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Available online at www.jpsscientificpublications.com
Volume – 3; Issue - 5; Year – 2017; Page: 1228 – 1250
DOI: 10.22192/iajmr.2017.3.5.2
Indo – Asian Journal of Multidisciplinary Research (IAJMR)
ISSN: 2454-1370
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
COMMERCIAL PRODUCTION AND APPLICATION OF BACTERIAL
ALKALINE PROTEASE: A REVIEW
P. Saranraj1*, A. Jayaprakash
2 and L. Bhavani
2
1Department of Microbiology, Sacred Heart College (Autonomous), Tirupattur – 635 601, Tamil Nadu,
India. 2Department of Biochemistry, Sacred Heart College (Autonomous), Tirupattur – 635 601, Tamil Nadu,
India.
Abstract
Microbial proteases are among the most important hydrolytic enzymes and have been extensively
since the advent of enzymology. They are essential constituents of all forms of life on earth. They can be
cultured in large quantities in relatively short time by established fermentation methods and produce an
abundant, regulate supply of the desired product. In recent years there has been a phenomenal increase in
the use of alkaline protease as industrial catalysts. Proteases are enzymes occurring everywhere in nature
be it inside or on the surface of living organisms such as plants, animals and microbes. Proteases are
ubiquitous being found in all living organisms and are essential for cell growth and differentiation. The
extracellular proteases are of commercial value and find multiple applications in various sectors. The
inability of the plant and animal proteases to meet current world demands has led to an increased interest in microbial proteases which account for the total worldwide enzymes sale.
Key words: Enzymes, Protease, Bacillus sp., Industrial application.
1. Introduction
Proteases are the group of enzymes that
have been found in several microorganisms like
bacteria and fungi which are involved in
breakdown of complex protein molecules into
simple polypeptide chains (Absida, 1985). The
induction of protease requires a substrate like
peptone, casein and other proteins. The ammonia
as final product of enzymatic reaction of
substrate hydrolysis, responses enzyme synthesis
by a well known mechanism of catabolite
repression. This extracellular protease has also
been commercially exploited to assist protein
degradation in various industrial processes
(Srinubabu et al., 2007).
*Corresponding author: Dr. P. Saranraj Received: 18.06.2017; Revised: 24.07.2017; Accepted: 30.08.2017. E.mail: [email protected].
Extracellular protease high commercial
value and multiple application in various
industrial sectors, such as detergent, food,
pharmaceutical, leather, diagnostic, waste
management and silver recovery industries
(Godfrey and West, 1996). Among proteases,
alkaline proteases are defined as enzymes that
are active from the neutral to the alkaline pH
range (Gupta et al., 2002). These enzymes are
generally active between pH 9.0 and 11.0 with
the exception of a few higher pH values of about
12.0 and 13.0 (Kumar and Takagi, 1999). The
microbial protease deals with very large group of
enzymes from the complete diversity of
microorganisms. Microbial proteases are
ubiquitous in all microorganisms where they
have a variety of biochemical, physiological, and
regulatory functions. Microbial proteases,
especially from Bacillus sp. have traditionally
held the predominant share of the industrial
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1229
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
enzyme market of the worldwide (Beg and
Gupta et al., 2003).
Bacteria belonging to Bacillus sp. are by
far the most important source of several
commercial microbial enzymes. They can be
cultivated under extreme temperature and pH
conditions to give rise to products that are in turn
stable in a wide range of harsh environments.
Bacillus is a rod shaped, Gram positive, spore
forming, aerobic, usually catalase positive,
chemoorganotropic bacterium. Alkaliphilic
Bacillus sp. can be found mostly in alkaline
environments such as soda soils, soda lakes,
neutral environments and deep - sea sediments.
Animal manure, man - made alkaline
environments such as effluents from food,
textile, tannery and potato processing units,
paper manufacturing units, calcium carbonate
kilns and detergent industry are also good
sources (Akbalik, 2003; Siva Sakthi et al., 2011;
Saranraj et al., 2012; Geetha et al., 2012).
Protease are among the most valuable
catalysts used in food, pharmaceutical and
detergent industries because they hydrolyze
peptide bonds in aqueous environments and
synthesize peptide bonds in microaqueous
environments (Ogino et al., 1999). Microbial
proteases dominate the commercial applications
with large market share taken from Bacillus
subtilis. For laundry detergent applications, a
major requirement for commercial applications
is thermal stability because thermal denaturation
is a common cause of enzyme inactivation (Kavi
Karunya et al., 2011; Senthilkumar et al., 2012;
Naidu and Saranraj, 2013).
Considering the commercial significance
of proteases, there were some attempts to study
and maximize protease production and
economize them in detergents (Chauhan and
Gupta et al., 2004). For the prospective uses of
proteases and their high demand, the need exists
for the invention of new strains of marine
bacteria that produce enzymes with novel
properties and the development of low cost
industrial media formulations (Esakkiraj et al.,
2011; Annamalai et al., 2013). Optimization of
media components by classical methods which
involves the change of single variable
optimization strategy has some disadvantages,
such as time consuming, requirement of more
experimental data sets, and missing the
interactions among variables (Cazetta et al.,
2007; Li et al., 2008). Owing to these
disadvantages, it has been replaced by statistical
optimization such as response surface
methodology, which is an efficient experimental
strategy to seek optimal conditions for the multi-
variable system. This method has been
successfully applied for the optimization of
multiple variables in many fermentation
processes and showed satisfactory results
(Montgomeryd and Runger, 2002).
Enzyme cost is also the most critical
factor limiting wide use of alkaline proteases for
different applications. A large part of this cost is
accounted for the production cost of the enzyme
which includes cost of media components as
well as downstream processing. In submerged
fermentation up to 40 % of the total production
cost of enzymes was due to the production on
the growth substrate (Enshasy et al., 2010; Siva
Sakthi et al., 2012; Saranraj and Stella, 2013).
The protease production mainly requires
the appropriate substrates. There are many
substrates used for protease production, which
include skim milk, milk, peptone and casein.
Some of the agricultural wastes, animal wastes,
and plant wastes are also used as substrates for
the production of protease, because they are
readily available and economically very cheap
and also they have high protein content. Yang et
al. (1999) stated that whey is one of the good
substrates used for protease production due to its
high protein content. The experiments showed
that the whey produced in dairies constituted a
large amount of protein and consequently the
study of its utilization by fermentation process
could be of greater significance (Romero et al.,
1998; Saranraj and Naidu, 2014).
The thermostable proteases are
advantageous in some applications, due to
employing higher processing temperatures, thus
yielding faster reaction rates, increasing
solubility of nongaseous reactants and products
and reducing incidence of microbial
contamination by mesophilic organisms.
Proteases secreted from thermophilic bacteria
are unique and have become increasingly useful
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1230
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
in a wide range of commercial applications
(Adams and Kelly et al., 1998).
The potential use of thermostable
enzyme in range of biotechnological applications
is widely acknowledged. Thermostable proteases
are advantageous in some applications because
higher processing temperature can be employed,
resulting faster reaction rates due to a decrease
in viscosity and increase in diffusion co-efficient
of substrates. Furthermore, higher processing
temperature will also increase the solubility of
nongaseous reactants and products as well as
reduce the incidence of microbial contamination
by mesophilic organisms (Olajuyigbe and Ajele,
2005). It was expected that the applications will
keep increase in the future as will the need for
stable biocatalysts capable of withstanding harsh
conditions of operation which occurred normally
in industry (Beg and Gupta, 2003; Ellaiah et al.,
2003; Nascimento and Martins, 2004).
2. Alkaline Protease
Alkaline proteases are one of the most
important groups of microbial enzymes that find
varied uses in various industrial sectors such as
leather, detergents, textile, food and feed etc.
Industrially important alkaline proteases from
bacterial sources have been studied extensively,
of which Bacillus sp. was most reported. Most of
the alkaline proteases that play a role in
industries are thermostable as their optimal
activity lies between 50 ˚C to 70 ˚C. The
recently used statistical methods have given way
to a more rapid optimization process for alkaline
protease production. Other than traditional
industrial uses, alkaline proteases have
promising application in feather degradation and
feather meal production for animal feed (Singhal
et al., 2012).
An effective proteolytic enzyme
producing microbial strain has been isolated
from marine soil banana tree and evaluated its
extracellular protease production properties with
respect to different fermentative physiological
parameters. The strain has been identified based
on biochemical tests according to Bergey’s
Manual of systematic Bacteriology as Bacillus
sp and designated as SVN12. This strain has
potential to hydrolyse Starch, Tributyrin, Gelatin
and Casein revealing its industrial potential for
production of multi-enzyme complex. Since the
isolated strain which is not inhibited by EDTA
suggesting the enzyme not belongs to the
metalloprotease. But the produced enzyme is
inhibited by phenyl methyl sulfonyl fluoride
(PMSF) suggesting the enzyme belongs to the
serine type of protease. The maximum enzyme
production is observed at pH 8.0 and incubated
at 37˚C under aerated environment. Analysis of
the pH profile before and after fermentation
depicted that irrespective of initial medium pH,
it is shifted to pH 9.0 after fermentation
suggesting the enzyme produced is alkaline in
nature. The strain Bacillus SVN12, showed the
maximum growth at 37˚C with alkaline protease
production of 9900 U/ml in 72h of incubation at
pH 8.0 and at rpm 150 with 1.0 % inoculums.
Several carbon and nitrogen sources were
screened to understand their impact on growth
and subsequent production of enzyme (Srinivas
et al., 2013).
Microorganism was found to be closely
related to Bacillus cereus based on 16S
ribosomal DNA sequencing. The culture
conditions for higher protease production were
optimized with respect to carbon and nitrogen
sources, metal ions, pH and temperature.
Maximum protease production was obtained in
the medium supplemented with 1 % skim milk, 1
% starch and 0.6 % MgSO4.7H2O, initial pH 8.0
at 35 °C. The best enzyme production was
obtained during the stationary phase in which the
cell density reached to 1.8 × 108 cells/ml. The
level of protease was found to be low in the
presence of inorganic nitrogen sources. The
protease production was diminished in the
presence of sucrose and lactose. The extreme
stability towards Triton X-100, Tween 20 and
SDS was observed by Bacillus sp. CA15
alkaline protease. The enzyme activity was
inhibited by PMSF suggested that presence of
serine residues at the active sites (Fikret et al.,
2011).
Roja et al. (2012) isolated and identified
the Bacillus licheniformis and used to examine
the changes in alkaline protease production
following UV irradiation. Induction of mutation
in Bacillus licheniformis strain was carried out
by 0, 3, 6, 9, 12, 15, 18 and 20 min with 30-W
germicidal lamp that has radiation at 2540 –
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1231
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
2550 A0 at a distance of 15 cm in dark and
irradiated. A total of 17 mutants were selected.
They were designated as Bl1 to Bl9 and Bl10 to
Bl17. Among these, only three strains viz., Bl2,
Bl11, and Bl16 did exhibit high efficiency in
production on the basis of relative growth
production. Of the seventeen mutants of Bacillus
licheniformis, ten were chosen to assay their
productivity. Mutants like Bl8, Bl3, Bl16 were
the most effective in enzyme production under
submerged conditions being 180, 140, 128 U/ml
respectively. Results of their research revealed
that the alkaline protease activity assay under
submerged culture conditions was more accurate
than the relative growth production method
because there is no correlation between zone
diameter and the ability to produce the enzyme
in submerged cultures. High level of
productivity was increased with Bl8 mutant of
Bacillus licheniformis, indicating that the
enzyme is to be thermo-alkaliphilic protease.
Among the various protease producing
isolates, two species namely Bacillus
licheniformis and Bacillus coagulans efficiently
produced alkaline protease in glucose extract –
asparagine (GYA) medium. The protease
production efficiency of these organisms was
measured with different carbon sources,
incubation time, pH and temperature. Enzyme
production was better in Bacillus licheniformis
than in Bacillus coagulans. From the above
investigations, it was concluded that the protease
production by these microbes at wide
temperature and pH ranges could be explored for
varied industrial applications (Asokan and
Jayanthi, 2010).
The protease enzyme was found to be a
thermostable alkaline serine protease with
optimal activity at 75 °C and pH 10. The enzyme
had a half life of 45 min at 80 °C and 12 hrs at
70 °C. It was stable over the pH range of 5.0 to
11.0. The enzyme was inhibited by
phenylmethane - sulfonyl fluoride and EDTA
but not by N-Tosyl-L phenanyl alanine
chloromethyl, iodoacetamide and o-
phenathroline. The ions Ca2+
and Fe2+
at 0.5 and
2.5 mM concentration were stimulatory, while
Mg2+
and Mn2+
had little effect on the enzyme
activity. The enzyme produced by bacterium
Bacillus sp. was concluded to be an alkaline
protease that requires calcium and iron ions for
its activity (Parawira and Zvauya, 2012).
3. Production of Alkaline Protease from
Proteolytic Bacillus isolates
Proteolytic enzymes can be produced by
submerged and solid state fermentation. For the
growth of fungi, Solid state fermentation is most
appropriate method because it resembles the
natural habitat of the fungi. Some characteristics
make Sold state fermentation (SSF) more
attractive than Submerged fermentation (SMF):
simplicity, low cost, high yields and
concentrations of the enzymes and the use of
inexpensive and widely available agricultural
residues as substrates (Chutmanop et al., 2008).
Solid state fermentation (SSF) is
preferred over Submerged fermentation (SMF)
since it exhibit advantages such as; reduced
production cost, higher yield and less energy
consumption (Pandey, 2003). Proteases are also
envisaged as having extensive applications in
development of eco-friendly technologies as
well as in several bio-remediation processes
(Bhaskar et al., 2007; Wang et al., 2008). Most
of the studies on microbial proteases are
confined to characterization of enzymes with
relatively fewer reports on optimization of
enzyme production (Bajaj and Sharma et al.,
2011).
Proteases can resist extreme alkaline
environments produced by a wide range of
alkalophilic microorganisms. Different isolation
methods are discussed which enable the
screening and selection of promising organisms for industrial production. Further, strain
improvement using mutagenesis and
recombinant DNA technology can be applied to
augment the efficiency of the producer strain to
a commercial status. The various nutritional and
environmental parameters affecting the
production of alkaline proteases are delineated.
The purification and properties of these
proteases was also discussed by various
researchers, and the use of alkaline proteases in
diverse industrial applications was highlighted
(Ganesh and Hiroshi, 1999).
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1232
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
Li et al. (2008) isolated a 41 Bacillus
subtilis from a raw milk sample. Forty-one
isolates with a clear zone surrounding a colony
were primary selected and identified by using
staining techniques, biochemical characteristics
and growth of bacteria at 50 ˚C. Ten out of 41
isolates showing a clear zone diameter of more
than 10 mm were selected and evaluated for the
presence of protease activity. The BA26 and
BA27 gave high levels of protease activity with
12 U/ml protein towards 1.5 % casein at 50 ˚C
for 10 min. Based on the biochemical and
physiological characteristics, BA26 and BA27
were classified as Brevibacillus non reactive.
However, their 16S rRNA gene sequence
showed 99 % identity to that of Bacillus subtilis.
The enzymes were more specific to 1 % casein
than 1 % gelatine. Moreover, the selected
bacteria selected extracellular protease upon
incubation at 50 ˚C and 121 ˚C. This confirmed
that the enzyme proteases produced by Bacillus
sp. are thermotolerant proteases.
Randa et al. (2009) isolated thermostable
organic solvent-tolerant protease producer and
identified as Bacillus subtilis strain, based on the
morphological characteristics, biochemical
properties and 16S rRNA analysis. The
production of the thermostable organic solvent
tolerant protease was optimized by varying
various physical culture conditions. Inoculation
with 5.0 % (v/v) of inoculum size, in a culture
medium (pH 7.0) and incubated for 24 hrs at 37
°C with 200 rpm shaking, was the best culture
condition which resulted in the maximum
growth and production of protease (444.7 U/ml;
4042.4 U/mg). The protease was not only stable
in the presence of organic solvents, but it also
exhibited a higher activity than in the absence of
organic solvent, except for pyridine which
inhibited the protease activity. The enzyme
retained 100 %, 99 % and 80 % of its initial
activity, after the heat treatment for 30 min at 50
°C, 55 °C and 60 °C respectively.
Debananda et al. (2010) analyzed the
biochemical, physiological characterization and
acid production from various carbohydrates by
API 50 CHB tests led to its identification as
Bacillus subtilis and it was designated as
Bacillus subtilis strain. Corn starch (1 %) and
peptone (0.2 %) was as optimal C and N sources
for protease production. The enzyme was active
over a wide range of temperatures and pH with
optima at 500 °C and pH 8. It was inhibited by
PMSF as well as EDTA and seems to be a
metal-activated serine protease or a mixture of
enzymes. SH1, interestingly, was stimulated by
FeSO4.
Geethanjali and Anitha (2011) screened
the best protease producing Bacillus subtilis.
Then, production medium for Bacillus subtilis
were optimized by using different pH,
temperature, carbon and nitrogen sources for 48
hours fermentation period. The findings of their
study revealed that the protease production can
be optimized at pH – 9.0, temperature 40 ˚C by
utilizing carbon as glucose and nitrogen source
as peptone.
Sharma and Aruna (2011) carried out the
primary screening for protease production by
observing the zone of clearance on Skim milk
agar, GYEA milk agar and Gelatin plates.
Different parameters like temperature, pH,
incubation time and aeration studies were
initially done to get maximum protease
production. A temperature of 55 ºC and pH 9
gave maximum production in 24 hours under
shaker conditions. Different carbon and nitrogen
source in the form of fine powder of organic and
inorganic meals were studied to select a suitable
substrate for protease production. The highest
level of protease was obtained to be the best
inducer while inorganic source in the form of
ammonium salts repressed the enzyme activity.
Media components at 0.2 % MgSO4, 0.05 % KCl
was found to give maximum enzyme activity.
The substrates with highest water
absorption index and more heterogeneous
granulometric distribution have positively
influenced on protease production. Some
cultivation parameters were studied by Ruann
Janser Soares and Helia Harumi (2013) and the
results showed that the optimum fermentation
medium was composed of wheat bran, 2.0 %
(w/w) peptone and 2.0 % (w/w) yeast extract,
and the conditions for maximum protease
production were an initial moisture content of
50.0 %, an inoculums level of 107 spores g
-1 and
an incubation at 23 °C. The biochemical
characterization using experimental design
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1233
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
showed that the enzyme was most active over
the pH range 5.0 – 5.5 and was stable from pH
4.5 to 6.0, indicative of an acid protease. The
optimum temperature range for activity was 55 –
60°C and the enzyme was stable at 35 – 45°C.
The results showed that wheat bran have great
potential as support matrix for protease
production by Aspergillus oryzae in Solid State
Fermentation (SSF).
Mrunmaya et al. (2013) tested the ability
of the bacterium to tolerate high temperatures
and identified as Bacillus amyloliquefaciens by
morphology, biochemistry and sequencing of its
16S rRNA gene. BLAST search analysis of the
sequence showed maximum identity with
Bacillus amyloliquefaciens. The identified strain
exhibited considerable protease activity.
Phylogenetic analysis of the isolate revealed
close affiliation with thermophilic Bacillus
species. The G + C content were found to be
54.7 %.
Marcela et al. (2013) isolated hundred
and fifty six isolates and type strains Bacillus
subtilis and Bacillus amyloliquefaciens were
classified according to phenotypic and molecular
characteristics. Only differences in growth
temperature could be used to distinguish isolates
among the phenotypic traits tested and these
distinctions were supported by molecular
analysis. Randomly amplified polymorphic
DNA analysis (RAPD) analysis was shown to be
a friendly, technically simple and accurate
method for rapid screening and identification of
Bacillus subtilis and Bacillus amyloliquefaciens.
Further analysis of 16S rRNA, rpoB and gyrA
gene sequences of the isolates was done to
confirm species identification. Sequences from
the isolates and type strains showed between
96.5 – 100 % (16S rRNA), 94.8 – 100 % (rpoB)
and 80.6 - 99.6 % (gyrA) similarity, thus
allowing for more refined distinction using the
rpoB and gyrA genes. In addition, gyrA gene
sequences had greater discrimination potential in
having higher divergence between species (18.2
± 0.7 %) than did rpoB sequences (4.9 ± 0.3 %).
BOX PCR fingerprinting was shown to have the
potential for analysis of genotypic diversity of
these species at the strain level.
Lakshmi and Prasad (2013) examined the
changes in alkaline protease production by
Bacillus licheniformis following UV irradiation.
Induction of mutation in Bacillus licheniformis
strain was carried out by 0, 3, 6, 9, 12, 15, 18
and 20 min with 30-W germicidal lamp that has
radiation at 2540 – 2550 A0 at a distance of 15
cm in dark and irradiated and then total of 17
mutants were selected. They were designated as
Bl 1 to Bl 9 and Bl 10 to Bl 17. Among these
Bacillus licheniformis isolates, only three strains
viz., Bl2, Bl11 and Bl16 did exhibit high
efficiency in production on the basis of relative
growth production (C/G). Of the seventeen
mutants of Bacillus licheniformis, ten were
chosen to assay their productivity. Mutants no
Bl8, Bl3, Bl16 were the most effective in
enzyme production under submerged conditions
being 180, 140, 128 U/ml respectively. Results
of their study revealed that the alkaline protease
activity assay under submerged culture
conditions was more accurate than the relative
growth production (C/G) method because there
was no correlation between zone diameter and
the ability to produce the enzyme in submerged
cultures. High level of productivity increased
with Bl8 mutant of Bacillus licheniformis,
indicating that the enzyme is to be thermo-
alkaliphilic proteae.
4. Factors influencing Protease production by
Bacillus species
Microbes which produces alkaline
protease needs to be screened and should be
optimized to produce substantial amount of protease by adapting favourable conditions like
optimal pH, temperature and favourable media
should be demonstrated to increase its yield.
Alkaline protease from extreme organisms
should be produced commercially in high yield
at a low-cost method (Rajesh et al., 2005).
Although, there are many microbial sources
available for producing proteases, only a few are
recognized as commercial producers. A large
proportion of the proteases are derived from
Bacillus strains (Wang et al., 2006).
All microorganisms have their optimal
conditions for their growth, reproduction and
other physiological activities. Depending upon
the nutritional factors such as carbon and
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1234
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
nitrogen sources, environment factors like
incubation temperature and cultural conditions
like pH their growth, reproduction and
physiological activities showed significant
different in growth and enzyme production.
Valerie et al. (2009) quantitatively
assessed and showed that the strains of Bacillus
subtilis, the Bacillus cereus group, Paenibacillus
polymyxa and Bacillus amyloliquefaciens are
strongly proteolytic, along with Bacillus
licheniformis, Bacillus pumilus and
Lysinibacillus fusiformis to a lesser extent.
Lipolytic activity could be demonstrated in
strains of Bacillus subtilis, Bacillus pumilus and
Bacillus amyloliquefaciens. Qualitative
screening for lecithinase activity was also
revealed that Paenibacillus polymyxa strains
produce this enzyme besides the Bacillus cereus
group that was well known for causing a ‘bitty
cream’ defect in pasteurized milk due to
lecithinase activity. They found a strain of
Paenibacillus polymyxa were able to reduce
nitrate. A heat-stable cytotoxic component other
than the emetic toxin was produced by strains
identified as Bacillus amyloliquefaciens,
Bacillus subtilis, Bacillus pumilus and the
Bacillus cereus group. Variations in expression
levels between strains from the same species
were noticed for all tests. The importance of
aerobic spore forming bacteria in raw milk as the
species that are able to produce toxins and
spoilage enzymes are all abundantly present in
raw milk. Moreover, some strains are capable of
growing at room temperature and staying stable
at refrigeration temperature.
Nisa et al. (2010) optimized the protease
production by bacterial strain, seven
fermentation variable were screened using a
Placket-Burman design, and were then further
optimized via Response surface methodology
(RSM) based on a Central composite design
(CCD). Three significant variables, i.e., soy
flour, skimmed milk and shaker speed were
selected for their study. The optimal values were
2.0 % soy flour, 0.1 % skimmed milk and a
shaker speed of 280 rpm. The experimental
result (1537 units/ml) in a medium optimized for
protease production was in good agreement with
the predicted value of a quadratic model (1576
units/ml), thus confirming its validity. In
addition, the adequacy of the model was
supported by a coefficient of determination (R2)
of 0.912. protease production in the optimized
medium (1537 units/ml) in the shaken flask
culture, when the experiment was scaled up in a
stirred tank reactor, 1891 units/ml protease
activity was achieved at 27 hrs of cultivation,
which was an overall 2.6 fold increase over the
basal medium.
Gitishree and Prasad (2010) identified
the Bacillus subtilis and the isolated bacterial
were positive on Skim milk agar (1 %) and
selected as protease producing strain. The
Bacillus subtilis were tested for various
biochemical tests, which lead to the production
of Bacillus subtilis producing protease enzyme.
These Bacillus subtilis could group up to 40 ºC
and pH range 6 - 9 with optimal growth
temperature and pH at 37 ºC and 8.0
respectively. It was also optimized for carbon
test and nitrogen test with optimal growth in
dextrose and peptone respectively. Enzyme
production was carried in 1 litre of optimized
media in the fermented at 37 ºC for 48 hours at
pH 8.0. Harvested protease product was purified
by salt precipitation method. The enzyme
protease was purified by Column
chromatography. The protein was characterized
using SDS-PAGE. The results of their study
showed that the Bacillus subtilis was a good
producer of extra cellular protease, which can be
beneficial for industries.
Ozgur and Nilufer (2011) detected the
protease production from 15 bacteria isolated
from soil samples and the one showed the
highest protease activity was selected. The strain
was identified and determined as Bacillus cereus
by 16S rRNA phylogenetic analysis. After
optimization of protease production from the
novel medium, the Michaelis - Menten kinetics
was also studied. Temperature, pH and, time
parameter of protease incubation was determined
and maximum temperature was detected at 50 ˚C
as 5.15 IU/ml. The optimum pH range of the
enzyme was in between pH 7-9. The crude
enzyme was approximately 2 - fold purified by
dialysis.
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1235
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Ibrahim Noor and Yusoff (2013) isolated
the bacteria and identified as Bacillus subtilis
and Bacillus licheniformis on the basis of the
16S rRNA gene sequencing. The effect of
temperature, pH and inhibitors on enzymes
activity and stability were investigated. The
crude proteases for both isolates displayed
maximal activity at 70 °C and showed
characteristic pH optima at pH 9.0. Enzymes
activities were totally inhibited by phenyl methyl
sulphonyl fluoride (PMSF) suggested that the
protease from Bacillus subtilis and Bacillus
licheniformis belongs to the family of serine
protease. The thermostability profile exhibited
the protease from Bacillus subtilis was very
stable at 50 °C (maintain 100 % relative
activity) and the protease activity retained 89
% of its original activity after heat treatment at
60 °C for 30 min. Meanwhile, protease activity
for Bacillus licheniformis retained 96 and 72 %
of the original activity after heat treatment at 50
and 60 °C, respectively. Considering their
promising properties, Bacillus subtilis and
Bacillus licheniformis could be a potential
source of enzymes for industrial applications.
Effect of different carbon and nitrogen source
on the Alkaline protease production
Protease production was enhanced 2.3
fold by optimizing the culture conditions. The
nutritional factors such as carbon and nitrogen
sources and also physical factors like pH,
temperature, agitation speed, inoculums level
and incubation period were optimized for the
maximum yield of protease. Studies on the effect of different carbon and nitrogen sources revealed
that lactose and combination of yeast extract and
soya bean meal enhances the enzyme
production. The bacterium Bacillus
stratophericus produced the maximum amount
of enzyme when allowed to grow for 48 hrs at
35 ˚C and pH 10 (Raga et al., 2013). Substantial
level of protease enzyme activity for Bacillus sp.
AGT isolate was achieved at 40 °C, pH 9.0
during 18 hours incubation in our production
medium containing maltose as carbon source
and 0.5 % gelatine as nitrogen source (Ashok et
al., 2012).
Glucose has been reported to be the best
carbon source for protease production by
Bacillus subtilis (Gomma et al., 1990), though
high levels of glucose are also found to repress
protease synthesis in some cases (Battaglino et
al., 1991; Sen and Satyanarayana, 1993).
Similarly, starch has been reported as a good
source of alkaline production by Bacillus
licheniformis (Sinhan and Satyanarayana, 1991).
Among the various nitrogen compounds tested
in early research, 0.5 % (w/w) urea was found to
be the best one followed by Tryptone, Yeast
extract, Organic nitrogen, Ammonium nitrate,
Ammonium sulphate and Potassium nitrate for
protease production by Bacillus sp. Among the
tested carbon compounds, 0.5% (w/w) lactose
was observed as the best followed by fructose
and glucose for protease production by Bacillus
sp. While growth and protease production was
optimum at 5% (w/v) NaCl, only marginal
growth without enzyme production was evident
in the absent of salt. The protease had to highest
activity at pH 8.0 and 35°C for 48 hours
incubation and inoculum level played a vital role
in a protease production was found to be
associated with the growth of the bacterial
culture (Kuberan et al., 2010).
Reddy et al. (2007) selected four
significant variables (corn starch, yeast extract,
corn steep liquor and inoculum size) for the
optimization studies. The statistical model was
constructed via central composite design (CCD)
using three screened variables (corn starch, corn
steep liquor, and inoculum size). An overall 2.3
fold increase in protease production was
achieved in the optimized medium as compared
with the unoptimized basal medium. Enzyme
activity increased significantly with optimized
medium (939 U ml-1
) when compared with
unoptimized medium (417 U ml-1
).
The maximum alkaline protease activity
was 6.376 U/ml in medium M 6 using casein as
substrate. Temperature of 60 ˚C was found to be
optimum for enzyme production in medium M 3.
Similarly, maximum protease activity was found
at pH 10 in production medium. Among the
different sources, glucose was found to be best
carbon source for production of alkaline protease
and gelatin was found to be the optimum
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1236
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
nitrogen source for protease enzyme production
by Bacillus subtilis (Verma et al., 2011).
The highest protease production was
attained with casein, peptone and mung
seedlings as nitrogen sources. The extracellular
protease production and mycelial growth were
influenced by the concentration of casein. Other
protein sources (yeast extract) supported growth
but did not induce such excellent protease
synthesis and ammonia as end product repressed
it, indicating catabolite repression in this
microorganism. Optimal protease production
was obtained at final pH 5.3 (Arun Kumar et al.,
2011).
Nihan and Elif Demirkan (2011)
estimated the production of protease and the
effects of major medium ingredients such as
carbon, nitrogen sources and metal ions on the
production of the enzyme were investigated.
Among the carbon sources used, fructose
showed the highest potential for the production.
The best organic nitrogen source was skim milk.
Inorganic nitrogen sources were not as effective
as organic sources. Addition of combine metal
ions minimized the enzyme production.
Combinations of Ca2+
and Mg2+
in medium were
the best. Both ions were not effective alone.
Increased production (51 %) of the enzyme was
obtained by manipulating the medium
composition. The optimum pH and temperature
for the purified enzyme activity were 7.0 and 55
°C, respectively. On their research, stability
showed that the enzyme was stable in the
alkaline pH range 6.0 - 9.0 and at temperatures
between 40 and 70 °C. The enzyme was also
thermostable (77 % at 55 °C for 3 hrs). The
enzyme activity was stimulated by Mn2+
and
Ca2+
.
The best source found was glucose for
Bacillus thuringiensis. Effect of glucose
concentration and initial pH on cell and alkaline
production was studied by Sugumaran et al.
(2012). Based on the optimum condition,
alkaline protease production was investigated in
submerged batch fermentation process. The
crude enzyme obtained from fermentation was
subjected to acetone precipitation. Then,
partially purified enzyme was collected. Effect
of temperature, pH and substrate concentration
on alkaline protease activity was studied under
various conditions. The enzyme showed
maximum activity at 50 ºC and at pH 10.
Krishnan et al. (2012) analyzed the
microbiological, biochemical characterization
and 16S rRNA phylogenetic analysis of the
isolated bacterium was Bacillus subtilis with an
optimum alkaline protease producing
temperature, 37 °C and pH 9.0. The maximum
alkaline protease production was achieved at 24
hrs of incubation period. Among various
nitrogen (organic and inorganic) sources, beef
extract was found to be the best inducer for
alkaline protease in the concentration of 1.5 % as
was reported for the maximum alkaline protease
production. Effect of carbon sources for example
xylose, on protease production proved high
protease production than the other tested carbon
sources and subsequently 2 % concentration
registered an optimum to enhance the protease
production. The halotolerancy of Bacillus
subtilis for alkaline protease production
indicated that 3 % of sodium chloride was
optimum to yield maximum protease activity.
During production, agitation rate was 250 rpm at
air flow rate of 1 VVM. Maximum protease
activity of 42.7556 U/ml was observed at the end
of 24 hrs cell free supernatant of fermentation
broth. Crude alkaline protease was most active at
55 °C, pH 9 with casein as substrate. The
produced enzyme could be effectively used to
remove hair from goat and sheep hide indicating
its potential application in leather processing
industry.
Prabhavathy et al. (2013) isolated and
identified the Bacillus subtilis by the sequencing
of 16S rRNA gene and BLAST. In their study,
protease production was optimized with wheat
bran substrate, glucose (carbon source) and
peptone (nitrogen source) with optimum pH 7.0,
temperature of 45˚C and incubation time 96 hrs.
The activity of the enzyme was checked by the
DNS method.
Medium components and culture
conditions for alkaline protease production were
optimized using statistical optimization. Plackett
– Burman design was employed to find out the
optimal medium constituents and culture
conditions to enhance protease production.
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© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
Central composite design revealed that four
independent variables, such as NaCl (60.53 g/L),
beef extract (14.73 g/L), CuSO4 (4.73 g/L) and
pH (10.7) significantly influenced the protease
production. Protease production obtained
experimentally coincident with the predicted
value and the model was proven to bead equate.
The enhancement of protease from 298.34 U/ ml
to 982.68 U/ml was achieved with the
optimization procedure (Annamalai et al., 2013).
Mohamad et al. (2013) isolated and
identified two bacterial isolates viz., Bacillus
amyloliquefaciens and Bacillus subtilis based on
morphological, biochemical characteristics and
16S rRNA gene sequencing. Bacillus
amyloliquefaciens and Bacillus subtilis produced
alkaline keratinolytic serine protease when
cultivated in Mineral medium containing 1 % of
wool straight off sheep as sole carbon and
nitrogen source. The two strains were observed
to degrade wool completely to powder at pH 7
and 37 °C within 5 days. Under these conditions
the maximum activity of proteases produced by
Bacillus amyloliquefaciens and Bacillus subtilis
was 922 U/ml and 814 U/ml respectively. The
proteases exhibited optimum temperature and
pH at 60 °C and 9 respectively. However, the
keratinolytic proteases were stable in broad
range of temperature and pH values towards
casein Hammerstein. Furthermore, the protease
inhibitor studies indicated that the produced
proteases belong to serine protease because of
their sensitivity to PMSF while they were
inhibited partially in presence of EDTA. The
two proteases are stable in most of the used
organic solvents and enhanced by metals
suggesting their potential use in biotechnological
applications such as wool industry.
Effect of pH on the Alkaline protease
production
The enzyme Alkaline protease was stable
in the alkaline pH range (8.0 - 12.0), with the
optimum temperature and pH range of the
proteases being 70 ºC and 6.0 - 12.0,
respectively. All three proteases were also highly
stable at 70 ºC. After 60 min of incubation at 70
ºC, the enzymes retained 100 % of their original
activities. Enzymes were mostly inhibited by
Phenyl methyl sulfonyl fluoride (PMSF),
however 80 – 90 % enzyme activities were
retained in presence of 2-mercaptoethanol and
iodoacetate. Addition of SDS and ethylene
diamine tetra acetic acid (EDTA) also
marginally influenced protease activities, but
addition of Ca2+
to the proteases did not bring
about any change (Li et al., 2008).
The optimum protease activity at pH 9
was 34 Unit/ml at 70 °C for Geobacillus sp. and
46 Unit/ml at 60 °C for Bacillus licheniformis.
The apparent lipase activity for Geobacillus sp.
was 30.4 Unit/ml and 25.86 Unit/ml for Bacillus
licheniformis. Lipase or proteases that produced
from these two Bacillus strains are tested on
artificial fat and protein dirt clothes in presence
and absence of commercial powder detergent to
investigate their cleaning effect. The enzyme
activity of each has been determined and the
results of Amro et al. (2009) proved the
possibility to use the crude enzymes alone or in
combination with the powder detergent in
washing purposes.
The enzyme was active in pH range 7 – 9
and temperature 20 – 50 °C with optimum pH of
8 and temperature 35 °C. Moreover, the enzyme
activity of PA02 protease was not strongly
inhibited by specific inhibitor showing the novel
nature of enzyme compared to serine, cysteine,
aspartyl and metalloproteases. Kinetic studies
indicated that substrate specificity of PA02
protease was towards various natural and
synthetic proteolytic substrates but inactive
against collagen and keratin. These findings
suggest protease secreted by Pseudomonas
aeruginosa MCMB-327 may have application in
dehairing for environment-friendly leather
processing (Vasudeo et al., 2011).
Vidhya et al. (2011) selected the strains
positive on Skim Milk Agar (1 %) as protease
producing strains and biochemically
characterized. The strains were found capable of
growth at temperature >40 ºC and in wide pH
range of 7.0 - 12.0. The enzyme assay of strains
revealed maximum activity at 50 ºC and pH 10.
The enzyme production was carried out at 37 ºC
for 48 hrs in fermentor containing 1 L medium
having pH 8.0. The molecular weight of enzyme
determined through SDS-PAGE, was 6000 kDa.
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1238
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
The optimum pH and temperature for
maximal protease activity was 9.0 and 40 ºC,
respectively. The optimum protease production
was achieved with 0.5 % lactose and 0.5 % yeast
extract added medium. Among the inorganic
nitrogen sources used, the protease production
was supported by the addition of potassium
nitrate. In experimentation with metal ions, the
maximum protease production was observed
(863.44 ± 1.63 U/ml) in the media supplemented
with magnesium chloride. The maximum
amount of protease production was obtained in
Triton X 100 (309.275 ± 1.63 ml) added medium
when compared to the other tested surfactants
(Suppiah Sankaralingam et al., 2012).
Georage et al. (2012) selected Bacillus
sp. which demonstrated the highest protease
activity and used for protease production by
Shake - flask fermentation technique at 180 rpm.
The maximum protease yield for 72 hrs (2.697 +
0.19 IU mc-1
) was achieved under optimized
culture conditions of pH 9.0, temperature of 45
°C and 5 % inoculums density with soy meal (1
%) and sugar cane bagasse (1 %) as nitrogen and
carbon sources of the fermentation medium. the
protease at 72 hrs incubation was significantly (p
>0.05) higher that obtained from expensive
substrates. The protease achieved > 85.7 = 0.08
% hydrolytic activities on the tested nitrogen
wastes with soybean waste being the mostly
hydrolyzed (96.3 = 0.13 %). Their results
indicated the use of soy meal and sugar cane
bagasse as rich substrates for maximum protease
yield and the enzyme hydrolytic activity on
nitrogen wastes suggests its application in
environmental waste degradation.
Effect of different incubation and
temperature on the Alkaline protease
Temperature has a profound influence on
protease production by microorganisms. The
mechanism of temperature control on enzyme
production is not well understood (Chaoupka,
1985). A link also exists between the enzyme
synthesis and energy metabolism in Bacillus,
which is controlled by temperature and oxygen
uptake (Frankena et al., 1986). The
microorganism utilized several carbon sources
for the production of protease. Starch was the
best substrate, followed by trisodium citrate,
citric acid and sucrose. Among the various
organic and inorganic nitrogen sources,
ammonium nitrate was found to be the best.
Studies on the protease characterization revealed
that the optimum temperature of this enzyme
was 60 ºC. The enzyme was stable for 2 hrs at 30
ºC, while at 40 ºC and 80 ºC, 14 % and 84 % of
the original activities were lost, respectively.
The optimum pH of the enzyme was found to be
8.0. After incubation of crude enzyme solution
for 24 hrs at pH 5.5, 8.0 and 9.0, a decrease of
about 51 %, 18 % and 66 % of its original
activity was observed respectively (Wellingta et
al., 2004).
A higher enzyme secretion by Bacillus
licheniformis in the alkaline protease of Bacillus
megaterium was studied by Borriss (1987).
Similarly, pH 6.5 to 7.5 has been reported to be
optimum for neutral proteases of Bacillus
megaterium (Fartima et al., 1989). Jen-Kuo et al.
(1999) optimized conditions for protease
production was found when the culture was
shaken at 30°C for 3 days in 100 ml of medium
(phosphate buffer adjusted to pH 6.0) containing
7 % shrimp and crab shell powder (SCSP), 0.1
% K2HPO4, 0.05 % MgSO4, 1.0 % arabinose,
1.5 % NaNO3, and 1.5 % CaCl2. Under such
conditions, the protease of Bacillus subtilis
attained the highest activity. It was as high as
20.2 U/ml. The protease was purified in a three-
step procedure involving ammonium sulfate
precipitation, DEAE-Sepharose CL-6B ionic
exchange chromatography, and Sephacryl S-200
gel permeation chromatography. The enzyme
was shown to have a relative molecular weight
of 44 kDa by SDS polyacrylamide gel
electrophoresis. The protease was most active at
pH 8.0 and 50 °C with casein as substrate. The
protease was activated by Mn, Fe, Zn, Mg and
Co but inhibited completely by Hg. The protease
was also inhibited by metal-chelating agent such
as EDTA, sulfhydryl reagents as b-
mercaptoethanol, and by cysteine hydrochloride,
Histidine and glycerol. The EDTA was the most
effective inhibitor that caused complete
inhibition of protease. They concluded that this
enzyme is a metal-chelator-sensitive neutral
protease.
The bacterium produced protease at
maximum rate after 48 hrs of incubation at 37 °C
with agitation speed of 170 rpm and 4 % (v/v)
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1239
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
starter culture. The best carbon and organic
nitrogen sources for this bacterium were glucose
and beef extract, respectively. While, the most
effective inorganic nitrogen sources were urea
and lysine. Supplementation of the culture
medium with Mn2+
improved the protease
production substantially. Under these conditions,
Bacillus cereus strain was found to produce
alkaline protease at a maximum rate of
approximately 2.0 μg/ml/min (Norazizah et al.,
2005).
Bacillus subtilis gives the maximum
enzyme production by using papaya peel as the
substrate with the optimized conditions of
incubation time 24 hrs, temperature 300 ˚C,
moisture content 40 % w/v, and inoculums level
of 0.8 % w/v and with substrate concentration of
10 g and pH 8.0, glucose concentration 2.0 %
w/v. The maximum production of protease
enzyme considering all optimum conditions of
various parameters was found to be 0.69 mg/ml
(Meena et al., 2012).
5. Alkaline Protease Extraction and Recovery
Enzyme extraction refers to liberation of
enzymes from cells or cellular constituents.
Extraction may first require mechanical,
physical, chemical or combination of these
methods to disrupt the cell wall or membrane.
For either intra or extra cellular enzyme it may
be necessary to modify the nature of liquid
medium to complete the dissociation (Coxon et
al., 1991). The release of intracellular enzymes
from microorganisms requires violent method of
cell breakage, while extra cellular enzymes from
microbial cells do not require cell disruptions
(Peck et al., 1990).
Calcium alginate was found to be an
effective and suitable matrix for higher alkaline
protease productivity compared to other matrices
studied. All the matrices were selected for
repeated batch fermentation. The average
protease production with calcium alginate was
585 U/ml which is 70 % higher production over
the convention free cell fermentation. Similarly,
the protease production by related batch
fermentation was 380 U/ml with
polyacrylamide, 498 U/ml with agar-agar and
438 U/ml with gelatine respectively (Ram et al.,
2012).
Sadia et al. (2013) selected fifteen
positive mutants on Skim milk agar plates for
shake flask experiments. The Bacillus
licheniformis mutant strain showed 81.21± 3.24
PU/mL alkaline protease activity higher than
parent strain (23.57 ± 1.19 PU/mL) in optimized
fermentation medium. The fermentation profile
like pH (9), temperature (45 °C), inoculum size
(2 ml), incubation time (24 hrs, and kinetic
parameters such as U h-1
, Yp/s, Yp/x, Yx/s, qs,
Qs, qp also confirmed the hyper proteolytic
activity of alkaline protease produced from
Bacillus licheniformis mutant strain over parent
strain and other mutants. Finally, the Bacillus
licheniformis mutant strain was immobilized by
entrapping it in calcium alginate beads and agar.
Alkaline protease production and stability of
biocatalyst were investigated in both free and
immobilized cells. It was concluded that the
immobilized cells were more efficient for
enzyme production then free cells when used
repeatedly.
In the cell immobilization technique, the
free movement of microorganisms is restricted
in the process and a continuous system of
fermentation can be used. This technique has
been used for alkaline protease production using
different carriers such as chitosan, corn cob and
corn tasse. Enzyme activity before
immobilization (72 hrs) was 78.3 U/ml. Corn
cob with 65 % immobilization capacity and the
highest enzyme activity was selected as the best
carrier by various researchers. After
immobilization on the corn cob enzyme, activity
was obtained (119.67 U/ml) (Vida Maghsoodi et
al., 2013).
6. Purification of Protease
The protease enzyme was purified by
Ammonium sulfate precipitation and Sephadex
G 200 filtration. A trial for the purification of
protease resulted in an enzyme with specific
activity of 6381.75 (units/mg prot/ml-1
) with
purification folds 7.87 times. The protease
activity increased as the increase in enzyme
concentration; optimum substrate concentration
(gelatin) was 0.5 % (w/v); an optimum
incubation temperature was 35 ºC. Purified
protease enzyme had a maximum activity at pH
7.0 of phosphate buffer, and the optimum
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1240
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
incubation time was 24 hrs. Data emphasized the
possibility of the production and purification
microbial protease enzyme for application under
industrial scale (El-Safey and Abdul-Raouf,
2005).
The enzyme was purified by precipitation
with 55 – 60 % Ammonium sulfate, Gel
filtration on Sephadex G-100 and DEAE ion
exchange chromatography. The enzyme was
purified 53-fold with 2 % yield. The optimum
pH and temperature for catalytic activity of
protease was pH 6.8 and 80 ºC respectively and
31 % activity of protease remained even after
heat treatment at 100 ºC for 60 min. The relative
activity of the enzyme was highly stable (90 %)
at 50 ºC for 2 hrs. The half-life of the enzyme at
90 ºC, 80 ºC and 70 ºC was estimated to be 3, 4
and 6 hrs, respectively. The activation energy of
denaturation of purified enzyme was 21.7 kJ
mol-1
. Iron, sodium, calcium, and manganese
increased protease activity. On the other hand,
magnesium, cobalt and zinc variably decreased
the residual activity. But, cadmium and copper
drastically inhibited the enzyme activity. The
enzymatic activity was highly stable in the
presence of 1 and 2 mM EDTA at pH 6.8 and 80
ºC. The neutral protease therefore could be
defined as a highly thermostable with new
properties make the present enzyme applicable
for many biotechnological purposes (Hazem et
al., 2012).
Sathyaguru et al. (2011) showed that all
the organisms were capable of producing
maximum Alkaline protease at pH 6 (8.533 to
10.133 IU/ml) and at 50 °C (8.666 to 10.666
IU/mL). The crude enzymes produced by the
tested organisms were individually purified by
two different methods viz., sodium alginate and
ammonium sulphate-butanol methods. The
purity of the protease determined in these two
methods was ranged between 3.24 to 5.44 IU/ml
and 3.13 to 5.55 IU/ml respectively. The
partially purified enzymes were further analyzed
through SDS-PAGE; accordingly the molecular
weight of protein produced by the test organisms
was determined in between 49.44 kDa and 50.98
kDa.
Studies of various researchers involved
partial purification of the isolated Bacillus
protease by protein separation technique and
application of crude enzyme in detergent
formulation and deharing technique. It was
found that pH 9, 37 ˚C, fructose, yeast extract
jack fruit seed, zinc sulphate is optimum for
protease production in the fermentation medium.
The protein profile in sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE)
revealed protein bonds around 50-75 kDa. The
partially purified enzyme showed its distaining
capability against blood stained cloth and
deharing capability on cow skin (Mukesh et al.,
2012).
Aqel (2012) showed the variation
between two Bacillus strains based on their
ability to grow at different pH values and
temperatures, pH 5 - 11 and 28 - 73 ˚C
(HUTBS71) and pH 5 - 7 and 37 - 63 ˚C
(HUTBS62), respectively. The purified enzyme
from the two different strains also showed
variation in purification folds and % yields in
different steps of purification methods.
Ammonium sulfate fractionation was achieved at
75 - 80 % for HUTBS71 and 55 – 60 %
concentrations for HUTBS62. The purification
fold and yield was 10 fold and 67 % for strain
HUTBS71 and 6.5 fold and 61 % for strain
HUTBS62, respectively. Sephadex G-100
purification step achieved 40-fold purification
and 16.7 % yield from strain HUTBS71 and 32-
fold purification and 12 % yield of protease from
strain HUTBS62. DEAE ion exchange
chromatography step achieved 60 fold
purification and 1.7 % yield for strain HUTBS71
and 53 - fold purification and 2 % yield for
strain HUTBS62. The molecular weight of
purified proteases from HUTBS71 and
HUTBS62 was 49 kDa and 48 kDa, respectively.
The target enzyme was purified using a
one-step Aqueous two-phase systems (ATPS)
protocol involving 22 % (w/w) polyethylene
glycol (PEG)-10,000 and 18 % (w/w) citrate
with a yield of 39.7 %, specific activity of 2600
U/mg and purification factor of 4.8. It was
shown to have a molecular weight of 40 kDa by
(SDS-PAGE). The purified thermophile enzyme
was stable in alkaline pH range (9.0 - 11.0) with
the optimum pH of 9.0. It was highly stable at
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1241
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
60 °C and retained 100 % activity even after 90
minutes, suggesting that it belong to the family
of Thermophilus. Collectively, our obtained data
revealed that the thermophilic protease produced
by Bacillus subtilis has the potential application
in industrial processes under high temperature
(Mashayekhi et al., 2012).
Microbes serve as a preferred source for
proteases and a large proportion of the proteases
are derived from Bacillus strains. To purify
protease from Bacillus subtilis and also looked
for its potential application in leather making
process. The results of Sathiya (2013) revealed
that the bacterial strain Bacillus subtilis is a
potent source for protease enzyme. The
purification techniques have proceeded
successfully without any major difficulties and
resulted in an increase in protein concentration.
7. Application of Alkaline Protease
Alkaline proteases are one of the most
important groups of industrial enzymes widely
used in detergent, food and leather tanning
industries. Alkaline proteases can also be used
on the hydrolysis of fibrous proteins such as
horn, feather and hair for converting them into
useful biomass other potential industrial
application of alkaline protease include its
utilization in peptide synthesis, resolution of
racemic mixture of amino acids, hydrolysis of
gelatin laws of X-ray films and also in the
recovery of silver (Anwar and Saleemuddin,
1998; Kumar and Takagi, 1999).
Application of Alkaline Protease in Industries
Food Processing Industries
In food industry, protease helps in
processing and production of food products such
as meat, milk products and beverages which
requires series of enzyme treatment and alkaline
protease is an important enzyme among all.
Proteases are used as tonic for proper digestion
for children. Protease also plays an important
role in processing tea, coffee and coco by
oxidizing for producing complete product.
Microbe helps in sugar fermentation for ethanol
production, along with other enzymes, alkaline
protease also aids in fermentation. Hence,
alkaline protease plays an effective role in
various streams of food processing industries
which also includes meat tenderization.
Leather Making
India is one of the major countries in
leather production and in Tamil Nadu, Vellore
district is well known for its Leather industries.
It stands at second place worldwide. Leather
production involves a complex process such as
soaking, dehairing, bating and tanning.
Traditional method of carrying out of processing
leather was done by treating with chemicals, it
was less efficient and requires huge amount of
chemical and also it produces enormous amount
of toxic compounds to the environment so
biological mean of leather processing was
focused, that is treatment of raw material with
enzymes. One of the major enzyme employed in
this case was alkaline protease. This
conventional method is environmental friendly
and doesn’t cause pollution. Both fungal and
bacterial proteases are used for leather
processing, protease helps in hydrolysis of non
collagenous part of the skin non fibrillar protein.
The leather sample processed by using alkaline
protease was found to have maximum softness.
Thus, the use of protease in leather processing
could eliminate the use of pollution causing
chemicals such as sodium, lime and solvents and
greatly help to prevent environmental pollution.
Currently, alkaline protease with hydrated lime
and sodium chloride are used for dehairing and it
also aids significant low waste production.
Textile Industry
Silk production is the back bone of textile industry, quality of silk determines the
quality of a fabric. Alkaline protease plays a
major role in production of quality silk by
removing gum and other impurities produced
along with silk’s native form, even synthetic
fabric also treated with protease for complete
smooth finish. Indian sericulture field is growing
enormously and hence use of protease is also
been increased. Moreover, protease treatment is
an environmental friendly process rather than
employing chemicals for silk treatment which
causes environment pollution.
The proteolytic enzyme have been used
to solve this problem and shown promising
results not only in the production level but also
Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1242
© 2017 Published by JPS Scientific Publications Ltd. All Rights Reserved
quality of silk. Since, alkaline protease based
degumming was eco-friendly which will be an
additional advantage. Though, the conventional
protease are quite efficient for degumming but
having some disadvantage like thermal and
chemical stability which was one drawback has
to solved, also alkaline protease can hamper the
quality and physical appearance of silk as silk is
quite sensitive to alkali and alkaline protease.
The thermostable protease basically forms
Geobacillus genus has been used for the
enzymatic degumming of silk which are quite
resistant to various chemicals and temperature
(Annavarapu et al., 2011).
Detergent Additives
Enzymes used as detergent was in
practice from long back. The two German
scientists namely, Rohm and Haas used human
protease and sodium carbonate in washing
detergents. Proteinaceous dirt binds strongly to
fabric even after washing without protease.
Protease helps removal of blood and other
proteinaceous compounds. protease has a great
role in industries as detergent for sterilization
since chemical steriliants fails to remove minute
trapped dirts. Hence, microbial protease
commercially produced are used for cleaning
large industrial boilers, surgical instruments and
also for various domestic purposes
Medical applications of Alkaline protease
Alkaline proteases shows a large variety
of functions in medical field, which includes
from basic molecular level to whole organism
therapeutic use such as haemostasis and
inflammation. Alkaline proteases are used
extensively in the pharmaceutical industry for
preparation of medicines such as ointments for
debridement of wounds.
Anti-Inflammatory Activity
Inflammation is the physiological
condition occurs as a result of microbial invasion
or infection which results in accumulation of
immune cell along with plasma. Usual ways of
treating inflammation was treatment with non-
steroidal drug. However, they show several side
effects. To overcome this, COXII targeting
drugs were produced but thou, these specific
drugs are costly. Hence, alkaline protease are
used nowadays used especially Serratio
pepetidase is most effective alkaline protease.
Alkaline protease is available for use in
management of inflammation. Additionally, a
group of serine protease from Indian Earthworm
has been studied for its anti-inflammatory
potential.
Anti-Cancer Activity
Many alkaline protease enzymes plays a
role in normal multiplication of cell count in
biological process, many protease present in its
inactive form ymogen requires activation by
cleavage of small portion of native protein, any
imbalance in this process leads to cancer. On the
other hand, enzymes like caspase primarily
involves in killing of abnormal cells, caspase is
alkaline protease enzyme which aids in proper
immune system. Advantage of using enzyme in
cancer management over chemotherapeutic
agents is to reduce toxicity impart by chemical
based drugs. In the year 2014, a serine protease
from Indian earthworm was evaluated for its
antitumor activity against breast cancer cell lines
and result shown tremendous scope for protease
in development of anti-cancer therapeutics. In
future, enzymatic treatment of cancer can be an
effective remedy over other methods of
treatments.
Clot Dissolving Agent
Blood and thrombus clotting is an natural
phenomenon which occurs as a result of hurt,
were aggregation of thrombus occurs, blood clot
can also be found in many cases like blood
vessel disorder and it ultimately leads to severe
complications. To combat these vascular hurdles, an external clot dissolving agent needed
to perfuse in vascular pipeline. The available
external clot dissolving agents called as
thrombolytic are basically protease. May
recombinant variants like Tissue plasminogen
activators (t-PA), Urokinase (u-PA),
Streptokinase (SK), Staphylokinase (SAK),
Earthworm fibrinolyitc Enzyme (EFE) are
developed for the clinical purpose. Alkaline
protease plays a vital role in external protease
production because of their stability and
substrate selectivity.
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Research Applications of Alkaline proteases
Nucleic Acid Isolation
Cell composed of complex structure with
rigid cell membrane. In order to isolate nucleic
acid from the cell its membrane has to be lyzed
and all other molecules, contaminants has to be
removed. Alkaline proteases are like proteolytic
enzyme aid in obtaining protein free nucleic acid
proportion. The most widely used proteolytic
enzyme in nucleic acid purification is Proteinase
K. The Proteinase K also quickly inactivates the
nucleases which might degrade the nucleic acids
present in the sample. It also helps in preventing
degradation of DNA or RNA and hence, high
yield can be achieved.
Cell Isolation and Tissue Dissociation
Cytology studies rely primarily on
isolation of desire cell which are surrounded by
other molecules and extracellular matrix. The
commonly used method of cell isolation is done
by treating with enzymes there are several
enzymes available in market for the detachment
of cultured cells, cell dissociation and cell
component or membrane -associated protein
isolation. Besides the polysaccharidases,
nucleases and lipases, the proteases are the most
important enzymes used widely to dissociate
cells from tissues, depending on desire type of
cell, enzyme with high specificity are employed.
Collagenase, elastase, amidase, chymotrypsin
and trypsin are some of the proteolytic enzymes
used in cell isolation process.
Cell Culturing
Cell adhered to the culture plate during
cell culture can be separated by treating the
culture with trypsin i.e., trypsinization.
However, trypsin treatment can lead to cleavage
of membrane proteins and receptors, which can
cause significant changes in the expression level
of different proteins so the effect should be
considered and minimized.
Alkaline Proteases in Effluent treatment
One of major cause of water and soil
pollution of this modern era is because of
improper waste water management and
ineffective method of treating solid waste
processing and industrial effluent waste. The
better way of treating this waste can be done by
microbes with xenobiotic property, alkaline
protease plays a major role in waste
management. Kumar and Takagi (1999) reported
an enzymatic process using a Bacillus subtilis
alkaline protease in the processing of waste
feathers from poultry slaughter houses.
Alkaline Proteases in Silver recovery
Silver recovery from photographic films
and x-ray films involves burning the films
directly oxidation of metallic silver followed by
electrolysis stripping the silver-gelatin layer
using microbial enzymes especially protease
which breaks the gelatin layer embedded with
silver in films approximately 1.5 % to 2.0 % (by
weight) silver in its gelatin layers. By using this
method, pollution free stripping can be done.
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DOI Number DOI: 10.22192/iajmr.2017.3.5.2
How to Cite this Article:
P. Saranraj, A. Jayaprakash and L. Bhavani. 2017. Commercial production and
application of bacterial Alkaline protease – A Review. Indo - Asian Journal of
Multidisciplinary Research, 3(5): 1228 – 1250.
DOI: 10.22192/iajmr.2017.3.5.2