indo asian journal of multidisciplinary research (iajmr ...€¦ · saranraj et al., 2012; geetha...

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

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Saranraj/Indo – Asian Journal of Multidisciplinary Research (IAJMR), 3(5): 1228 – 1250 1229


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



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 –



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).



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



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



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.



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



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.



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.



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)



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



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



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



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.



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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P. Saranraj, A. Jayaprakash and L. Bhavani. 2017. Commercial production and

application of bacterial Alkaline protease – A Review. Indo - Asian Journal of

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