Partial purification of protease by a novel bacterium, Bacillus cereus and enzymatic properties

Proteazlar (E.C. 3.4.X.X) proteinlerdeki peptid bağlarının hidrolizinden sorumlu enzimlerdir. Mikrobiyal proteazlar, etkide bulundukları bölge ve biyokatalitik mekanizmalarına göre sınıflandırılırlar. Bu proteazlar hücre beslenmesi, fizyolojisi, düzenlenmesi ve mikrobiyal patogenezde rol oynamaktadırlar. Toprak örneklerinden izole edilmiş 15 bakterinin proteaz aktiviteleri saptanmış ve en yüksek aktiviteye sahip suş seçilerek stoklanmıştır. Bu suşun 16S rRNA filogenetik analizi yapılarak Bacillus cereus olduğu saptanmıştır. Proteaz üretiminin optimizasyonu sonrasında bu suşun Michaelis-Menten kinetiği hesaplanmıştır. Proteaz inkübasyonunun sıcaklık, pH ve zaman parametreleri araştırılmış ve maksimum sıcaklık aktivitesi 50oC’de 5.15 IU/ml olarak saptanmıştır. Enzimin optimum pH aralığı 7-9’dur. Ham enzim, diyaliz yöntemiyle yaklaşık olarak 2 kat saflaştırılmıştır.

Yeni izole Bacillus cereus proteazının kısmi saflaştırılması ve enzimolojik özellikleri

Proteases (E.C. 3.4.X.X) catalyze the hydrolysis of peptide bonds in proteins. Microbial proteases are classified based on their mode of action and biocatalytic mechanisms. They participate in most aspects of cell nutrition, physiology, and regulation, and in microbial pathogenesis. Protease production was detected 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 studied. Temperature, pH and, time parameters of protease incubation was determined and maximum temperature was detected at 50oC 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.

PDF

___

1. W. Aehle, Enzymes in Food Applications, Enzymes In Industry – Production and Applications., Third, Completely Revised Edition, Wolfgang Aehle, (Ed), WILEY-VCH Verlag GmbH & Co. KgaA, 2007.
2. O.P. Ward, M.B. Rao, A. Kulkarni, Proteases, Production, Encyclopedia of Enzymes, Third Edition, Editor in Chief: Moselio Schaechter, Elsevier Inc., 2009.
3. R. Gupta, Q.K. Beg, P. Lorenz, Bacterial alkaline proteases: molecular approaches and industrial applications, Appl. Microbiol. Biotechnol., 59 (2002) 15.
4. F. Hasan, A.A. Shah, A. Hameed, Industrial applications of microbial lipases, Enzyme Microb. Technol., 39 (2006) 235.
5. K.B. Maal, G. Emtiazi, and I. Nahvi, Production of alkaline protease by Bacillus cereus and Bacillus polymixa in new industrial culture mediums and its immobilization, Afr. J. Microbiol. Res., 3 (2009) 491.
6. M.A. Ferrero, G.R. Castro, C.M. Abate, M.D. Baigori, F. Sineriz, Thermostable alkaline proteases of Bacillus licheniformis MIR 29: Isolation, production and characterization, Appl. Microbiol. Biotechnol., 45 (1996) 327.
7. R.S. Boethling, Regulation of protease secretion in P. maltophila, J. Bacteriol., 123 (1975) 954.
8. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265.
9. N. Cihangir ve N. Aksöz, Bacillus sp. proteazının sentezi ve etkili bazı kültürel parametrelerinin saptanması, Kükem Dergisi, 11 (1988) 27.
10. E.M. Upton, W.M. Fogarty, Production and purification of thermostable amylase and protease of Thermomonospora viridis, Appl. Environ. Microbiol., (1977) 59.
11. S. Mehrotra, P.K. Pandey, R. Gaur, N.S. Darmwal, The production of alkaline protease by a Bacillus species isolate, Bioresource Technol., 67 (1999) 201.
12. S. Dube, L. Singh, S.I. Alam, Proteolytic anaerobic bacteria from lake sediments of Antarctica, Enzyme Microb. Technol., 28 (2001) 114.
13. J.F. Hawumba, J. Theron, V.S. Brözel, Thermophilic protease-producing Geobacillus from Buranga Hot Springs in West Uganda, Curr. Microbiol., 45 (2002) 144.
14. K. Aikat, B.C. Bhattacharyya, Protease extraction in solid state fermentation of wheat bran by a local strain of Rhizopus oryzae and growth studies by the soft gel technique, Process Biochem., 35 (2000) 907.
15. M.A. Bjurlin, S. Bloomer, C.J. Nelson, Characterization of proteolytic activity of proteases, Biotechnol. Lett., 24 (2002) 191.
16. P.P. Kanekar, S.S. Nilegaonkar, S.S. Sarnaik, and A.S. Kelkar, Optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in India, Bioresource Technol., 85 (2002) 187.
17. M.A. Abdel-Naby, A.M.S. Ismail, S.A. Ahmed, A.F. Abdel-Fattah, Production and immobilization of alkaline protease from Bacillus mycoides, Bioresource Technol., 64 (1998) 205.
18. J.K. Yang, I.L. Shih, Y.M. Tzeng, S.L. Wang, Production and purification of protease from a Bacillus subtilis that can deproteinase crustacean wastes, Enzyme Microb. Technol., 26 (2000) 406.
19. D.R. Durham, D.B. Stewart, and E.J. Stellwag, Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638, J. Bacteriol., 169 (1987) 2762.
20. R. Gupta, K. Gupta, R.K. Saxena, S. Khan, Bleachstable, alkaline protease from Bacillus sp., Biotechnol. Lett., 21 (1999) 135.
21. H.R. Horton, L.A. Moran, R.S. Ochs, J.D. Rawn, K.G. Scrimgeour, Principles of Biochemistry, Neil Patterson Publishers/Prentice-Hall Inc., 1993.
22. T. Palmer, Understanding Enzymes, 4th Edition, Prentice Hall / Ellis Horwood Ltd., 1995.
23. F. Bono, P. Savi, A. Tuong, M. Maftouh, J-M. Pereillo, J. Capdevielle, J.C. Guillemot, J.P. Maffrand, J.M. Herbert, Purification and characterization of a novel protease from culture filtrates of a Streptomyces sp., FEMS Microbiol. Lett., 141 (1996) 213.