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Research scholar, 4Professor, Department of Microbiology, Andhra University, Visakhapatnam, Andhra Pradesh,
India
Abstract
Bacillus strains isolated from the salteren pond (Kakinada) were screened and identified for high alkaline protease activity. The
isolates which were positive on skim milk agar (1%) were selected as protease producing strains. Of the ten bacterial isolates
screened, isolate S-8 was observed as a potential haloalkaline protease producer and it was identified as Bacillus cereus strain S8
(MTCC NO: 11901) by 16S rRNA gene sequencing, phylogenetic tree analysis and by different biochemical tests. Protease production
was enhanced by optimizing the culture conditions. The nutritional factors such as carbon and nitrogen sources, NaCl and also
physical parameters like temperature, incubation time, pH, inoculum size were optimized for the maximum yield of protease. Studies
on the effect of different carbon and nitrogen sources revealed that maximum protease production was obtained in the medium
supplemented with Molasses,1%(w/v); Potassium nitrate, 0.75%(w/v); salt solution- 5%(v/v) {MgSo4.7H2O, 0.5%(w/v); KH2PO4,
0.5%(w/v)}; FeSO4.7H2O, 0.01%(w/v) and CaCO3, 0.5% respectively. Thus, with selected carbon and nitrogen sources along with 1 %
NaCl and 2% inoculum the maximum protease production (205.0 U/ml) was obtained in the period of 72 h incubation at pH-12.0
under 160 rpm when compared to the initial enzyme production (165.0 U/ml). The crude enzyme extract of this strain was also
characterized with respect to temperature, pH, incubation period and different concentrations of casein which was used as enzyme
substrate. This study shows that the enzyme has wide range of pH stability from 8 to 11 with optimum activity at pH-10.0. It is
thermostable with optimum activity at 70C (392U/ml) with 1h incubation of enzyme with 1% casein as its substrate. From the above
investigations it was concluded that the protease production by these microorganisms at wide temperatures and pH ranges could be
explored for varied industrial applications.
Keywords: Bacillus cereus strain S8, Alkaline protease, Optimization, Protease production, 16S rRNA gene sequencing,
phylogenetic tree analysis, Enzyme characterization.
----------------------------------------------------------------------***----------------------------------------------------------------------1. INTRODUCTION
Proteases are one of the most important classes of enzymes,
occupying a major share of 60% of total enzyme market [1].
Proteolytic enzymes are ubiquitous in occurrence, being found
in all living organisms, are essential for cell growth and
differentiation. Proteases represent one of the three largest
groups of industrially important enzymes [2]. Alkaline
proteases are of great interest because of their high proteolytic
activity and stability under alkaline conditions [3, 4]. These
enzymes find applications in detergents, feather processing,
food
processing,
silk
gumming,
pharmaceuticals,
bioremediation, biosynthesis, biotransformation,
silver
recovery from photographic film, production of digestive and
certain medical treatments of inflammation and virulent
wounds [5, 6, 7, 8, 9].The majority of commercial alkaline
proteases are produced by bacteria, especially Bacillus sps. [9].
Since the first alkaline protease Carlsberg from Bacillus
licheniformis was commercialized as an additive in detergents
in 1960s [4], a number of Bacillus derived alkaline proteases
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Protease
4. RESULTS
4.1 Isolation and Screening Of Protease Producing
Bacterial Strain
In the present study, a total of 17 halotolerant bacteria from the
soil of saltern pond have been screened for the presence of
protease production on skim milk agar plates. Ten strains were
identified as protease producers by zone of hydrolysis around
the colonies were quantified their activity. Out of all the strains,
strain S8 showed highest diameter of zone of hydrolysis
(4.6cm) (Fig. 1) and production (165.0 U/ml) with respect to
other strains and it was used for further protease optimization
studies.
ENZYME
The crude protease obtained from the strain S8, which showed
the highest potential for proteolytic activity, was further
subjected to preliminary characterization study. Therefore, the
effects of pH, incubation period, temperature and substrate
concentration on enzyme activity were studied. The procedures
are outlined in detail below.
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4.2.
Morphological
Characterization
and
Isolated strain S8
Rods
Gram Positive
Positive
Positive
Motile
Negative
Positive
Positive
Positive
Negative
Negative
Negative
Negative
Positive
Positive
Positive
Negative
Negative
Positive
Negative
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200
150
100
50
0
20
100
50
-50
2 3 4 5 6 7 8 9 10 11 12
.
250
200
150
100
50
0
1%
2%
3%
4%
Inoculum Size
5%
.
70
.
Protease Production
The effect of salt on protease production showed optimum
protease production in the medium containing 1% NaCl
(109.52 U/ml) after 72 h of incubation with growth tolerance
up to 3% NaCl. The growth and production of protease was
reduced when salt concentration increases above 1% NaCl
(Chart.7). Among various carbon sources used, protease
production was highest in the medium containing molasses
(205.00 U/ml) followed by maltose, rice bran and wheat flour
as shown in (Chart. 8). Molasses can be easily utilized by B.
cereus strain S8 when compared to other carbon sources.
Various nitrogen sources were investigated for protease
production. High yield of protease production was observed in
potassium nitrate (198.00 U/ml) (Chart. 9) followed by peptone
and skim milk powder as nitrogen sources. Effects of different
concentrations of carbon and nitrogen sources were carried out.
It shows that maximum production was achieved at a
concentration of Molasses (1%) and Potassium nitrate (0.75%).
Protease Activity ( U/ml)
200
150
100
50
0
48
72
96
120
Incubation Period(h)
37 40 50 60
Temperature(0C)
250
24
30
200
150
pH
140
120
100
80
60
40
20
0
NaCl concentration
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200
Protease Activitty(U/ml)
150
100
50
Mol
R.H
R.B
S.B
W.F
S.B
Man
Mal
0
Glu
250
Carbon Source
500
400
300
200
100
0
30 35 40 45 50 55 60 65 70 75 80
Temperature(0C)
250
200
150
100
50
70
60
50
40
30
20
10
0
2 3 4 5 6 7 8 9 10 11 12
pH
P.N
Pep: Peptone, Cas: Casein, SKM: Skim milk powder, BE: Beef
extract, YE: Yeast extract, A.C: Ammonium chloride, A.N:
Ammonium nitrate, A.S: Ammonium sulphate, P.N: Potassium
nitrate.
Fig 10 Effect of different Organic and Inorganic nitrogen
sources on protease production in Bacillus cereus strain S8
isolated from saltern sediments. The bars indicate the standard
deviation of three replicates analyzed.
140
120
100
80
60
40
20
0
5 10 15 20 25 30 35 40 45 50 55 60 65
from Strain S8
Bacillus cereus Strain S8 demonstrated a higher potential for
alkaline protease activity. Therefore, a preliminary study on the
characterization of this enzyme was carried out. According to
this study, it was observed that, this enzyme was thermostable
with an optimum temperature of 700C (Fig 11) at a pH 10.0
(Fig 12) with 1h incubation (Fig 13) of enzyme reaction
mixture. Casein was used as substrate for this enzyme and
A.S
A.N
A.C
YE
BE
SKM
Cas
0
Pep
Incubation Period(minutes)
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440
Protease Activity(U/ml)
250
200
150
100
50
0
5. DISCUSSION
One of the main concerns of this study was to isolate and
identify alkalophilic Bacillus sp. having a vital tendency to
secrete extra-cellular proteolytic enzyme. Accordingly, 17
bacterial strains were isolated from saltern pond soil sample
suspension processed on nutrient agar medium. Out of these
isolates, isolate S8 represents genus Bacillus and exhibited
vivid zone of clearance (4.6 cm) on skim milk agar medium at
pH 10. The use of alkaline skim milk agar for the isolation of
alkaline protease producing bacteria has earlier been reported
by some workers [23, 24, 25]. In fact these bacteria are known
for their abilities to secrete large amounts of alkaline proteases
having significant proteolytic activity and stability at
considerably high pH and temperatures [26, 27]. In the present
study, the proteolytic activities of the isolate S8 were tested
under extreme alkaline conditions and it was confirmed as
Bacillus cereus isolate S8 by 16S r RNA gene sequencing.
In the present work, the self constructed medium of pH-12.0
supported maximum enzyme production (205.00 U/ml) after
72h of incubation. Similar results were also reported in
Bacillus odyssey with 72 h incubation period [28]. The medium
provided primarily adequate amount of nutrients required for
the production of alkaline protease. Carbon and nitrogen
sources, inorganic salts and other growth factors are important
variables that affect the growth and products of microbes [29,
30].
Requirement for specific carbon and nitrogen source differs
from organism to organism, or even among the same species
isolated from different sources [26]. Among various carbon and
nitrogen sources used molasses (1%) and potassium nitrate
(0.75%) supported maximum enzyme production. Both organic
and inorganic nitrogen compounds were utilized by this strain.
A decrease in enzyme production was observed at lower and
higher concentrations. The results indicated that proper
concentration level of molasses and potassium nitrate played a
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441
6. CONCLUSIONS
Alkaline protease producing strain Isolate S8 from the soil of
saltern pond was isolated and identified as Bacillus cereus
strain S8 by 16s rRNA gene sequencing and phylogenetic tree
analysis. Optimization of the fermentation conditions
(temperature, inoculum concentration, carbon and nitrogen
sources, incubation time and initial media pH) were optimized.
Based on the results, maximum protease production was
obtained in the medium supplemented with Molasses,1%(w/v);
Potassium nitrate, 0.75%(w/v); salt solution- 5%(v/v)
{MgSo4.7H2O, 0.5%(w/v); KH2PO4, 0.5%(w/v)}; FeSO4.7H2O,
0.01%(w/v) and CaCO3, 0.5% at 370C after 72h incubation.
Production of protease was achieved with cheap carbon sources
like molasses in a cost effective manner and showed halo
tolerance. Characterization studies of the crude enzyme showed
that the enzyme was thermostable, showing activity at wide pH
ranges. These promising features of the isolated Bacillus cereus
strain S8 might increase the scope of the strain to meet
industrial demands of various sectors.
ACKNOWLEDGEMENTS
The authors would like to acknowledge UGC-MRP, New
Delhi, India, for providing financial support to carry out this
work.
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BIOGRAPHIES
B.K.M.Lakshmi,
Research
scholar
Department of Microbiology College of
science and technology Andhra University
Visakhapatnam- 530 003 Andhra Pradesh,
India, Kamalab42@gmail.com
P.V. Ratna sri, Research scholar
Department of Microbiology College of
science and technology Andhra University
Visakhapatnam,-530 003 Andhra Pradesh,
India, ratnasrii@gmail.com
K. Ambika Devi, Research scholar
Department of Microbiology College of
science and technology Andhra University
Visakhapatnam,-530 003 Andhra Pradesh,
India, Ambicadevi24@gmail.com
Prof.K.P.J.Hemalatha,
Head
of
the
Department of Microbiology College of
science and technology Andhra University
Visakhapatnam,-530 003 Andhra Pradesh,
India, hemalathakpj@gmail.com
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