Experimental Preliminary Study for Production of Recombinant Subtilisin Enzyme by pET28b Cloning Vector

Öz

Enzymes are biological catalysts and allow reactions to occur 103 – 108 times faster than reactions without enyzmes. Because of these, they are widely used in industry and the use of proteases accounts for 60 % of all enyzmes. It is quite hard and costly to obtain from biological resources. Therefore, it is more attractive to produce them by recombinant DNA technology that enables the production of many proteins. Subtilisin is a serine protease enzyme and it has a broad range of applications in detergent, food, textile, pharmaceutical industries. Thus, its continuous production has a large importance. In this study originated from this point; subtilisin gene belonging to Bacillus subtilis was cloned in pET28b vector and then expression was performed. Subtilisin enzyme was produced and it was clearly observed in the 35000-55000 Da band range by SDS-PAGE analysis, which has 41646.82 Da molecular mass.

Anahtar Kelimeler

• Recombinant Protein
• Subtilisin
• pET28b

Kaynakça

1. Anonymous, 2020a. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC668139 9/#mbt213469-sec-0002title
2. Anonymous, 2020b. http://www.addgene.org/vectordatabase/2566/
3. Billman-Jacobe, H., Wang, L., Kortt, A., Stewart, D., &Radford, A. (1995). Expression and secretion of heterologous proteases by Corynebacterium glutamicum. Applied and environmental microbiology, 61 (4), 1610-1613.
4. Bryan, P. N. (2000). Protein engineering of subtilisin. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1543 (2), 203- 222.
5. Damhus, T., Kaasgaard, S., Lundquist, H. and Olsen, H.S., (2008). All of Novozymes A/S. “enzymes at work – novozymes” 3rd edition 2008. D'Souza NM, Mawson AJ (2005). "Membrane cleaning in the dairy industry: A review. Crit. Rev. Food Sci. Nutr. 45: 125-134
6. Falch, E. A. (1991). Industrial enzymes—developments in production and application. Biotechnology advances, 9 (4), 643-658
7. Ghasemi, Y., Dabbagh, F., & Ghasemian, A. (2012). Cloning of a fibrinolytic enzyme (subtilisin) gene from Bacillus subtilis in Escherichia coli. Molecular biotechnology, 52 (1), 1-7.
8. Gupta, R., Beg, Q., Khan, S., & Chauhan, B. (2002). An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Applied microbiology and biotechnology, 60 (4), 381-395.

9. Hanahan, D. (1985). Techniques for transformation of E. coli. DNA cloning: a practical approach, 1, 109-135. Hasan, F., Shah, A. A., Javed, S., & Hameed, A. (2010).Enzymes used in detergents: lipases. African journal of biotechnology, 9 (31), 4836-4844.
10. Joshi, S., & Satyanarayana, T. (2013, September).Heterologous expression of P. anomala phytase in P. pastoris and recombinant enzyme production. In YEAST (Vol. 30, pp. 136-136). 111 RIVER ST, HOBOKEN 07030-5774, NJ USA: WILEY-BLACKWELL.
11. Kalisz, H. M. (1988). Advances in BiochemicalEngineering/Biotechnology. ed. Fiechter A, Springer, Berlin, 1-65.
12. Kumar, C. G., & Takagi, H. (1999). Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnology advances, 17 (7), 561-594.
13. Laemmli, U. K. (1970). Denaturing (SDS) discontinuous gel electrophoresis. Nature, 277, 680-685.
14. Mienda, B. S., Yahya, A., Galadima, I. A., & Shamsir, M. S. (2014). An overview of microbial proteases for industrial applications. Res J Pharm Biol Chem Sci, 5 (1), 388-396.
15. Okeley, N. M., Paul, M., Stasser, J. P., Blackburn, N., & Van Der Donk, W. A. (2003). SpaC and NisC, the cyclases involved in subtilin and nisin biosynthesis, are zinc proteins. Biochemistry, 42 (46), 13613-13624.
16. Outtrup, H., & Boyce, C. O. L. (1990). Microbial proteinases and biotechnology. In Microbial enzymes and biotechnology (pp. 227-254). Springer, Dordrecht.
17. Porro, D., Sauer, M., Branduardi, P., & Mattanovich, D.(2005). Recombinant protein production in yeasts. Molecular biotechnology, 31 (3), 245-259
18. Rao, M. B., Tanksale, A. M., Ghatge, M. S., & Deshpande, V. V. (1998). Molecular and biotechnological aspects of microbial proteases. Microbiology and molecular biology reviews, 62 (3), 597-635.
19. Saeki, K., Ozaki, K., Kobayashi, T., & Ito, S. (2007). Detergent alkaline proteases: enzymatic properties, genes, and crystal structures. Journal of bioscience and bioengineering, 103 (6), 501-508.
20. Saul, D. J. (2010). Biocatalysis: Industrial Enzymes and the Exploitation of Micro-Organisms. New Zealand Institute of Chemistry. Ward, O. P. (1985). Proteolytic enzymes. Blanch HW, Drew S, Wang DI (Eds),
21. Comprehensive Biotechnology. Yang, Y., Jiang, L., Yang, S., Zhu, L., Wu, Y., & Li, Z. (2000). A mutant subtilisin E with enhanced thermostability. World Journal of Microbiology and Biotechnology, 16 (3), 249-251