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Page 1
Journal of Applied Sciences Research, 3(11): 1363-1368, 2007
© 2007, INSInet Publication
Corresponding Author: ​Abdelnasser S.S.Ibrahim, Department of Chemistry of Natural and Microbial Products, National
Research Centre, Dokki, Cairo, Egypt.
1363
Isolation and Identification of Alkaline Protease Producing
Alkaliphilic Bacteria from an Egyptian Soda Lake
Abdelnasser S.S.Ibrahim, Nefisa M.A. EI-Shayeb and Sohair S. Mabrouk
Department of Chemistry of Natural and Microbial Products,
National Research Centre, Dokki, Cairo, Egypt
.
Abstract: ​Screening of water and soil samples collected from Wadi El-Natrun, an Egyptian soda lake,
for alkaline proteases producing bacteria, resulted in isolation of 15 alkaline proteases producing
alkaliphilic strains. WN-SK5 showed the highest enzyme production (61.0 U/ml) after 48h. This isolate
was gram positive and was able to grow in the presence of NaCl up to 15 %. Growth was observed at
25 ºC, 37 ºC, 45 ºC and 55 ºC but no growth was seen at 60 ºC. It could grow at pH value from 8 to
11. No growth was detected at pH 7 after 48 h incubation at 55 ºC, which indicted this strain to be
moderate halophilic thermophilic ​alkaliphiles​. It was found that 16S-rDNA sequence of strain WN-SK5
had 97.35 % identity with the corresponding sequence of ​Bacillus halodurans ​(Acc. Nr. gb10172612). The
crude alkaline ​protease​ showed reasonable activity at temperature range of 65 to 75 °C with maximum
activity at 70 ºC and had a relatively wide pH range of activity between pH 8 to 11, with maximum
enzyme activity at pH 10 in 50 mM Tris -HCl buffer maximum activity at 70 °C and pH 10.0, indicating
the enzyme to be thermo- alkaline proteases.
Keywords: Alkaliphiles​, alkaline proteases, Soda lake isolation, 16S rDNA
INTRODUCTION
Proteolytic enzymes are degradative enzymes which
catalyze the cleavage of peptide bonds in other proteins.
Currently, proteases are classified on the basis of three
major criteria, type of reaction catalyzed, chemical
nature of the catalytic site and evolutionary relationship
with reference structure. Alkaline proteases are
referring to proteolytic enzymes which work optimally
in alkaline pH (1, 12). The vast diversity of proteases,
in contrast to the specificity of their action, has
attracted worldwide attention in attempts to exploit their
physiological and biotechnological applications,
[6,22, 12]
e. g. food and feed industry, peptide synthesis, leather
industry, management of industrial household waste,
photographic industry, medical usage, silk gumming
and detergents industry.
Alkaliphiles​ are defined as organisms that grow
optimally at alkaline pH, with pH optima for growth
being in excess of pH 8 (usually between 9 and 10),
and some being capable of growing at pH > 11
.
[9,14]
Although they were once considered to be curiosities,
awareness of ​alkaliphiles​ has blossomed in recent years
due to an interest in their physiological adaptation to
high pH and their potential uses in biotechnological
applications. Soda lakes and soda deserts represent the
major types of naturally occurring highly alkaline
environments, in which the indigenous microflora is
subjected to number of extreme ecological pressures.
They represent the most stable high pH environments
on Earth, where large amounts of carbonate minerals
can generate pH values >11.5. Soda lakes are widely
distributed throughout the world; however as a result of
their inaccessibility, few of such lakes have been
explored from the microbiological point of view
.
[9,18]
One of those environmental niches which have not
been studied in details is the Wadi El-Natrun soda
lakes in northern Egypt. The aims of this work were
isolation of aerobic alkaliphilic bacteria from some
Wadi Natrun soda lakes and screening for and alkaline
protease​ producing alkaliphilic bacteria, characterization
and identification of the interested strains and
preliminary investigation of the alkaline ​protease​.
MATERIAL AND METHODS
Soil and Water Samples: ​Soil and water samples were
taken from Wadi Natrun in northern Egypt. Wadi
Natrun extends in a northwest by southeast direction
between latitudes 30º 15 north and longitude 30º 30
east. The bottom of the Wadi Natrun area is 23 m
below sea-level and 38 m below the water-level of
Rosetta branch of the Nile. The lowest part of the
depression, encircled by contour zero, covers an area
of about 272 km2 .
[24]

 
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J. Appl. Sci. Res., 3(11): 1363-1368, 2007
1364
Soil and water samples were collected from the
different locations of Wadi Natrun soda lakes: Hamara,
Bani salama, Dawood, Elbida lakes. Samples were kept
in sterile tubes in refrigerator, at 4 °C, and were
transferred to the laboratory within few hours
Enrichment and Isolation of Aerobic Alkaline
Protease Producing Alkaliphilic Bacteria: ​Isolation of
alkaline ​protease​ producing alkaliphilic bacteria was
carried out using rich alkaline agar medium containing
skimmed milk (27, modified). Aliquots (100 µl) of
different dilutions of soil suspensions and water
samples were plated and incubated at 37 °C, 50 °C and
60 °C for three days. Formation of halo zone around
the colonies, resulting from casein hydrolysis, was
taken as evidence of proteolytic activity. These colonies
were isolated and streaked in fresh plates until single
uniform
colonies were obtained. The medium
contained (g/l): Skimmed milk
100, yeast extracts
2
3
2
3
10, Na CO 15, agar 20. Skimmed milk, Na CO and
the other constituents were autoclaved separately
(at 121 °C for 20 min) and mixed after cooling.
Some Characterization of the Isolate: ​Classification
of the isolates as gram positive or gram negative was
done by Gram stain reaction
and KOH sensitivity
[7]
test . For Gram staining the Color Gram 2 kit of
[11]
bioMérieux (Marcy l’Etoile, France) was used. For
KOH sensitivity test a heavy mass of 24 h bacterial
cultures were picked up and transferred to glass slide
with 2-3 drops of 3 % (w/v) KOH solution and the
cells suspensions were agitated rapidly with circular
motion with toothpick for 15-30 seconds. The
formation of a string (DNA) in 3 % (w/v) KOH
indicates that the isolate is a gram negative organism.
E. coli ​and ​Bacillus megaterium w​ ere used as gram
negative and gram positive control, respectively.
The effect of temperature on growth was studied
by plating out the cells on alkaline agar medium and
incubated at different temperatures: 30 °C, 37 °C, 45
°C, 50 °C, 55 °C and 60 °C, respectively, for 48 h. To
study the influence of salinity on cells growth, alkaline
agar medium containing different concentrations of
NaCl (w/v) 0 %, 5 %, 10 %, 15 % and 20 %,
respectively, was inoculated with the cells and
incubated at 37 °C for 48 h. For the alkaliphily test,
the isolates were inoculated to agar medium adjusted at
different pH values: 7, 8, 9, 10 and pH 11 and
incubated at 37 °C for 48 h.
Analyses of 16S rDNA Gene Sequences: ​For the
sequence analysis, bacterial genomic DNA was
extracted and purified using a Wizard Genomic DNA
Prep. Kit (Promega Co., Madison, USA). Two primers
annealing at the 5 and 3 end of the 16S rDNA were
5 -GAGTTTGATCCTGGCTCAG-3 (positions 9–27
[​Escherichia coli 1​ 6S rDNA numbering]) and 5 -
AGAAA GGAGG TGATC CAGCC-3
(positions
1542–1525 [​E. coli ​16S rDNA
numbering]),
respectively. (Chun and Bae, 2000). PCR amplification
was performed in a final reaction volume of 100 ìl,
and the reaction mixture contained each primer at a
concentration of 0.5 ìM, each deoxynucleoside
triphosphate at a concentration of 200 ìM, 50 mM
2
KCl, 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl ,
0.01% (w/v) gelatin, and 2.5 U of ​Taq ​DNA
polymerase. The PCR reaction was run for 35 cycles
in a DNA thermal cycler (Model No. 9700,
Perkin–Elmer Co. Wellesley, USA). The following
thermal profile was used for the PCR: denaturation at
94 °C for 1 min, primer annealing at 60 °C for 1 min
and extension at 72 °C for 2 min. The final cycle
included extension for 10 min at 72 °C to ensure full
extension of the products. The amplified PCR products
were then analyzed in a 1.0% (w/v) agarose gel,
excised from the gel, and purified. The purified
products were cloned into pGEM-T Easy vector
(Promega Co., Madison, USA) and subsequently
sequenced using ALF Red automated DNA sequencer
(Pharmacia, Sweden).The 16S rDNA sequence of the
isolate was aligned with those in the GenBank
database. Multiple alignment of sequences and
calculations of levels of sequence similarity were
performed by using CLUSTAL W . The neighbor-
[26]
joining phylogenetic analysis was carried out with
MEGA program .
[19]
Production of Alkaline Proteases: ​A loopful of
culture from agar plate was inoculated into 50 ml-glass
tube containing 5 ml of alkaline ​protease​ production
medium, and incubated overnight at 180 rpm and 50
°C. This culture was then inoculated into 500 ml
capacity Erlenmeyer flask containing 95 ml of the same
medium and incubated at 50 °C for 48 h. Cells and
insoluble materials were removed by centrifugation at
10 000 g for 10 min at 4 °C and the cell-free
supernatant was filtered through a 0.45-µm pore-size
membrane filter and was used as the source of crude
alkaline ​protease​ enzyme. The production medium
contained (g/l) (Jasvir ​et al​. 1999, modified): Glucose
2
4
4
10, Peptone 5, yeast extract 5, K HPO
1, M gSO
2
3
2
3
0.2, Na CO 15, pH 10.5. Na CO , glucose and the
other constituents were
autoclaved separately (at
121 °C for 20 min) and mixed after cooling.
Alkaline Protease Assay: ​Proteolytic activity was
assayed by a modified method of Kunitz . Samples
[20]
containing 400 ìl of 0.5 % (w/v) casein in 50 mM
Tris –HCl buffer, pH 10, with 100 ìl enzyme sample
were incubated in a water bath at 50 °C for 20 min.

 
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J. Appl. Sci. Res., 3(11): 1363-1368, 2007
1365
The enzyme reaction was terminated by addition of 500
ìl of 10 % (w/v) trichloroacetic acid and was kept at
room temperature for 10 min. The reaction mixture
was centrifuged at 10 000 g for 10 min at 4 °C
and
the absorbance was measured against a blank
(non-incubated sample) at 280 nm. One unit of
proteases was defined as the amount of the enzyme
releasing the equivalent of one ìmol of tyrosine per
minute under the defined assay conditions. Standard
curve of tyrosine was done using 10, 20, 30, 40, 50,
60, 70, 80 and 100 mg/ml tyrosine in 50 mM glycine-
NaOH buffer, pH 10,
Protein Determination: ​Protein concentration was
determined according to the method described by
Bradford . One ml of Bradford reagent was added to
[3]
50 ìl of sample and the extinction was measured after
5 min at 595 nm. Different concentrations of bovine
serum albumin (BSA) were used as a protein standard:
10, 20, 40, 60, 80, and 100 ìg/ ml distilled water.
One ml of Bradford reagent was added to 50 ìl BSA
standard and the extinction was measured after 5 min
at 595 nm.
Effect of Temperature on the Activity of the Crude
Alkaline Protease: ​The crude alkaline ​protease​ was
prepared as described above. The influence of
temperature on the catalytic activity of the crude
alkaline ​protease​ was determined by measuring the
enzyme activity at temperatures range from 25 °C to
90 °C under the standard assay conditions.
Effect of pH on the Activity of the Crude Alkaline
Protease: ​The influence of pH on the alkaline ​protease
activity was determined by measuring the enzyme
activity at varying pH values ranging from 5 to 12 at
70 ºC using different suitable buffers, 50 mM sodium
acetate (pH 5.0 and 6.0), 50 mM sodium phosphate
buffer (pH 7.0 and 8.0), 50 mM Na2HPO4-NaOH
buffer (pH 9.0, 10.0 and 11.0) and 50 mM KCl-NaOH
(pH 12), respectively.
RESULTS AND DISCUSSIONS
Isolation of Alkaline Protease Producing Alkaliphilic
Bacteria: ​Soda lakes are characterized by the presence
of a high concentration of sodium carbonate formed by
evaporative concentration, and are also associated with
varying degree of salinity and low concentration of
both Mg
and Ca
ions
. Wadi EL-Natrun
2+
2+
[10,8,18]
(Wadi: Arabic for Valley) and its alkaline inland saline
lakes is an elongated depression about 90 km northwest
of Cairo. Its average length is about 60 km and
average width about 10 km. The bottom of the
Wadi Natrun area is below sea-level and below the
Fig. 1: ​Screening of alkaliphilic bacteria for alkaline
proteases production. Isolate are streaked on
alkaline agar plate containing skimmed milk,
incubated for 72 h at 55 °C. The clear zone
indicated the hydrolysis of casein as a result
of alkaline ​protease​ production
Table 1: ​Production of alkaline ​protease​ by different isolated
strains. Isolates were grown in alkaline medium, pH 10.4,
at 50 ºC in shaking incubator at 180 rpm for 48 h; W for
Wadi, N for Natrun, SK for skimmed milk.
Alkaline ​protease​ activity (U/ml)
Strains
-----------------------------------------------------
24 h
48 h
WN- SK1
15.15
19.18
WN SK2
5.51
10.12
WN-SK3
5.11
0.91
WN-SK4
22.15
29.11
WN-SK5
50.16
61.00
WN-SK6
41.12
31.51
WN-SK7
38.91
39.15
WN-SK8
0.90
3.50
WN-SK9
11.11
19.50
WN-SK10
8.88
16.11
WN-SK11
19.11
3.05
WN-SK12
6.55
19.12
WN-SK13
31.15
19.12
WN-SK14
6.16
11.15
WN-SK15
19.11
15.12
water-level of Rosetta branch of the Nile
The
[24]
features of Wadi Natrun area created an ecosystem
which considers as rich sources for isolation of
different extremophiles, including halophilic, alkaliphilic
in addition to thermoalakiliphilic microorganisms, due
to the sun-heated alkaline soil of this area which are
not well studied up to date
. Isolation of alkaline
[24,17]
protease​ producing alkaliphilic bacteria was carried out
using rich alkaline agar medium containing skimmed

 
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J. Appl. Sci. Res., 3(11): 1363-1368, 2007
1366
milk. Formation of clear zone around colonies was
considered as indication of alkaline ​protease​ production
(Figure). 15 isolates, showing large clear zone around
their colonies, were isolated. It has been established
that there is not necessarily good correlation between
zones of clearing around colonies on milk-agar plates
and levels of proteinase activity . Therefore, all the
[5]
positive isolates were cultivated in the alkaline
production medium and the proteolytic activity was
measured. The results shown in Table 1 indicated that
some isolates with considerable proteolytic activity
could be isolated, WN-SK5, WN-SK6 and WN-SK7.
The
strain WN-SK5
showing
the
highest
alkaline ​protease​ activity (61.0 U/ml) was selected
for further characterization.
Characterization and Identification of the Alkaline
Protease Producing Strains: ​The alkaline ​protease
producing isolate, termed WN-SK5, showed white
mucoide colonies with circular margin. Cells grown in
liquid medium for 24 h showed non-motile long rod-
shaped cells under light microscope. This isolate was
gram positive using KOH sensitivity test. Strain WN-
SK5 was able to grow in the presence of NaCl up to
15 %. Growth was observed at 25 ºC, 37 ºC, 45 ºC
and 55 ºC but no growth was seen at 60 ºC after 48 h
at pH 10.5. It could grow at pH value from 8 to 11.
No growth was detected at pH 7 after 48 h incubation
at 55 ºC which indicted this strain to be moderate
halophilic thermophilic ​alkaliphiles​ .
[14]
Identification of the Strain WN-SK5 by 16S-rDNA
Sequencing: ​Genomic DNA of the strains was isolated
and the PCR amplified 16S-rDNA was cloned into
E. coli
and
sequenced as already described in
material and method section. By using six primers
(three forward and three reverse), 1461 bp of the
16S-rDNA were sequenced with minimum mistakes.
The 16S-rDNA sequence was aligned with the known
16S-rDNA sequences of other bacteria. It was found
that 16S-rDNA sequence of strain WN-SK5 had 97.35
% identity with the corresponding sequence of ​Bacillus
halodurans (​ Acc. Nr. gb10172612). Considering the
exactly identified bases, there were 14 differences
between the base sequences of strain WN-SK5 and ​B.
halodurans.​ One base differences were found to be in
the universal region (U5), six in semivariable regions
(S 1, 2, 3, 5 and 6) and the other seven differences
were found to be in the variable regions V1 and V5,
but mainly in V6
. The difference of 2.65 % is the
[21]
sum of these well identified mismatches and other
unspecific. For differentiation of a unique species the
study has to continue in more detail. An alkaliphilic
bacterium, strain C-125 (JCM9153), isolated in 1977,
Fig. 2: ​Effect of temperature on the activity of crude
alkaline ​protease​ from B. halodurans SK5. The
alkaline ​protease​ activity was measured using
0.5 % casein Tris -HCl buffer, pH 10, at
different temperatures. Standard deviations of
the relative activities were in the range of
1.0-3.5 %.
Fig. 3: ​Effect of pH on the activity of B. halodurans
WN-SK5 alkaline ​protease​. Activity was
measured under the standard assay conditions
at various pH values. Standard deviations
of
the relative activities
were in
the
range of 1-4 %.
was recently identified as ​Bacillus halodurans ​based
on
1 6 S -r D N A
se q u e n c e
and
D N A -D NA
hybridization . It is reported as a â-galactosidase
[25]
[16]
and xylanase producer .
[13]
Effect of Temperature on the Activity of the Crude
Alkaline Protease from ​B. Halodurans W ​ N-SK5:
Cell-free supernatant was prepared as described in
material and methods section and was filtered through
a 0.45-µm pore-size membrane filter and used as the
source of crude alkaline ​protease​. For determination of
the optimum temperature of the crude alkaline ​protease​,
the enzyme activity was measured at different
temperatures at pH 10. The results illustrated
graphically in Figure 2 indicated that the crude alkaline
protease​ showed reasonable activity at temperature

 
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J. Appl. Sci. Res., 3(11): 1363-1368, 2007
1367
range of 65 to 75 °C with maximum activity at 70 ºC.
A rapid decrease of enzyme activity was detected
above 80 ºC and the enzyme was completely
inactivated at 90 ºC.
Effect of pH on Activity of Crude Alkaline Protease
from ​B. Halodurans W ​ N-SK5: ​The effect of different
pH values of the reaction mixture on the activity of the
crude alkaline ​protease​ was investigated in pH range
from 5 to 11 at 70 ºC. It was found that the crude
alkaline ​protease​ had a relatively wide pH range of
activity between pH 8 to 11, with maximum enzyme
activity at pH 10 in 50 mM Tris -HCl buffer (Fig. 3).
These preliminary properties of the enzyme are
considered to be interested in comparison to other
alkaline proteases
.
[15,12,2,23]
The results of this work indicated that the Wadi
Natrun soda lakes, in Egypt, are a rich source of many
alkaliphilic bacteria which could be a good source of
many interested enzymes from the industrial point of
view and further studies are recommended on this soda
lakes including study of microbial biodiversity and the
biotechnological potent of the isolated strains.
REFERENCES
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Brazilian Journal of Microbiology (2007) 38:766-772
ISSN 1517-8382
766
SECRETION OF AN ALKALINE PROTEASE FROM A SALT- TOLERANT AND ALKALIPHILIC,
STREPTOMYCES CLAVULIGERUS ​STRAIN MIT-1
Jignasha T. Thumar; Satya P. Singh*
Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India.
Submitted: December 19, 2006; Returned to authors for corrections: April 02, 2007; Approved: November 19, 2007.
ABSTRACT
An alkaliphilic and salt- tolerant actinomycete, ​Streptomyces clavuligerus s​ train Mit-1, was isolated from
Mithapur, the western coast of India. The organism was Gram-positive, having filamentous, long thread like
structure. The sporulation started after two days of growth and the optimum level of alkaline ​protease​ (130 U/
ml) was produced during the early stationary phase. The strain could grow and produce ​protease​ with 0-10%
NaCl (w/v), the optimum being 5% NaCl (w/v). Growth and ​protease​ production was optimum at pH 9 with
substantial decline at neutral pH. Sucrose and gelatin were the best carbon and nitrogen sources respectively,
whereas gelatin broth was the preferred medium for ​protease​ production. Mit-1 produced substantial ​protease
with various amino acids, when employed as the sole nitrogen sources. Crude substrates, such as molasses,
whey and wheat flour had significant effect on enzyme production. The results are quite valuable, as only few
actinomycetes, particularly salt-tolerant alkaliphilic ones, have so far been explored for their enzymatic potential
and process optimization.
Key words​: ​Streptomyces clavuligerus, s​ alt-tolerant actinomycetes, alkaline ​protease​, enzymatic potential,
protease​ optimization
*Corresponding Author. Mailing address: Department of Biosciences, Saurashtra University, Rajkot - 360005, Gujarat, India. Tel.: + 91 281
2586419.
E-mail: satyapsingh@yahoo.com
INTRODUCTION
Besides antibiotics, enzymes are the bioactive compounds
that have focused attention. The demand for microbial enzymes
in industrial fields is increasing day by day due to their
applications in clean, environment-friendly and cost effective
biotechnological processes. The present global market for the
industrial enzymes is around $ 2 billion and expected to rise at
an average annual growth rate of 3.3%. But most of the enzymes
are derived from microbes thriving in ambient temperature,
neutral pH and other modest conditions. In view of the above
realization, exploration of extremophiles and their biocatalytic
machinery has been attracting the greater attention. Among the
various groups of extremophiles, halo-tolerant/halophilic
organisms are the major categories with greater biocatalytic
potential (17,32).
Proteases are among the most important class of industrial
enzymes, which constitute >65% of the total industrial
applications, such as laundry detergent, leather preparation,
meat tenderization and peptide synthesis (11). During last several
years, few haloalkaliphilic bacteria have been studied for their
ability to secrete alkaline ​protease​ (5,13,16,26-28); however,
alkaliphilic actinomycetes are relatively less explored in this
context.
While most of the studies are concerned with the molecular
phylogeny of halo-tolerant and alkaliphilic actinomyces
(6,10,14,18,19), the similar attention has not been paid to their
enzymology. However, the ability to produce a variety of
enzymes may be an attractive phenomenon in these
prokaryotes. Recently, alkaline ​protease​ from ​Nocardiopsis
sp. NCIM 5124 (8) has been purified and characterized.
Similarly, an alkaline ​protease​ was purified and crystallized
from ​Nesterenkoni s​ p. (2). Several species of the ​Streptomyces
are among the most important industrial microorganisms
because of their capacity to produce numerous bioactive
molecules, particularly antibiotics and enzymes (7,22). A

Page 2
Alkaline proteose from ​S. clavuligerus
767
keratinolytic serine proteinase was characterized from
Streptomyces pactum ​DSM 40530 (4).
While there are few reports on the secretion of alkaline
proteases from alkaliphilic actinomycetes, the studies on the
factors affecting the production of the enzyme are rare. The
investigations on salt- tolerant alkaliphilic actinomycetes with
respect to their enzymatic potential are particularly limited. Thus,
process optimization for the enzyme production from such
organisms would be an alternative to traditionally used ​protease
producers. Therefore, the present study aims at the factors that
affect alkaline ​protease​ production from salt- tolerant alkaliphilic
actinomycetes.
MATERIALS AND METHODS
Microorganism
A halo-tolerant and alkaliphilic actinomycete, Mit-1, identified
as ​Streptomyces clavuligerus,​ was isolated from saline soil
collected from coastal region of Mithapur, Gujarat, India. The
saline soil (10g) was incubated at 45ºC with CaCl
2
(1g) for one
week. The treated soil was enriched in Actinomyces broth (Hi
Media Ltd.) with NaCl (5%, w/v)
.
The pH of the medium was
adjusted to 9 by adding separately sterilized Na
2
CO
3
(20%, w/v).
The enriched culture was streaked on the Actinomyces agar (5%
w/v NaCl, pH 9). After the incubation of 6 days at 30ºC, a typical
chalky white colony was picked up and re-streaked to ensure the
purity of the colony. The organism was gram-positive, with
filamentous structure. The organism was identified as ​Streptomyces
clavuligerus b​ ased on the morphological, physiological and
biochemical characteristics. The culture was assigned MTCC
7037 as accession/strain number by Microbial Type Culture
Collection and ​Gene Bank, I​ MTECH, Chandigarh (India).
Protease production in liquid culture medium
Inoculum was prepared by transferring a loop full of culture
from the slant into the 25 ml of sterile gelatin broth containing
(g/l): gelatin, 10; peptone, 5; yeast extract, 5; NaCl, 50 and pH 9.
The inoculum was incubated at 30ºC on a rotary shaker (100
rpm) for 48 h. The inoculum at 10% was added into the gelatin
broth followed by incubation at 30ºC under shaking conditions.
After the incubation for 110 h, the cultures were harvested and
mixed to homogeneity by Vortex mixer followed by measurement
of growth at 540 nm. The crude enzyme was harvested by
filtration with cellulose filter and the filtrate was used as crude
enzyme preparation.
Enzyme Assay
The enzyme was assayed by the Anson- Hagihara method
(12) using casein as a substrate. One unit of alkaline ​protease
activity (U) was taken as the amount of enzyme liberating 1 µg
of tyrosine per min under the assay conditions. The estimations
were based on a tyrosine calibration curve.
Growth kinetics of Mit-1 with reference to protease
production
After activation in sterile gelatin broth (5% w/v NaCl, pH 9),
10% of inoculum was added into the gelatin broth and incubated
at 30ºC under shaking conditions (100 rpm). Cultures were
withdrawn aseptically at 6 h interval and growth and enzyme
activity were monitored as described above.
Effect of media on growth and protease production
The growth and ​protease​ production was compared in
different media such as gelatin, starch, complete medium, gelatin
casaminoacid, glucose asparagine, starch casein, glycerol
asparagine, glycerol glycine and Actinomyces broth. The culture
media after inoculation were incubated at 30ºC under shake
flask conditions for 110 h followed by the measurement of growth
and ​protease​ production.
Effect of NaCl and pH
The effect of NaCl on ​protease​ production was carried out
in gelatin broth at varying concentrations of salt (0-10%, w/v)
at pH 9. The growth and enzyme activity were monitored after
110 h of growth under shaking conditions. Similarly, the growth
and enzyme production were also monitored at pH in the range
of 7-10 with 5% w/v NaCl.
Effect of cations on protease production
Different salts; CaCl
2
, MgCl
2
· ​7H
2
O, MnCl
2-
and KCl were
included in gelatin broth at the concentration of 0.5%, w/v for
Ca
++
, Mg
++
, Mn
++
and K
+
respectively. After the incubation of
110 h, growth and ​protease​ secretion were measured.
Effect of carbon and nitrogen sources
In order to study the effect of carbon sources on ​protease
production, gelatin broth was supplemented with various sugars
viz. xylose, glucose, maltose, lactose, sucrose and starch at 1%
(w/v). After 110 h growth at 30ºC under shaking conditions,
growth and ​protease​ activity were measured.
Correspondingly, various organic and inorganic nitrogen
sources were investigated for their effect on the growth and
protease​ production. The organic nitrogen sources were
gelatin, casein, peptone and yeast extract, while inorganic
nitrogen sources included ammonium nitrate, ammonium
oxalate, ammonium sulphate, ammonium molybdate and
ammonium acetate; all at 1% (w/v). The respective nitrogen
sources were included as the sole source of nitrogen in minimal
medium (5% NaCl, w/v, pH 9), which contained; solution A
(800 ml) + solution B (200 ml). Solution Aincluded (g/l): KH
2
PO
4
,
3; Na
2
HPO
4
, 6; NH
4
Cl, 2; NaCl; 50. Solution B contained (g/l):
glucose, 8; MgSO
4
· ​7H
2
O, 0.1. The culture media after
inoculation were incubated at 30ºC for 110 h under shake flask
conditions followed by the estimation of growth and ​protease
activity.

Page 3
768
Thumar, J.T. and Singh, S.P​.
Protease production with amino acids as the sole source of
nitrogen
Experiments were designed to investigate the effect of amino
acids as the sole nitrogen source. Methionine, alanine, histidine,
tyrosine, arginine, phenylalanine, aspartic acid and leucine were
selected from the five classes of amino acids based on the
polarity and R group. The respective amino acids, at the
concentration of 1% w/v, were included in minimal medium
(5%, w/v, NaCl, pH 9). Incubation was carried out at 30ºC for
110 h under shaking conditions followed by the measurement
of growth and ​protease​ activity.
Protease production with crude sources
Molasses and whey in the range of 0 - 2% v/v and wheat
flour in the range of 0-2% w/v were included in minimal medium
(5% w/v NaCl and pH 9) as the sole source of both, carbon and
nitrogen. After the incubation of 110 h at 30ºC, the culture
samples were withdrawn and monitored for growth and enzyme
activity.
All the experiments described have been done in triplicates
and average values are presented in the results.
RESULTS AND DISCUSSION
The present study relates to the production and optimization
of an alkaline ​protease​ from a halo-tolerant, alkaliphilic
actinomycete. The growth kinetics of Mit-1 was followed up to
156 h in gelatin broth (5% w/v, NaCl, pH 9). The organism entered
in the exponential phase after 26 h that continued up to 100 h
followed by stationary phase (Fig. 1). ​Protease​ production was
optimum at 110 h (130 U/ml) during early stationary phase (Fig.
1). This result is in agreement with Bascaran ​et al.​, (3) and
Moreira ​et al.,​ (25), who showed that synthesis of ​protease
from ​Streptomyces clavuligerus ​starts in the early stationary
phase of growth. Similarly, in ​Streptomyces rimosus, t​ he optimum
protease​ production was recorded at 166 h (36).
The study on enzymes usually involves a search for optimal
media for their production. The comparative production of
enzyme in different media revealed that while growth was nearly
similar, the enzyme production varied. While the growth of Mit-
1 was optimum in Actinomyces broth, the enzyme production
was optimum (135 U/ml) in the gelatin broth, which may be due
to induction of enzyme secretion by peptone and gelatin (Fig.
2). In ​Streptomyces clavuligerus​, the enzyme production varied
greatly with the culture media used (30). Recently, a newly
isolated haloalkaliphilic ​Bacillus ​sp. was reported to produce
optimum level of alkaline ​protease​ in gelatin broth (29).
Salt had pronounced effect on growth and enzyme
production. Mit-1 grew up to 10% w/v salt, the optimum being
at 5% w/v, which is quite similar to that of ​Nocardiopsis
kunsanensis ​sp. nov., a moderately halophilic actinomycete (6).
Similarly, ​Kocuria marina ​sp. nov., a novel actinomycete,
tolerated up to 15% NaCl in growth media, although its presence
was not essential for growth (18). Mit-1 was also capable to
grow with 0% salt indicating the halo-tolerant nature of the
organism (Fig. 3). The ​protease​ production was optimum (158
U/ml) with 5% w/v NaCl with a sharp decrease at 10%. The salt
requirement of our isolate, however, was much less compared
to ​Streptomonospora alba s​ p. nov., a truly halophilic
actinomycete (20).
The effect of pH was investigated on growth and enzyme
production. Mit-1 grew optimally at pH 9 with slow growth at
neutral pH (Fig. 4). Recently, a novel alkaliphilic actinomycetes
Streptomyces sodiiphilus s​ p. nov. was reported to grow with
Figure 1. ​Growth kinetics of Mit-1 with references to ​protease
production by ​Streptomyces clavuligerus.​ Growth (λ
540
)(
l
)
and ​protease​ activity (p).
Figure 2. ​Effect of various media on growth (
n
) and ​protease
production (o) in ​Streptomyces clavuligerus.​ GB: Gelatin Broth,
SB: Starch Broth, CMB: Complete Medium Broth, SCB: Starch
Casein Broth, AB: Actinomyces Broth, GAA: Glucose
Asparagine Broth, GAB: Glycerol Asparagine Broth, GCB:
Gelatin Casaminoacid broth, GGB: Glycerol Glycine Broth.

Page 4
Alkaline proteose from ​S. clavuligerus
769
an optimum pH of 9 -10 with scant growth at pH 7 (21). Similarly,
Nocardiopsis alkaliphila ​also grew optimally at pH 9.5-10
(14). Mit-1 displayed optimum ​protease​ activity (180 U/ml) at
pH 9. Although the growth was almost similar at pH 9 and 10,
protease​ production varied greatly. The enzyme secretion was
adversely affected at lower pH compared to that at pH 10,
confirming the alkaliphilic nature of the enzyme. Similar
response has also been observed in alkaliphilic ​Nocardiopsis
sp. TOA-1 in which alkaline ​protease​ was produced optimally
at pH 9-10 (24).
In general, cations are known to induce enzyme secretion
and increase the thermo stability of the enzyme (33). While
growth was optimum with Ca
2+
and Mg
2+
, the organism secreted
alkaline ​protease​ optimally (310 U/ml) with Mg
2+
(Fig. 5). The
enzyme production was comparable with Ca
2+
and K
+
, and was
completely suppressed with Mn
2+
. The results are similar to
those from ​Streptomyces rimosus ​where calcium carbonate
enhanced ​protease​ production (35).
Various sugars were investigated for their effect on ​protease
production. Glucose was the best carbon source for growth
whereas ​protease​ production was optimum with sucrose (410
U/ml). While other sugars, such as maltose, xylose, lactose,
glucose, supported ​protease​ production; starch affected it
adversely (Fig. 6). Contrary to our results, in ​Streptomyces
rimosus​, ​protease​ production was optimum with starch (35).
In most microorganisms, the nitrogen source (both organic
and inorganic forms) is metabolized to produce primarily amino
acids, nucleic acids, proteins and cell wall components. Alkaline
protease​ is comprised of 15.6% nitrogen and its production
depends heavily on the availability of nitrogen sources in the
medium. In our study, the growth, as compared to enzyme
production, was marginally affected by organic nitrogen
sources. ​Protease​ production was optimum with gelatin (100 U/
ml) and peptone (100 U/ml) followed by yeast extract and casein
(Fig. 7). Although a reduced growth was observed with yeast
extract, ​protease​ production was enhanced significantly in its
Figure 3. ​Effect of salt on growth (
n
) and ​protease​ production
(o) in ​Streptomyces clavuligerus​.
Figure 4. ​Effect of pH on growth (
n
) and ​protease​ production
(o) in ​Streptomyces clavuligerus​.
Figure 5. ​Effect of cations (0.5%) on growth (
n
) and ​protease
production (o) in ​Streptomyces clavuligerus.​
Figure 6. ​Effect of carbon sources on growth (
n
) and ​protease
production (o) in ​Streptomyces clavuligerus.​

Page 5
770
Thumar, J.T. and Singh, S.P​.
presence. Similarly, ​Nocardiopsis metallicus ​sp. nov., a metal-
leaching actinomycete, has been reported to utilize various
organic nitrogen sources for growth and enzyme production
(31).
In the presence of inorganic nitrogen sources such as
ammonium nitrate, ammonium sulfate, ammonium molybdate,
ammonium oxalate and ammonium acetate, the growth and
enzyme production were adversely affected as compared to
organic nitrogen supplements. Among the inorganic nitrogen
sources, however, ammonium nitrate emerged as the best in
supporting enzyme production (100 U/ml) (Fig. 8). Yang and
Lee (35) reported ammonium sulphate as the best nitrogen
source for ​protease​ production in ​Streptomyces rimosus​.
Interestingly, in ​Streptomyces cremeus​, the enzyme production
was not supported by any single nitrogen source, instead a
combination of peptone, soy flour and ammonium sulphate was
most suitable (34).
Amino acids are known to induce ​protease​ production (23).
Growth and ​protease​ production greatly vary in response to
different amino acids. Among the amino acids employed,
asparagine was the best for growth; however, it did not support
the enzyme production at all. Significant growth was also evident
with arginine and phenylalanine (Fig. 9). Although the growth
was quite less, the ​protease​ production was optimum with
leucine (130 U/ml). The leucine supported ​protease​ production
was quite comparable to those obtained with complex media.
Except histidine and phenylalanine, ​protease​ production was
significantly retarded in the presence of other amino acids. The
results are comparable to a neutrophilic ​Streptomyces
clavuligerus t​ hat was capable to grow with asparagine and
glutamine (1). Similarly, ​Streptomycespristinaespiralis​has also
been reported to use alanine and glutamate as the sole source
of nitrogen (9). Bascaran ​et al.​, (3) stated that better ​protease
production could be obtained with nitrogen-free medium or
poorly utilized amino acids.
Among various cheap sources, industrial by-products such
as molasses and other cheaper components like whey and
wheat flour have focused considerable attention, as they
support both cell mass and enzyme production (17). The crude
sources, employed in the present study, supported growth
and ​protease​ synthesis significantly. Molasses, when used
as the sole carbon and nitrogen source, induced ​protease
production up to 1% v/v above which it gradually repressed
(Fig. 10). Apart from molasses, wheat flour (1.5 %, w/v, Fig. 11)
and whey (1-1.5%, v/v, Fig. 12) also enhanced the enzyme
level significantly. These findings are in agreement with an
alkaline ​protease​ from ​Bacillus J​ B 99 (15). The ability of the
organism in this study to grow and produce enzyme with amino
acids as well as cheaper agro-industrial by-products assumes
significance. The production of the enzyme with these sources
would be economically attractive prepositions. Besides, the
regulation of enzyme synthesis in response to various carbon
and nitrogen sources would be interesting to investigate
further.
Figure 8. ​Effect of various inorganic nitrogen sources on growth
(
n
) and ​protease​ production (o) in ​Streptomyces clavuligerus.​
Figure 7. ​Effect of various organic nitrogen sources on growth
(
n
) and ​protease​ production (o) in ​Streptomyces clavuligerus.​
Figure 9. Protease​ production on amino acids as the sole source
of nitrogen. Growth (
n
) and ​protease​ production (o) in
Streptomyces clavuligerus​. Met: Methionine; Ala: Alanine; His:
Histidine; Tyr: Tyrosine; Phe: Phenylalanine; Arg: Arginine;
Leu: Leucine; Asn: Asparagine.

Page 6
Alkaline proteose from ​S. clavuligerus
771
Figure 10. ​Effect of molasses on growth (
l
) and ​protease
production (p) in ​Streptomyces clavuligerus.​
Figure 12. ​Effect of whey on growth (
l
) and ​protease
production (p) in ​Streptomyces clavuligerus.​
Figure 11. ​Effect of wheat flour on growth (
l
) and ​protease
production (p) in ​Streptomyces clavuligerus.​
Production of the enzyme at large scale is an important step
to realize the actual potential of the organisms. However, the
remarkable opportunities that the uncommon and relatively less
explored actinomycetes present for biotechnological applications
might not be realistic in actual economic sense. The results
describe the optimization of the parameters for alkaline ​protease
production at lab-scale in a salt-tolerant and alkaliphilic
Streptomyces clavuligerus​. The organism produced ​protease
optimally with 5% w/v NaCl at pH 9 during early stationary phase
in the gelatin broth. Organic nitrogen sources were better for
growth and enzyme production compared to inorganic ones.
The growth and ​protease​ secretion with molasses, whey and
wheat flour - the cheaper carbon and nitrogen sources, appear
to be a commercially attractive feature. The results in general
reflect on the enzymatic potential and its optimization among
the relatively less explored group of actinomycetes.
RESUMO
Secreção de uma protease alcalina por uma cepa
halotolerante e alcalifílica de ​Streptomyces
clavuligerus, M​ it-1
Uma cepa halotolerante e alcalifílica de ​Streptomyces
clavuligerus,​ Mit-1, foi isolada em Mithapur, na costa oeste da
Índia. Esse microrganismo é Gram positivo e apresenta estrutura
filamentosa na forma de longas cordas. A esporulação iniciou
após dois dias de cultivo e o nível ótimo de produção de ​protease
alcalina (130 U/ml) foi atingido no início da fase estacionária de
crescimento. Acepa foi capaz de multiplicar com 0-10% NaCl
(w/v), com um ótimo de 5% NaCl (w/v). O ótimo de crescimento
e produção de ​protease​ foi atingido em pH 9, apresentando
declínio substancial em pH neutro. Sacarose e gelatina foram as
melhores fones de carbono e nitrogênio, respectivamente,
enquanto o caldo gelatina foi o melhor meio para produção de
protease​. A cepa Mit-1 produziu bastante ​protease​ quando
vários aminoácidos foram empregados como única fonte de
nitrogênio. Substratos crus, como melaço, soro de leite e farinha
de trigo, tiveram um efeito significativo na produção da enzima.
Os resultados são bastante interessantes, considerando que
somente poucos actinomicetos, especialmente os
halotolerantes, já foram explorados por seu potencial de
produção de enzimas e otimização de processos.
Palavras-chave: ​Streptomyces clavuligerus, ​actinomicetos
halotolerantes, ​protease​ alcalina, potencial enzimático,
otimização de ​protease
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ACKNOWLEDGEMENT
Financial Assistance from University Grants Commission
(New Delhi, India) and Saurashtra University, Rajkot (India) is
acknowledged.

 
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