A mutant subtilisin E with enhanced thermostability
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A mutant subtilisin E with remarkably thermostability is reported. It is more active against the typical substrate s-AAPF-pna than the wild-type subtilisin E. The time required for getting 50% residual activity of Ser236Cys subtilisin E at 60 °C in aqueous solution was approximately 80 min which is 4 times longer than that of wild-type subtilisin E. Similar to the wild-type subtilisin E, the amidase activity of Ser236Cys subtilisin E is dramatically reduced in the presence of dimethylformamide (DMF).
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- Bryan, P.N., Rollence, M.L., Pantoliano, M.W., Wood, J., Finzel, B.C., Gilliland, G.L., Howard, A.J. & Poulos, T.J. 1986 Protease of enhanced stability: characterization of a thermostable variant of subtilisin. Proteins: Structure, Function & Genetics 1, 326-334.Google Scholar
- Chen, K. & Arnold, F.H. 1991 Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media. Biotechnology 9, 1073-1077.Google Scholar
- Estell, D.A., Graycar, T.P. & Wells, J.A. 1985 Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation. Journal of Biological Chemistry 260, 6518-6521.Google Scholar
- Guo, X.H., Xiong, Z., Zhou, M. & Jia, S.F. 1991 The construction shuttle vectors of Bacillus subtilis-Escherichia coli. Chinese Journal of Biotechnology 7, 224-229.Google Scholar
- Kawamura, H. & Doi, R.H. 1984 Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. Journal of Bacteriology 160, 442-444.Google Scholar
- Kidd, R.D., Yennawar, H.P., Sears, P., Wong, C.H. & Farber, G.K. 1996 A weak calcium binding site in subtilisin BPN' has a dramatic e.ect on protein stability. Journal of the American Chemical Society 118, 1645-1650Google Scholar
- Pantiliano, M.W., Whitlow, M., Wood, J.F., Dodd, S.W., Hardman, K.D., Rollence, M.L. & Bryan, P.N. 1989 Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding. Biochemistry 28, 7205-7213.Google Scholar
- Takagi, H. 1993 Protein engineering on subtilisin. International Journal of Biochemistry 25, 307-312.Google Scholar
- Takagi, H., Arafuka, S., Inouye, M. & Yamasaki, M. 1992 The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis. Journal of Biochemistry 111, 584-588.Google Scholar
- Takagi, H., Morinaga, Y., Ikemura, H. & Inouye, M. 1988 Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis. Journal of Biological Chemistry 263, 19592-19596.Google Scholar
- Takagi, H., Takahashi, T., Momose, H., Inouye, M. Maeda, Y., Matsuzawa, H. & Ohta, T. 1990 Enhancement of the thermostability of subtilisin E by introduction of a disul®de bond engineered on the basis of structural comparison with a thermophilic serine protease. Journal of Biological Chemistry 265, 6874-6878.Google Scholar
- Takagi, H., Yamamoto, M., Ohtsu, I. & Nakamori, S. 1998 Random mutagenesis into the conserved Gly154 subtilisin E: isolation and characterization of the revertant enzymes. Protein Engineering 11, 1205-1210.Google Scholar