Isolation, purification, characterization and applications of serine protease from Bacillus megaterium
Article Main
Abstract
Bacillus megaterium isolated from poultry farm soil was identified by standard biochemical tests and screened for the production of serine protease. Production of serine protease was done using 5 different medias by varying the type of amino acid added. The purification was done by salt precipitation, dialysis and DEAEcellulose ion exchange chromatography. The proline containing media obtained the highest fold purification out of the five different medias (leucine, lysine, proline, tryptophan and methionine cotaining media). The enzyme showed
an optimal activity at the temperature 37°C and the pH 6 which are known as its optimum temperature and pH respectively. The enzyme was proved as a Mn2+ dependent serine protease as it was activated by Mn2+ ions and inhibited by PMSF. The molecular weight of the enzyme was determined by SDS-PAGE technique as around 30kDa. It showed an excellent detergent activity on the blood stains and a very good stability in presence of locally available detrgents. The enzyme acted on the keratin protein of the chicken feather and showed a degrading capacity on the protein. So it was proved that the recently studied serine protease has a keratinase activity also. From these datas I conclude that the protease isolated from Bacillus megaterium is a Mn2+ dependent serine protease which has both keratinase and detergent activity.
Article Details
Article Details
Keywords
Bacillus megaterium, Keratinase, serine protease, phenylmethanesulfonylfluoride(PMSF)
References
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Robert S. Boethling (1975). Purification and properties of serine protease from Pseudomonas maltophilia. J. Bacteriol., 121(3): 933-941.
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Sigma, D. S. and Mooser, G. (1975). Chemical studies of enzyme active sites. Ann. Rev. Biochem., 44: 889-931.
Tsuchida, O., Yamagota, Y., Ishizuka, J., Arai, J., Yamada, J., Takeuchi, M. and Ichishima, E. (1986). An alkaline protease of an alkalophilic Bacillus sp. Curr. Microbiol., 14:7-12.
Usharani, B. and Muthuraj, M. (2009). Production and characterization of protease enzyme from Bacillus laterosporus, Global Journal of Molecular Sciences, 4 (2): 180-186.
Wang, R. B., Yang, J. K., Lin, C., Zhang, Y. and Zhang, K.Q.(2006). Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Dactylella shizishanna, Letters in applied Microbiology, 42: 589-594.
Ward, O. P. (1985). Proteolytic enzymes, In:Blanch H W, Drew S, Wang DI, eds. Comprehensive Biotechnology, 3; 789-818.
Yamagata, Y. and Ichishima, E. (1989). A new alkaline proteinase with PI 2,8 from alkalophilic Bacillus species, Curr. Microbiol., 19: 259-264.
Anson, M. L. (1938). The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin Journalernal of Gen. Physiology, 22: 79-89.
Asokan, S. and Jayanthi, C. (2010). Alkaline protease production by Bacillus licheniformis and Bacillus coagulan, Journal of Cell and Tissue Research, 10 (1): 2119-2123.
Bennett, T. P. and Frieden (1969). Modern Topics in Biochemistry, pg. 43-45, Macmillan, London.
Bergkvist, R. (1963). The proteolytic enzymes of Aspergillus oryzae. I. Methods for estimation and isolation of the proteolytic enzymes, Acta Chem. Scand. 17; 1521-1540.
Bjorklind and S. Arvidson (1978). Influence of amino acids on the synthesis of an extracellular proteinase from Staphylococcus aureus. Journal of General Microbiology, 107: 367-375.
Bockle, B., Galunsky and R. Muller (1995). Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530. Appl. Environ. and Microbiol., 63: 55-61.
Charles, V. Devanathan, Periasamy Anbu, M. N. Ponnuswamy1, P. T. Kalaichelvan and Byung-Ki Hur (2008). Purification, characterization and crystallization of an extracellular alkaline protease from Aspergillus nidulans HA-10, Journal of Basic Microbiology, 48: 347–352.
Cheng, S. W., H. M., Hu, S. W. Shen, H. Takagi, M. Asano and Y. C. Tsai (1995). Production and characterization of keratinase of feather degrading Bacillus licheniformis PWD-1, Biosci. Biotech. Biochem, 59;2239-2243.
Dayanandan, A., J. Kanagaraj, L. Soundarray, R. Govindaraju and G. S. Rajkumar (2003). Application of alkaline protease in leather processing: an eco-friendly approach, J. Cleaner Production, 11: 533-536.
Huang Guangrong, Ying Tiejing, Huo Po and Jiang Jiaxing (2006). Purification and characterization of a protease from Thermophilic bacillus strain HS08, African Journal of Biotechnology, 5 (24): 2433-2438.
Jackie Reynolds (2005). Serial Dilution Protocols, Microbe Library (Journal of Microbiology & Biology Education).
Kelly C.T. and Fogarty W. M.(1976) Microbial alkaline enzymes. Process Biochem., 11:3–9
Kida K, S. Morimura, J. Noda, Y. Nishida, T. Imai and M. Otagiri (1995). Enzymatic hydrolysis of horn and hoof of cow and buffalo. J. Ferment. Bioeng., 80: 478-484.
Kim, J. M., W. J. Lim and S. J. Suh (2001). Feather degrading Bacillus species from poultry waste. Process Biochem., 37: 287- 291.
Klemersrud, M. J., Klopfenstein, T. J. and Lewis, A. J. (1998). Complementary responses between feather meal and poultry by-product meal with or without rumminally protected methionine and lysine growing calves. J. Anim. Sci., 76: 1970-1975.
Kunamneni Adinarayana, Poluri Ellaiah and Davuluri Siva Prasad (2003). Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilisins PE-11, AAPS PharmSciTech, 4 (4) Article 56.
Lee, C. G., Forket, P. R. and Shih, J. C. H. (1991). Improvement of feather digestibility by bacterial keratinase as a feed additives, FASEB. J.,59:1312.
Lin, X., J. C. H. Shih and H. E. Swaisgood (1996). Hydrolysis of feather keratin by immobilized keratinase. Appl. Environ. Microbiol., 62: 4273-4275.
Mohsen Fathi Najafi, Dileep Deobagkar and Deepti Deobagkar (2005). Potential application of protease isolated from Pseudomonas aeruginosa PD100. Electronic Journal of Biotechnology, 8:2
Niu Qiuhong, Huang Xiaowei, Tian Baoyu, Yang Jinkui, Liu Jiang, Zhang Lin and Zhang Keqin (2006). Bacillus sp. B16 kills Nematodes with a serine protease identified as a pathologic factor. Appl. Microbiol Biotechnol., 69: 722-730.
Odetallah, N. H., Wang, J. J., Garlich, J. D. and Shih, J. C. H. (2003). Keratinase in starter diets improves growth of broiler chicks, Poultry Sci., 82: 664- 670.
Onifade, A. A., N. A. Al-Sane, A. A. Al- Musallam and S. Zarban (1998). A review: Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresource Technol., 66: 1-11.
Page, M. J. and Di Cera, E. (2006). Role of Na and K in enzyme function. Physiol. Rev., 86: 1049-1092.
Riffel, A., F. S. Lucas, P. Heeb and A. Brandelli (2003). Characterization of new keratinolytic bacterium that completely degrades native feather keratin. Archives Microbiol., 179: 258-265.
Robert S. Boethling (1975). Purification and properties of serine protease from Pseudomonas maltophilia. J. Bacteriol., 121(3): 933-941.
Saurabh, S., Jasmine, I., Pritesh, G. and Rajendra Kumar, S. (2007). Enhanced productivity of serine alkaline protease by Bacillus sp. using soybean as substrate. Malaysian Journal of Microbiology, 3(1): 1-6.
Sigma, D. S. and Mooser, G. (1975). Chemical studies of enzyme active sites. Ann. Rev. Biochem., 44: 889-931.
Tsuchida, O., Yamagota, Y., Ishizuka, J., Arai, J., Yamada, J., Takeuchi, M. and Ichishima, E. (1986). An alkaline protease of an alkalophilic Bacillus sp. Curr. Microbiol., 14:7-12.
Usharani, B. and Muthuraj, M. (2009). Production and characterization of protease enzyme from Bacillus laterosporus, Global Journal of Molecular Sciences, 4 (2): 180-186.
Wang, R. B., Yang, J. K., Lin, C., Zhang, Y. and Zhang, K.Q.(2006). Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Dactylella shizishanna, Letters in applied Microbiology, 42: 589-594.
Ward, O. P. (1985). Proteolytic enzymes, In:Blanch H W, Drew S, Wang DI, eds. Comprehensive Biotechnology, 3; 789-818.
Yamagata, Y. and Ichishima, E. (1989). A new alkaline proteinase with PI 2,8 from alkalophilic Bacillus species, Curr. Microbiol., 19: 259-264.
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Isolation, purification, characterization and applications of serine protease from Bacillus megaterium. (2010). Journal of Applied and Natural Science, 2(2), 313-317. https://doi.org/10.31018/jans.v2i2.141
