Bacillus subtilis RSKK246 şuşundan bir alfa amilaz geninin moleküler klonlanması ve Escherichia coli ve Bacillus subtilis şuşlarında ekspresyonu
Bacillus subtilis RSKK246 was found to produce approximately a 65-kDa $alpha$-amylase enzyme. A gene was isolated encoding $alpha$-amylase activity that corresponded to this size and was inserted into pUC18 plasmid which was transferred to Escherichia coli. An approximately 1.7kbp fragment, which contains a whole a-amylase gene, was excised and inserted into pUB110 and then transferred into the different B. subtilis strains including RSKK246, RSKK243, RSKK244, YB886 and ORBAM. The a-amylase gene was cloned into the plasmids and expressed with its own promoter, and this promoter sequence seemed to function in the E. coli and in all B. subtilis strains. Specific activity of the cloned enzyme was found to be higher than the native enzyme and molecular weight of the gene product remained the same in all other strains suggesting that it is resistant to the proteolytic attacks of these organisms.
Molecular cloning of an alpha amylase gene from Bacillus subtilis RSKK246 and its expression in Escherichia coli and in Bacillus subtilis
Bacillus subtilis RSKK246 was found to produce approximately a 65-kDa $alpha$-amylase enzyme. A gene was isolated encoding $alpha$-amylase activity that corresponded to this size and was inserted into pUC18 plasmid which was transferred to Escherichia coli. An approximately 1.7kbp fragment, which contains a whole a-amylase gene, was excised and inserted into pUB110 and then transferred into the different B. subtilis strains including RSKK246, RSKK243, RSKK244, YB886 and ORBAM. The a-amylase gene was cloned into the plasmids and expressed with its own promoter, and this promoter sequence seemed to function in the E. coli and in all B. subtilis strains. Specific activity of the cloned enzyme was found to be higher than the native enzyme and molecular weight of the gene product remained the same in all other strains suggesting that it is resistant to the proteolytic attacks of these organisms.
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- 1. Cornelis, P. Microbial amylases. Microbiol. Sci. 1987; 4: 342- 343.
- 2. Cornelis, P., Digneffe, C. and Willemot, K. Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli. Mol. Gen. Genet. 1982; 186: 507-511.
- 3. Ohdan, K., Kuriki, T., Kaneko, H., Shimada, J., Takada, T., Fujimoto, Z., Mizuno, H., and Okada, S. Characteristics of two forms of α-amylases and structural implication. Appl. Env. Microbiol. 1999; 65: 4652-4658.
- 4. Haddaoui, E., Chambert, R., Petit-Glatron, M-F., Lindy, O., and Sarvas, M. Bacillus subtilis α-amylase: the rate limiting step of secretion is growth phase-independent FEMS Microbiol. Let. 1999; 173 (1): 127-131.
- 5. Ozcan, N. Ethidium Bromid uygulamas› ile α-amylaz negatif Bacillus subtilis mutant flufllar›n›n oluflturulmas› ve karekterizasyonu. J. Agr. Fac. C. U. 1996; 11 (4): 87-96.
- 6. Maniatis, T., Fritsch, E. F. and Sambrook, J. Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y, 1982.
- 7. Hanahan, D. Techniques for transformation of Escherichia coli DNA cloning. A Practical Approach. (Glover, D. M. Eds.), IRL Press Inc., Oxford. 1985; 121-127.
- 8. Brigidi, P., De Rossi, E., Betarini, M.L., Ricardi, G. and Matteazzi, D. Genetic transformation of intact cells of Bacillus subtilis by electroporation. FEMS Microbiol. Let. 1990; 67: 135-138.
- 9. Cotta, M. A. Amylolytic activity of selected species of ruminal bacteria. Appl. Environ. Microbiol. 1988; 54: 772-776.
- 10. Cutting, S.M. and Van Der Horn, P.B. Genetic analysis in molecular biology methods for Bacillus (Eds. Hardwood, C.R. and Cutting S.M.) 1990; 27-74.
- 11. Barat-Gueride, M., Docherty, R. and Rickwood, R. DNA Replication and Transcription. Mitochondria: A Practical Approach. IRL Press Inc., Oxford. 1987; 247-250.
- 12. Satoh, E.; Niimura, Y.; Uchimura, T.; Kozaki, M. and Komagata, K. Molecular cloning and expression of two α-amylase gene from Streptococcus bovis 148 in Escherichia coli. Appl. Environ. Microbiol. 1993; 59: 3669-3673.
- 13. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature. 1970; 227; 680- 685.
- 14. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randal, R. J. Protein measurement with folin-phenol reagent. J. Biol. Chem. 1951; 193: 265-275.
- 15. Cotta, M. A. and Whitehead, T. R. Regulation and cloning of the gene encoding amylase activity of the ruminal bacterium Streptococcus bovis. Appl. Environ. Microbiol. 1993; 59: 189- 196.
- 16. Lacks, S. A. and Springhorn, S. S. Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. J. Biol. Chem. 1980; 255: 7467-7473.
- 17. Lee, S., Morrikawa, M., Takagi, M. and Imanaka, T. Cloning of the AapT gene and characterisation of its product, α-amylase pullanase (AapT) from thermophilic and alkaliphilic Bacillus sp. strain XAL601. Appl. Environ. Microbiol. 1994; 60: 3761-3773.
- 18. Teather, R. M., Wood, P. J. Use of Congo Red-polysaccharide interactions in enumeration and characterisation of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43: 777-780, 1982.
- 19. Flint, H. J., McPherson, C. A., Martin, J. Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13. J. Gen. Microbiol. 137: 123-129, 1991.
- 20. Birnboim, H. C. and Doly, J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acid Res. 1979; 7: 1513-1523,
- 21. Ikeda, T., Yamazaki, H. and Shinke, R. The tetracycline inducible expression of α-amylase in Bacillus subtilis. J. Ferm. Bioeng. 74, 58-60, 1992.
- 22. Ikuta, N., Souza, M. B. N., Valencia, F.F., Castro, M. E. B., Schenberg, A. C. G., Kleiner, A. P. and Filho, S. A. The α-amylase gene as a marker for gene cloning: Direct screening of recombinant clones. Biotechnology. 1990; 8: 241-242.
- 23. Freer, S.N. Purification and characterisation of the extra-cellular αamylase from Streptococcus bovis JB1. Appl. Environ. Microbiol. 1993; 59: 1398-1402.
- 24. Tanaka, T., Ishimoto, E., Shimomura, Y., Taniguchi. M. and Oi, S. Purification and some properties of raw starch-binding amylase of Clostridium butyricum t-7 isolated from mesophillic methane sludge. Agric. Biol. Chem. 1987; 51: 399-405.
- 25. De Mot, R. and Verachtert, H. Prufication and characterisation of extracellular amylolytic enzymes from the yeast Filobasidium capsuligenum. Appl. Environ. Microbiol. 1985; 50: 1474-1482.
- 26. Tsukagoshi, N., Iritani, S., Sasaki, T., Takemura, T., Ihara, H., Idota, Y., Yamagata, H. and Udaka, S. Efficient synthesis and secretion of a thermophilic α-amylase by protein producing Bacillus brevis 47 carrying the Bacillus stearothermophilus αamylase gene. J. Bacteriol. 1985; 164: 1182-1187.
- 27. Rothstein, D.M., Devlin, P.E., and Cate, R.L. expression of alphaamylase in Bacillus licheniformis. J. Bacteriol. 1986; 168: 839- 842.