Cover Image

Screening and Optimization of Physical Parameters for Enhanced Alkaline Protease Production by Alkaliphilic Bacillus Subtilis SH2 Isolate

Jeevan Chandra Sakinala, Anuradha BS, Krishna Reddy V, Pavan Kumar P, Ram Reddy S


The present investigations dealt with the optimization of the physical parameters for production of alkaline protease by alkaliphilic Bacillus subtilis SH-2 isolated from slaughter house soil of Warangal, Telangana State, India. Primary screening of four different samples revealed one potent isolate. Morphological and Biochemical characterization followed by Molecular signature of 16s rRNA homology confirmed that the isolate SH-2 belongs to Bacillus subtilis. Bacillus subtilis SH-2 was screened on four different reported mediums (M1213, M660, Horikoshi and Halophilic Bacillus medium) under shake culture conditions. Maximum alkaline protease production (500 EU/ml) obtained on M1213 and Horikoshi mediums. Further optimization of physical parameters by OVAT method revealed that mean generation time (41.18 min), 4% level inoculum, incubation time 72 hrs, pH 10, temperature 350C and agitation 150 rpm are ideal for enzyme production. OVAT method resulted in 2.2 fold increased production of alkaline protease production (1100 EU/ml).


Alkaliphiles; Bacillus subtilis SH-2; Alkaline protease; Physical parameters optimization and OVAT method

Full Text:



Barrett AJ, Rawlings ND, Woessner JF. (eds) The Handbook of Proteolytic Enzymes, 2nd ed., Academic Press. 2003.

Gaur S, Agrahari S, Wadhwa N. Purification of Protease from Pseudomonas thermaerum GW1 Isolated from Poultry Waste Site. Open Microbiol J. 2010, 4:67-74

Horikoshi K. Production and industrial applications of beta cyclodextrin. Proc Biochem. 1979, 14:26-30

Horikoshi K. Alkaliphiles: some applications of their products for biotechnology. Microbiol Mol Biol Rev. 1999, 63(4):735-750

Chandra SJ, Pallavi P, Anuradha BS, Reddy SR. Optimization of bioprocess for enhanced production of alkaline protease by a Bacillus subtilis SHmIIIa through Plackett-Burman design. Afr J Microbiol Res. 2015, 9(28):1738-1744

Holt JG, Krieg NR, Sneath P HA, Staley JT, Williams ST. Bergey’s manual of determinative microbiology. Williams and Wilkins, Maryland. 1994

Yang SS, Huang I. Protease production by amylolytic fungi in solid state fermentation. J Chinese Agric Chem Soc. 1994, 32:589-601

Sathya G, Naidu N, Panda T. Application of response surface methodology to evaluate some aspects on stability of pectolytic enzymes from Aspergillus niger. Bioche Eng J. 1998, 2(1):71-77

Rao K J, Kim CH,Rhee SK. Statistical optimization of medium for the production of recombinant hirudin from Saccharomyces cerevisiae using response surface methodology. Proc Bio. 2000, 35(7):639-647

Teng Y, Xu Y. Culture condition improvement for whole-cell lipase production in submerged fermentation by Rhizopus chinensis using statistical method. Biores Technol. 2008, 99(9):3900-3907

Satyanarayana T, Pradeep K. Microbial glucoamylases: characterstics and applications. Critic Rev Biotechnol. 2009, 29:225-255

Chu WH. Optimization of extracellular alkaline protease production from species of Bacillus. J Ind Microbiol Biotechnol. 2007, 34(3):241-245

Haddar A, Agrebi R, Bougatef A, Hmidet N, Sellami-Kamoun A, Nasri M. Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: purification, characterization and potential application as a laundry detergent additive. Biores Technol. 2009, 100(13):3366-3373

Jignasha T, Satya T, Singh P. Secretion of an alkaline protease from a salt tolerant alkaliphilic, Streptomyces clavuligerus strain MIT. Braz J Microbiol. 2007, 38:766-772

Moreira KA, Albuquerque BF, Teixeira MFS, Porto ALF, Lima Filho JL. Application of protease from Nocardiopsis sp. as a laundry detergent additive. World J Microbiol Biotechnol. 2002, 18(4):309-315

Kumar DM, Venkatachalam P, Govindarajan N, Balakumaran MD, Kalaichelvan PT. Production and purification of alkaline protease from Bacillus sp. MPTK 712 isolated from dairy sludge. Glob Vet. 2012, 8:433-439

Elibol M, Moreira AR. Optimizing some factors affecting alkaline protease production by a marine bacterium Teredinobacter turnirae under solid substrate fermentation. Proc Bio. 2005, 40(5):1951-1956

Hiraga K, Nishikata Y, Namwong S, Tanasupawat S, Takada K, Oda K. Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2-5. Biosci biotechnol biochem. 2005, 69(1):38-44

Gupta A, Roy I, Patel RK, Singh SP, Khare SK, Gupta MN. One-step purification and characterization of an alkaline protease from haloalkaliphilic Bacillus sp. J chroma. 2005, 1075(1):103-108

Patel RK, Dodia MS, Joshi RH, Singh SP. Production of extracellular halo-alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. isolated from seawater in Western India. World J Microbiol Biotechnol. 2006, 22(4):375-382

Kumar CG, Takagi H. Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol adv. 1999, 17(7):561-594

Esakkiraj P, Immanuel G, Sowmya SM, Iyapparaj P, Palavesam A. Evaluation of protease-producing ability of fish gut isolate Bacillus cereus for aqua feed. Food bioproc technol. 2009, 2(4):383-390

Moon SH, Parulekar SJ. A parametric study ot protease production in batch and fed‐batch cultures of Bacillus firmus. Biotechnol bioeng. 1991, 37(5):467-483

Khosravi-Darani K, Falahatpishe HR and Jalai M. Alkaline protease production on date waste by an alkalophillic Bacillus sp. 2-5 isolated from soil. Afr J Biotechnol. 2008, 7(10):1536-1542

Yu Y, Kim J, Hwang S. Use of real‐time PCR for group‐specific quantification of aceticlastic methanogens in anaerobic processes: population dynamics and community structures. Biotechnol bioeng. 2006, 93(3):424-433

Nadeem M, Qazi JI, Baig S. Effect of Aeration and Agitation Rates on Alkaline Protease Production by Bacillus licheniformis UV-9 Mutant. Turk J Biochem. 2009, 34(2):89-96

Dutta JR, Banerjee R. Isolation and characterization of a newly isolated Pseudomonas mutant for protease production. Braz arch of Biol Technol. 2006, 49(1):37-47

Aiba S, Humphrey AE, Millis NF. Biochemical Engineering 2nd edn, Academic Press Inc. New York. 1973, 358-362

Carvalho RV, Côrrea TL, Silva JC, Mansur LR, Martins ML. Properties of an amylase from thermophilic Bacillus SP. Braz J Microbiol. 2008, 39(1):102-107

Kim S, Kim Y, Rhee IK. Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674. Arch Microbiol. 2001, 175(6):458-461

Kumar CG, Joo HS, Koo YM, Paik SR, Chang CS. Thermostable alkaline protease from a novel marine haloalkalophilic Bacillus clausii isolate. World J Microbiol Biotechnol. 2004, 20(4):351-357

Joo HS, Chang CS. Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: optimization and some properties. Proc Bio. 2005, 40(3):1263-1270

Patil U, Chaudhari A. Optimal production of alkaline protease from solvent-tolerant alkalophilic Pseudomonas aeruginosa MTCC 7926. Ind J Biotechnol. 2011, 10(3):329-339

Nadeem M, Qazi JI, Baig S. Enhanced production of alkaline protease by a mutant of Bacillus licheniformis N-2 for dehairing. Braz Arch Biol Technol. 2010, 53(5):1015-1025


  • There are currently no refbacks.

AJCMicrob (ISSN 2572-5815) Copyright © 2012-2020. All rights reserved. Published by Ivy Union Publishing, 3204 Valley Rush Dr, Apex, North Carolina 27502, United States