PRODUCTION OF
PROTEASE:
Bacterial culture (Bacillus subtillis2724) was
collected and maintained by sub culturing on
selective growth media. Inoculum was prepared by
inoculating the culture in suitable nutrient broth for
future use. Vegetable waste juice was prepared and
used as a fermentation media. Media was optimized
by performing various estimation techniques. After
optimizing the media the innoculum was transferred
in the media under aseptic conditions in laboratory
batch fermenter and the process was carried at 37 0C
for 24 to 48 hrs for enzyme production with
continuous monitoring.
ENZYME PURIFICATION:
Enzyme purification was done by ammonium sulphate
precipitation by mixing the suspension in 40%
Ammonium Sulphate solution. The precipitated enzyme
was dissolved in 0.02M TrisHcl Buffer pH 8.5 for
purification using (High Performance Liquid
Chromatography).The Enzyme Characterization was
done by considering its properties like:
1. Degradation of gelatin from used X-ray
films.
2. Dehairing property.
3. Destaining property.
ENZYME PURIFICATION USING HPLC
(HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY)
ANALYSIS 1:
Carbohydrate Estimation
Sr.
N
o.
01
02
03
04
05
06
07
Carbohydrate was measured by dinitrosalisylic
(DNS) method as per the given protocol.
Workin Distille DNSA
The tubes Distill
O. D. at
g stock
d water Reagen covered
ed
540
(ml)
(ml)
t
with
water
nm
(ml)
marbles,
(ml)
are kept
02
01
03
0.00
Blank
in a
(00)
boiling
water
0.2
1.8
01
03
0.371
bath for
0.4
1.6
01
03
0.78
10 min,
0.6
1.4
01
03
1.16
cool to
0.8
1.2
01
03
1.58
room
1.0
1.0
01
03
2.25
temperat 03
Test
01
0.22
ure and
read the
extinction
at 540 nm
against
the blank
Chromatogram
TABLE II
ISSN: 2231-5381
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International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
Biochemistry,ISSN 0973-2691 Volume 6 Number 4
ANALYSIS 2:
(2010) pp. 493–504.
3.
O P Verma, PrashansaKumari, ShrutiShukla and Abha
Singh, Production of Alkaline Protease by Bacillus
subtilis(MTCC7312) using Submerged Fermentation
and Optimization of Process Parameters,European
Journal of Experimental Biology, 2011, 1 (3):124-129
4.
Aleksieva, P. and Peeva, L., (2000). Investigation of
acid
protinase
biosynthesis
bythe
fungus
HumicolaLutea120-5 in an airlift bioreactor. Enzyme
Microb.Technol., 26: 402-405.
5.
Boyer, H. W. and Carlton. B. C. (1968). Production of
two proteolytic enzymes by a transformable strain of
Bacillus subtilis. Arch. Biochem. Biophys.128:442-455.
6.
Chromatogram
FikretUyar,
IlknurPorsuk,
Göksel
Kizil,
EbruInceYilmaz,Optimal conditions for production of
RESULTS:
extracellular protease from newly isolated Bacillus
Amount of protein present in vegetable juice as
media (test sample) was 13.6 mg/ml, amount of
Carbohydrate present in vegetable juice media (test
sample) obtained was 3.8 mg/ml. Amount of
protease produced in vegetable juice after the
fermentation was 0.36μg/ml. in HPLC analysis. So
total concentration of protease present in 300mL
vegetable juice was 0.8gm and total amount of
Protease present in 3.4 Litre media was 9.06grams.
cereusstrain
7.
Journal
of
SadiaJaved,MunazzahMeraj, ShaziaAnwer Bukhari1,
RaoIrfan, and SaqibMahmood,Hyper-production of
Alkaline Protease by Mutagenic Treatment of Bacillus
subtilis M-9 using Agroindustrial Wastes in Submerged
Fermentation,Microbial& Biochemical Technology.
8.
K.
Krishnaveni,
Balakumaran,
CONCLUSION:
It was concluded that after inoculating the Bacillus
subtilis in a natural media that is in vegetable juice
the fermentation is carried out and protease was
successfully produced, means the means the media
used for fermentation has provided all the favourable
condition for the growth of bacteria to produce
desired product that is protease. So from this study it
was concluded that the process is economically
feasible because the vegetable waste are used as a
fermentation media which is optimized by carrying at
various kinds of parameter that satisfies all the
conditions for microbial growth (Bacillus subtilis) to
produce protease enzyme.
CA15.EurAsian
BioSciencesEurAsia J BioSci 5, 1-9 (2011).
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S.
J.
Mukeshkumar,
Ramesh
Kalaichelvan.Production
and
and
M.
D.
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Baby Joseph, SankarganeshPalaniyandi,Determination
of Alkaline Protease Production InSerratiaMarcescens
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Optimization
Of
Production
Parameters
And
Purification Of The Enzyme.
10. R. Gupta, Q. K. Beg, S. Khan, and B. Chauhan, “An
overview on fermentation, downstream processing and
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