Share This Article:

Optimized production and properties of thermostable alkaline protease from Bacillus subtilis SHS-04 grown on groundnut (Arachis hypogaea) meal

Abstract Full-Text HTML Download Download as PDF (Size:674KB) PP. 112-120
DOI: 10.4236/aer.2013.14012    3,750 Downloads   8,036 Views   Citations


Production of alkaline protease from Bacillus subtilis SHS-04 was investigated under different fermentation conditions involving low-cost substrates with the aim of optimizing yield of enzyme. Maximum enzyme production (1616.21 U/mL) was achieved using groundnut meal (0.75%) as nitrogen source and 0.5% glucose as carbon source at 48 h cultivation period, pH 9, 45 ° C and 200 rpm. The yield was 348% increase over comparable control samples. The alkaline protease had optimum temperature of 60 ° C and remarkably exhibited 80% relative activity at 70 ° C. It was highly thermostable showing 98.7% residual activity at 60 ° C after 60 minutes of incubation at pH 9.0 and was stable in the presence of organic solvents studied. These properties indicate the viability of the protease for biotechnological and industrial applications. The optimized yield of enzyme achieved in this study establishes groundnut meal as potential low-cost substrate for alkaline protease production by B. subtilis SHS-04.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Olajuyigbe, F. (2013) Optimized production and properties of thermostable alkaline protease from Bacillus subtilis SHS-04 grown on groundnut (Arachis hypogaea) meal. Advances in Enzyme Research, 1, 112-120. doi: 10.4236/aer.2013.14012.


[1] Maurer, K. (2004) Detergent proteases. Current Opinion in Biotechnology, 15, 330-334.
[2] Vellard, M. (2003) The enzyme as drug: Application of enzymes as pharmaceuticals. Current Opinion in Biotechnology, 14, 444-450.
[3] Breithaupt, H. (2001) The hunt for living gold. EMBO Reports, 2, 968-971.
[4] Rahman, R.N.Z.A., Geok L.P., Basri M. and Salleh A.B. (2005) Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K. Bioresource Technology, 96, 429-436.
[5] Joo, H.S., Kumar, C.G., Park, G.C., Kim, K.T., Paik, S.R. and Chang, C.S. (2002) Optimization of the production of an extracellular alkaline protease from Bacillus horikoshi. Process Biochemistry, 38, 155-159.
[6] Li, Y., Lin, J., Meng, D., Lu, J., Gu, G. and Mao, Z. (2006) Effect of pH, cultivation time and substrate concentrations on the endoxylanase production by A. awamori ZH-26 under submerged fermentation using central composite rotary design. Food Technology and Biotechnology, 44, 473-477.
[7] Joo, H.-S. and Chang, C.S. (2005) Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: Optimization and some properties. Process Biochemistry, 40, 1263-1270.
[8] Haddar, A., Hmidet, N., Ghorbel-Bellaaj, O., Fakhfakh-Zouari, N., Sellami-Kamoun, A. and Nasri, M. (2011) Alkaline proteases produced by Bacillus licheniformis RP1 grown on shrimp wastes: Application in chitin extraction, chicken feather degradation and as a dehairing agent. Biotechnology and Bioprocess Engineering, 16, 669-678.
[9] Agrebi, R., Hmidet, N., Hajji, M., Ktari, N., Haddar, A., Fakhfakhzouari, N., et al. (2010) Fibrinolytic serine protease isolation from Bacillus amyloliquefaciens An6 grown on Mirabilis jalapa tuber powders. Applied Biochemistry and Biotechnology, 162, 75-88.
[10] Bassam, N. El. (2010) Groundnut (Arachis hypogaea (L) Merr.). In: Hand-book of Bioenergy Crops: A Complete Reference to Species, Development and Applications, Earthscan Publishers, London, 199-202.
[11] Yaw, A.J., Richard, A., Osei, S., Kofi, A.H., Seth, O. and Adelaide, A. (2008) Chemical composition of groundnut, Arachis hypogaea (L) landraces. African Journal of Biotechnology, 7, 2203-2208.
[12] Ingale, S. and Shrivastava, S.K. (2011) Nutritional study of new variety of groundnut (Arachis hypogaea L.) JL-24 seeds. African Journal of Food Science, 5, 490-498.
[13] Savage, G.P. and Keenan, J.I. (1994) The composition and nutritive value of groundnut kernels. In: Smart, J., Ed., The Groundnut Crop: Scientific Basis for Improvement, Chapman and Hall, London, 173-213.
[14] Vos, P., et al. (2010) The Firmicutes. In: Vos, P., Garrity, G., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A., Schleifer, K. and Whitman, W.B., Eds., Bergey’s Manual of Systematic Bacteriology, 2nd Edition, Volume 3, Springer, New York, 4-325.
[15] Fujiwara, N., Masui, A. and Imanaka, T. (1993) Purification and properties of highly thermostable alkaline protease from an alkalophilic and thermophilic Bacillus sp. Journal of Biotechnology, 30, 245-256.
[16] Olajuyigbe, F.M. and Ajele, J.O. (2005) Production dynamics of extracellular protease from Bacillus species. African Journal of Biotechnology, 4, 776-779.
[17] Olajuyigbe, F.M. and Ajele, J.O. (2011) Thermostable alkaline protease from Bacillus licheniformis LBBL-11 isolated from traditionally fermented African locust bean (Parkia biglobosa). Journal of Food Biochemistry, 35, 1-10.
[18] Singh, S.K., Tripathi, V.R., Khare, S.K. and Garg, S.K. (2011) Comparative one-factor-at-a-time, response surface (statistical) and bench-scale bioreactor level optimization of thermoalkaline protease production from a psychrotrophic Pseudomonas putida SKG-1 isolate. Microbial Cell Factories, 10, 114.
[19] Sepahy, A.A. and Jabalameli, L. (2011) Effect of culture conditions on the production of an extracellular protease by Bacillus sp. isolated from soil sample of Lavizan Jungle Park. Enzyme Research, 2011, Article ID: 219628.
[20] Gouda, M.K. (2006) Optimization and purification of alkaline proteases produced by marine Bacillus sp. MIG newly isolated from Eastern Harbour of Alexandria. Polish Journal of Microbiology, 55, 119-126.
[21] Nascimento, W.C.A. and Martins, M.L.L. (2004) Production and properties of an extracellular protease from thermophilic Bacillus sp. Brazilian Journal of Microbiology, 35, 1-2.
[22] Bakermans, C. and Nealson, K.H. (2004) Relationship of critical temperature to macromolecular synthesis and growth yield in Psychrobacter cryopegella. Journal of Bacteriology, 186, 2340-2345.
[23] Chu, W.-H. (2007) Optimization of extracellular alkaline protease production from species of Bacillus. Journal of Industrial Microbiology and Biotechnology, 34, 241-245.
[24] Joshi, R.H., Dodia, M.S. and Singh, S.P. (2008) Production and optimization of a commercially viable alkaline protease from a haloal-kaliphilic bacterium. Biotechnology and Bioprocess Engineering, 13, 552-559.
[25] Abusham, R.A., Zaliha, R.N., Salleh, A.B. and Basri, M. (2009) Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halotolerant B. subtilis strain Rand. Microbial Cell Factories, 8, 20.
[26] Fang, H.H.P. and Liu, H. (2002) Effect of pH on hydrogen production from glucose by mixed culture. Bioresource Technology, 82, 87-93.
[27] Ellaiah, P., Srinivasulu, B. and Adinarayana, K. (2002) A review on microbial alkaline proteases. Journal of Scientific and Industrial Research, 61, 690-704.
[28] Zambare, V.P., Nilegaonkar, S.S. and Kanekar, P.P. (2004) Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent. World Journal of Microbiology and Biotechnology, 23, 1569-1574.
[29] Mehrotra, S., Pandey, P.K., Gaur, R. and Darmwal, N.S. (1999) The production of alkaline protease by a Bacillus species isolate. Bioresource Technology, 67, 201-203.
[30] Gupta, R., Beg, Q.K. and Lorenz, P. (2002) Bacterial alkaline proteases: Molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59, 15-32.
[31] Wan, M.-Y., Wang, H-Y., Zhang, Y.-Z. and Feng, H. (2009) Substrate specificity and thermostability of the dehairing alkaline protease from Bacillus pumilus. Applied Biochemistry and Biotechnology, 159, 394-403.
[32] Ghorbel, B., Sellami-kamoun, A. and Nasri, M. (2003) Stability studies of protease from Bacillus cereus BG1. Enzyme and Microbial Technology, 32, 513-518.
[33] Alexander, P.A., Ruan, B., Strausberg, S.L. and Bryan, P. N. (2001) Cation-dependent stability of subtilisin. Biochemistry, 40, 10640-10644.
[34] Nilegaonkar, S.S., Zambare, V.P., Kanekar, P.P., Dhakephalkar, P.K. and Sarnaik, S.S. (2007) Production and partial characterization of dehairing protease from Bacillus cereus MCMB-326. Bioresource Technology, 98, 1238-1245.
[35] Rai, S.K., Roy, J.K. and Mukherjee, A.K. (2010) Characterisation of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. AS-S24-II. Applied Microbiology and Biotechnology, 85, 1437-1450.
[36] Hadj-Ali, N.E., Agrebi, R., Ghorbel-Frikha, B., Sellami-Kamoun, A., Kanoun, S. and Nasri, M. (2007) Biochemical and molecular characterization of a detergent stable alkaline serine-protease from a newly isolated Bacillus licheniformis NH1. Enzyme and Microbial Technology, 40, 515-523.
[37] Kumar, D. and Bhalla, T.C. (2005) Microbial proteases in peptide synthesis: Approaches and applications. Applied Microbiology and Biotechnology, 68, 726-736.
[38] Gupta, A., Roy, I., Khare, S.K. and Gupta, M.N. (2005) Purification and characterization of a solvent stable protease from Pseudomonas aeruginosa PseA. Journal of Chromatography A, 1069, 155-161.

comments powered by Disqus

Copyright © 2018 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.