Available online at www.sciencedirect.

com

ScienceDirect
Journal of Taibah University for Science 9 (2015) 50–55

Production, optimization and partial purification of protease from

Bacillus subtilis
Gaurav Pant a,∗∗ , Anil Prakash a , J.V.P. Pavani a , Sayantan Bera a , G.V.N.S. Deviram a ,
Ajay Kumar a , Mitali Panchpuri b , Ravi Gyana Prasuna a,∗
aDepartment of Microbiology, GITAM Institute of Science, GITAM University, Visakhapatnam, Andhra Pradesh, India
b Department of Pharmaceutical Sciences, H.N.B. Garhwal University (Central University), Srinagar, Uttarakhand, India
Available online 10 July 2014

Abstract
Proteases have characteristics of biotechnological interest and have thus become important industrial enzymes. We report the
production of thermostable protease and its characterization in Bacillus species, which is a thermotolerant bacterium; Bacillus
subtilis is widely used for isolating protease enzyme. Gelatin was used as the substrate in nutrient agar medium for screening
and showed the maximum zone of activity (22 mm) after overnight incubation and addition of the indicator. Under submerged
fermentation conditions, a high level of protease production was found at 45 ◦ C after 36 h at pH 10, with continuous agitation
(180 rpm). The presence of galactose and peptone in the medium enhanced enzyme production by 0.5% when compared with other
carbon and nitrogen sources. Thus, such additions can augment protease production and their application in various industries.
© 2014 Taibah University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).

Keywords: Bacillus subtilis; Thermostable protease; Zone of inhibition; Non-proteolytic bacteria

1. Introduction

Microorganisms are known to play a vital role

in technology for the production of intracellular and
extracellular enzymes on an industrial scale [1,2]. For
∗ Corresponding author at: Department of Microbiology, GITAM maximum yield, selected organisms are grown in fer-
Institute of Science, GITAM University, Visakhapatnam, Andhra
menters under optimum conditions and can be further
Pradesh 530045, India. Tel.: +91 9703273038; fax: +91 891 2790032.
∗∗ Corresponding author at: Department of Microbiology, GITAM used to make products such as cheese, bread, wine and
Institute of Science, GITAM University, Visakhapatnam, Andhra beer [4–6]. Most reactions inside living cells require
Pradesh 530045, India. Tel.: +91 9014971616/8374600018; enzymes, which act as catalysts and are essential for life
fax: +91 891 2790032. [3,7,8].
E-mail addresses: [email protected] (G. Pant),
Bacillus species are the main producers of extra-
[email protected] (R.G. Prasuna).
Peer review under responsibility of Taibah University.
cellular proteases, and industrial sectors frequently use
Bacillus subtilis for the production of various enzymes.
Proteases are the main enzymes produced from micro-
bial sources, of which only few are recommended as
commercial producers. B. subtilis is found mainly in

http://dx.doi.org/10.1016/j.jtusci.2014.04.010
1658-3655 © 2014 Taibah University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
 G. Pant et al. / Journal of Taibah University for Science 9 (2015) 50–55 51

soil and is also known as hay bacillus and grass bacil- 2.2. Substrate screening and qualitative assay of
lus. It is a rod-shaped organism, which can form a tough, protease
protective endospore and can withstand extreme environ-
mental conditions. Bacillus species are obligate aerobes Gelatin was used as the substrate in nutrient agar
or facultative anaerobe and include both free-living and medium. The culture was streaked in a straight line and
pathogenic species [9,10]. In view of their wide applica- incubated at 37 ◦ C. After 12 h, the plates were flooded
tion in various industries, protease enzymes occupy an with 15% HgCl2 solution in 20% HCl for 5 min. Sub-
important position [11]. strate gelatin at 1% (w/v) was then added to nutrient agar
Alkaline proteases constitute 60–65% of the global medium, sterilized and poured into Petri plates. Wells
industrial market [12,13]. These proteases are the single were made with a sterile cork borer, and 100–200 ␮L of
class of enzymes widely used in detergents, pharma- cell-free culture filtrate were dispensed aseptically into
ceuticals, leather and the food and agriculture industries each well and incubated at 37 ◦ C. After 12 h, enzyme
[14,15]. B. subtilis is widely used for the production activity was visualized as clear zones around the wells
of specific chemicals and industrial enzymes [16,17,1]. due to hydrolysis of substrates in the presence of indi-
Proteases are important components of biopharma- cator solution, and the diameter of the proteolytic zone
ceutical products such as contact lens cleaners and was measured.
enzymatic debriders [18]. Proteolytic enzymes support
the natural healing process in local management of 2.3. Shake flask fermentation with selected medium
skin ulceration by efficient removal of necrotic material
[19]. Alkaline protease was produced in an optimized
Proteases catalyze or hydrolyze protein [20] and production medium of pH 8.0 containing 1% galac-
therefore play a vital role in various industrial applica- tose, 0.5% casein, 0.55% peptone, 0.2% KH2 PO4 , 1%
tions [21,22]. Hyperactive strains are being sought for Na2 CO3 and 0.2% MgSO4 ·7H2 O; 4% inoculum was
use in different industries [23]. Conversion of wastes into added to the production medium and incubated with
useful biomass by microorganisms and their enzymes is continuous shaking. Shaker fermentations were car-
a new trend, and new protease-producing microorgan- ried out at 37 ◦ C for 72 h with controlled agitation at
isms and perfected fermentation technology are needed 150–200 rpm. At the end of the fermentation period,
to meet the ever-growing demand for this enzyme [24]. the whole culture broth was centrifuged at 5000 rpm
The aim of the present study was to purify and char- for 30 min to remove debris, and the supernatant was
acterize alkaline protease from B. subtilis and optimize collected and used for further experiments.
the conditions for maximum production of extracellular
protease. 2.4. Effect of incubation time on protease
production

The optimized protease production medium was pre-

2. Materials and methods pared in six 100-mL conical flasks, into which 1% B.
subtilis culture broth was inoculated and kept for shaker
2.1. Microorganism and culture conditions fermentation. Every 12 h, each flask was filtered, fol-
lowed by centrifugation at 5000 rpm for 30 min. The
Soil samples were collected from side roads near culture filtrate solution obtained was used to assay pro-
Armats Biotek Institute in Chennai. Serial dilutions of tease by qualitative and quantitative methods.
the samples were made up to 10−5 dilutions, spread on
nutrient agar plates and incubated for 24 h at 37 ◦ C. The 2.5. Effect of carbon and nitrogen sources on
colonies were identified as B. subtilis on the basis of protease production
morphological and biochemical characteristics, includ-
ing endospore staining. Colonies were subcultured and The effect of carbon and nitrogen sources for protease
maintained to obtain a pure culture on nutrient agar production was determined with simple and complex
plates, which was transferred to a medium (pH 8.0) con- carbon sources (sucrose, maltose, xylose, galactose and
taining 1% glucose, 0.5% casein, 0.55% yeast extract, starch) instead of dextrose and with peptone, ammo-
0.2% KH2 PO4 , 1% Na2 CO3 , 0.2% MgSO4 ·7H2 O, nium nitrate, ammonium sulphate, urea and ammonium
pH 8 and incubated at 37 ◦ C with continuous shak- chloride as the nitrogen source instead of yeast extract.
ing. Each source was used at a concentration of 1% (w/v)
52 G. Pant et al. / Journal of Taibah University for Science 9 (2015) 50–55

and 0.55% (w/v) to replace carbon and nitrogen sources,

respectively. Protease yield was determined after 72 h of
incubation at 37 ◦ C with shaking at 150–200 rpm. After
production, the yield was estimated quantitatively.

2.6. Separation and purification of protease

The culture was harvested after 2 days’ growth at

room temperature. The crude enzyme preparation was
subjected to ammonium sulphate precipitation, and the
harvested culture was filtered through Whatman no. 1
filter paper of pore size 125 mm and centrifuged (REMI Fig. 1. Proteolytic activity of B. subtilis on (1) gelatin, (2) casein and
(3) skimmed milk.
C-24BL) at 5000 rpm for 30 min at 4 ◦ C. Ammonium
sulphate was added slowly to the cell-free culture fil- 2.8. Effect of pH on activity and stability of protease
trate at 75% saturation to precipitate the protein, with
continuous shaking and a magnetic stirrer. The precipi- Partially purified enzyme in a volume of 500 ␮L was
tated protein was separated by centrifugation at 5000 rpm made up to 1 mL with buffers of various pH, and 0.5 mL
for 30 min and was dissolved in 0.5 mmol/L phosphate gelatin was added. The buffers used were 0.1 mol/L
buffer, pH 7, dialysed and stored. This source of crude acetate buffer (pH 5, 5.4 and 5.8), 0.1 mol/L phosphate
enzyme was used for further enzyme characterization. At buffer (pH 6, 6.4, 6.8, 7.4 and 8.0) and 0.1 mol/L Tris
each step, the protease activity and total protein content buffer (pH 8.8, 8.6 and 9.0). The enzyme and sub-
were measured in an ultraviolet spectrophotometer. strate mixture was incubated at room temperature. After
60 min, the reaction was terminated by adding 5 mL of
110 mmol/L TCA. The enzyme and buffer mixture, with-
2.7. Quantitative assay of protease
out substrate, were pre-incubated for 30 min at room tem-
The protease activity in the culture filtrate of B. sub- perature; then, 5 mL of 0.65% gelatin were added to the
tilis was assayed in a mixture containing 0.5 mL of 0.65% suspensions, and the residual protease activity and stabil-
gelatin as the substrate and 1 mL of cell-free culture ity were estimated quantitatively in a spectrophotometer.
filtrate as the enzyme source. The mixture was incu-
2.9. Effect of temperature on activity and stability of
bated at 37 ◦ C for 10 min, and then 5 mL of 110 mmol/L
protease
trichloroacetic acid (TCA) were added to the reaction
mixture to terminate the enzyme reaction. The reaction Partially purified enzyme in a volume of 500 ␮L was
mixture was centrifuged (REMI C-24BL) at 5000 rpm made up to 1 mL with distilled water, to which 0.5 mL
for 30 min, and the supernatant was collected. To 2 mL of 0.65% gelatin was added. The enzyme and substrate
of supernatant, 5 mL of alkaline solution (500 mmol/L of mixture was incubated for 30 min at 40 ◦ C, 50 ◦ C, 60 ◦ C,
Na2 CO3 ) and 1 mL of 25% Folin phenol reagent (25 mL 80 ◦ C and 100 ◦ C, and the reaction was terminated by
of reagent diluted with 100 mL of glass-distilled water adding 5 mL of 110 mmol/L TCA to the suspension.
before use) were added and the mixture was incubated Residual protease activity was measured. As a control,
at room temperature. After 30 min, the protease activ- the enzyme and buffer mixture was incubated at the
ity was read at 650 nm in a spectrophotometer (U-2900 different temperatures without substrate; after 30 min,
UV/VIS). Simultaneous controls containing enzyme, 0.5 mL of 0.65% gelatin was added, and residual protease
heat-killed enzyme and substrate were maintained. One activity was assayed.
unit of protease activity was defined as the amount of
enzyme required to liberate 1 ␮mol tyrosine/mL per min. 3. Results
The protein content of the culture filtrate was estimated
by the dye-binding method of Bradford [34]. The production of enzymes from microorganisms is
Enzyme activity was calculated from the formula strongly influenced by the composition of the medium,

␮mol tyrosine equivalents released × total volume of assay

Units/mL enzyme =
volume of enzyme used × length of assay × volume used in colorimetric determination
 G. Pant et al. / Journal of Taibah University for Science 9 (2015) 50–55 53

Fig. 4. Effect of pH on activity of protease from B. subtilis.

Fig. 2. (A) Effect of different carbon sources on protease production

by Bacillus sp. (B) Effect of different nitrogen sources on protease
production by Bacillus sp.

Fig. 5. Effect of temperature on activity of protease from B. subtilis.

over the pH range 5–9 at room temperature. The high-

est protease activity was found at pH 7.4 (143.73 U/mL
per min; Fig. 4). Residual protease activity was deter-
mined at different temperatures and was maximal at
40–50 ◦ C, although considerable residual activity (50%)
was observed even after incubation at 80 ◦ C for 30 min
(Fig. 5). Hence, protease obtained from B. subtilis can
be considered a thermostable enzyme. To obtain proteins
from the cell-free culture filtrate, they were precipitated
Fig. 3. The incubation period for protease production from B. subtilis. with ammonium sulphate at 75% (w/v) saturation by
adding powdered ammonium sulphate slowly with con-
temperature, pH, percentage of culture inoculated and tinuous shaking and a magnetic stirrer to avoid bubble
length of incubation. B. subtilis showed higher proteo- formation. The protein precipitate was then dissolved
lytic activity on 0.03 (w/v) gelatin than on casein or and dialysed for 24 h against phosphate buffer. The
skimmed milk in nutrient agar medium (Fig. 1). Car- yield of protein was 2.31 mg/mL, with a total activity
bon and nitrogen sources are also important factors for of 475.56 U/mL per min.
the production of protease in culture medium. Of the
five carbon and nitrogen sources tested, galactose and 4. Discussion
peptone resulted in the highest protease activity (236.31
and 175.083 U/mL per min, respectively), while the least B. subtilis had high proteolytic activity, with a clear
enzyme activity was observed with starch and ammo- zone of 22 mm when gelatin was used as the matrix.
nium chloride (Fig. 2). Enzyme production began 6 h after cell entrapment of
In tests to optimize the time, protease production gelatin and increased gradually to a maximum level
increased gradually from 0 to 36 h, at which it was max- of 10.8 U/mL; thereafter, enzyme production decreased
imal, at 243.28 U/mL per min; it then decreased with [25].
time (Fig. 3). To determine the optimal H+ concentra- B. subtilis grew in the pH range 6–9, with optimum
tion for protease production, B. subtilis was cultivated protease secretion at pH 7.4 and galactose and peptone
54 G. Pant et al. / Journal of Taibah University for Science 9 (2015) 50–55

as the carbon and nitrogen source, respectively, at spe- of soybean hulls supplemented with wheat bran, Process
cific incubation times and temperature. It was observed Biochem. 45 (2010) 120–128.
previously that use of casein as the substrate under the [6] P.M. Schenk, S.R. Thomas-Hall, E. Stephens, U.C. Marx, J.H.
Mussgnug, C. Posten, O. Kruse, B. Hankamer, Second genera-
standard assay conditions gave the highest activity at tion biofuels: high-efficiency microalgae for biodiesel production,
pH 8.0 [26,33]. Other experiments showed that an incu- Bioenerg. Res. 1 (2008) 20–43.
bation temperature of 37 ◦ C, pH 9.0 and glucose and [7] L. Johnson, Symmetry at the molecular level in biology, Eur. Rev.
sodium nitrate as the carbon and nitrogen source, respec- 13 (2005) 77–95.
tively, resulted in the highest production of protease [8] A. Pingoud, M. Fuxreiter, V. Pingoud, W. Wende, Type II restric-
tion endonucleases: structure and mechanism, Cell. Mol. Life Sci.
[27,28]. Protease yields vary considerably with temper- 62 (2005) 685–707.
ature and pH [30,31], which may be attributed to other [9] S.A. Dubal, Y.P. Tilkari, S.A. Momin, I.V. Borkar, Biotechno-
components of the medium and their combined influence logical routes in flavour industries, Adv. Biotechnol. 6 (2008)
on the metabolism of the bacterial species [29,32]. In the 30–45.
present experiment, a high yield of protease was found [10] B.S. Sekhon, Food nanotechnology—an overview, Nanotechnol.
Sci. Appl. 3 (2010) 1–15.
at 50 ◦ C and when alternate nitrogen and carbon sources [11] P. Widsten, A.K. Laccase, Applications in the forest products
were used. industry: a review, Enzyme Microb. Technol. 42 (2008) 293–307.
[12] L.M. Zanphorlin, F.D.A. Facchini, F. Vasconcelos, R.C.B. Santos,
5. Conclusion A. Rodrigues, L.D. Sette, E. Gomes, G.O.B. Rodriguez, Pro-
duction, partial characterization, and immobilization in alginate
B. subtilis can be used for large-scale production beads of an alkaline protease from a new thermophilic fungus
Myceliophthora sp., J. Microbiol. 48 (2000) 331–336.
of alkaline protease to meet present-day needs in the
[13] S.A. Sawi, H.M. Labdane, P. Abiet, An editerpene from the fruits
industrial sector. In the present study, a natural poly- of Juniperus phoenicea L. grown in Egypt and their activities
mer gelatin was used with the nutrient agar medium to against human liver carcinoma, Aust. J. Med. Herbal. 2 (2008)
help in cell immobilization for maximum production of 115–122.
alkaline protease by strains of B. subtilis. We also deter- [14] M.B. Rao, A.M. Tanksale, M.S. Ghatge, V.V. Deshpande,
Molecular and biotechnological aspects of microbial protease,
mined the optimum growth parameters for cultivating the
Microbiol. Mol. Biol. 62 (1998) 597–635.
organism: maximum production of alkaline protease was [15] O.A.T. Azura, L.K. Abubakar, F. Hamid, N.S.A. Radu, S.S.
observed at pH 7.4 and a temperature of 50 ◦ C with galac- Nazamid, Phenotypic and molecular identification of a novel ther-
tose and peptone as the carbon and nitrogen sources, mophilic Anoxybacillus species: a lipase-producing bacterium
respectively. As alkaline protease has more applications isolated from a Malaysian hotspring, World J. Microbiol. Bio-
technol. 25 (2009) 1981–1988.
than protease in industry, alkaline protease production
[16] P. Esakkiraj, G. Immanuel, S.M. Sowmya, P. Iyapparaj, A.
from B. subtilis is recommended. Palavesam, Evaluation of protease-producing ability of fish gut
isolate Bacillus cereus for aqua feed, Food Bioprocess. Technol.
Acknowledgements 2 (2009) 383–390.
[17] W.H. Chu, Optimization of extracellular alkaline protease pro-
The authors are grateful to the management and duction from species of Bacillus, J. Ind. Microbiol. Biotechnol.
staff of GITAM University, Visakhapatnam, Andhra 34 (2007) 241–245.
[18] A. Anwar, M. Saleemuddin, Alkaline protease from Spilosoma
Pradesh, India, and H.N.B. Garhwal University (Central obliqua: potential applications in bio-formulations, Biotechnol.
University), Srinagar, Uttarakhand, India, for providing Appl. Biochem. 31 (2000) 85–89.
facilities for the research. [19] J. Sjodahl, A. Emmer, J. Vincent, J. Roeraade, Characterization
of proteinases from Antarctic krill (Euphausia superba), Protein
References Expr. Purif. 26 (2002) 153–161.
[20] M. Schallmey, A. Singh, O.P. Ward, Developments in the use of
[1] R. Gupta, Q.K. Beg, P. Lorenz, Bacterial alkaline proteases: Bacillus species for industrial production, Can. J. Microbiol. 50
molecular approaches and industrial applications, Appl. Micro- (2004) 1–17.
biol. Biotechnol. 59 (2002) 15–32. [21] R. Nijland, O.P. Kuipers, Optimization of protein secretion by
[2] M. Moo-young, Y. Chisti, Biochemical engineering in biotech- Bacillus subtilis, Recent Pat. Biotechnol. 2 (2008) 79–87.
nology, Pure Appl. Chem. 66 (1994) 117–136. [22] W. Li, X. Zhou, P. Lu, Bottlenecks in the expression and secretion
[3] J.K. Sierecka, Purification and partial characterization of a neutral of heterologous proteins in Bacillus subtilis, Res. Microbiol. 155
protease from a virulent strain of Bacillus cereus, Int. J. Biochem. (2004) 605–610.
Cell Biol. 30 (1998) 579–595. [23] L.L. Fu, Z.R. Xu, W. Li, J.B. Shuai, P. Lu, C.X. Hu, Protein
[4] Y. Lin, S. Tanaka, Ethanol fermentation from biomass resources: secretion pathways in Bacillus subtilis: implication for optimiza-
current state and prospects, Appl. Microbiol. Biotechnol. 69 tion of heterologous protein secretion, Biotechnol. Adv. 25 (2007)
(2006) 627–642. 1–12.
[5] K. Brijwani, H.S. Oberoi, P.V. Vadlani, Production of a cellu- [24] P. Rathakrishnan, P. Nagarajan, R.R. Kannan, Optimization of
lolytic enzyme system in mixed-culture solid-state fermentation process parameters using a statistical approach for protease
 G. Pant et al. / Journal of Taibah University for Science 9 (2015) 50–55 55

production by Bacillus subtilis using cassava waste, Int. J. Chem. [30] V. Maghsoodi, A. Kazemi, P. Nahid, S. Yaghmaei, M.A. Sabze-
Tech. Res. 4 (2012) 749–760. vari, Alkaline protease production by immobilized cells using B.
[25] R. Kumar, R. Vats, Protease production by Bacillus subtilis immo- licheniformis, Sci. Iran. 20 (2013) 607–610.
bilized on different matrices, N. Y. Sci. J. 3 (2010) 20–24. [31] B.K. Bajaj, G. Jamwal, Thermostable alkaline protease pro-
[26] J.K. Yanga, I.L. Shihb, Y.M. Tzengc, S.L. Wanga, Production duction from Bacillus pumilus D-6 by using agro-residues as
and purification of protease from a Bacillus subtilis that can substrates, Adv. Enzyme Res. 1 (2013) 30–36.
deproteinize crustacean wastes, Enzyme Microb. Technol. 26 [32] P. Vijayaraghavan, S. Lazarus, S. Gnana, P. Vincent, De-hairing
(2000) 406–413. protease production by an isolated Bacillus cereus strain AT under
[27] R.S. Rao, Y.D. Deshmukh, P.S. Borkar, C.N. Khobragade, Pro- solid-state fermentation using cow dung: biosynthesis and prop-
duction of alkaline protease from Bacillus subtilis using rice bran, erties, Saudi J. Biol. Sci. 21 (2014) 27–34.
J. Cell Tissue Res. 8 (2008) 1347–1350. [33] D. Karadag, A.E. Makinen, E. Efimova, J.A. Puhakka,
[28] A.S. Qureshi, M.A. Bhutto, I. Khushk, M.U. Dahot, Optimization Thermophilic biohydrogen production by an anaerobic heat
of cultural conditions for protease production by Bacillus subtilis treated-hot spring culture, Bioresour. Technol. 100 (2009)
EFRL 01, Afr. J. Biotechnol. 10 (2011) 5173–5181. 5790–5795.
[29] A. Sonnleitner, Biotechnology of thermophilic bacteria—growth, [34] B. Starcher, A ninhydrin–based assay to quantitate the total
products, and application, Adv. Biochem. Eng. Biotechnol. 28 protein content of tissue smaples, Anal. biochem. 292 (2001)
(1983) 69–138. 125–129.