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Changes in Optimum pH and Thermostability of $\alpha$-amylase from Bacillus licheniformis by Site-directed Mutagenesis of His 235 and Asp 328

  • Kim, Mi-Sook (Department of Chemistry, College of Natural Science, Seoul National University) ;
  • Lee, Sang-Kyou (Department of Chemistry, College of Natural Science, Seoul National University) ;
  • Jung, Han-Seung (Department of Chemistry, College of Natural Science, Seoul National University) ;
  • Yang, Chul-Hak (Department of Chemistry, College of Natural Science, Seoul National University)
  • Published : 1994.10.20

Abstract

The ${alpha}$-amylase gene of Bacillus licheniformis has been cloned and two mutant ${alpha}$-amylase genes of which histidine 235 was changed to glutamine (H235Q) and aspartic acid 328 to glutamic acid (D328E) have been produced by site-directed mutagenesis. The kinetic parameters, optimum pH and thermostability of wild type(WT) and these two mutant amylases expressed in E. coli MC1061 have been compared after purification. The $K_m$ values of WT, H235Q and D328E ${alpha}$-amylases were 0.22%, 0.73%, and 0.80% respectively, when using starch as the substrate. The $V_max$ values of wild type ${alpha}$ -amylase and mutant ${alpha}$-amylases were 0.6-0.7%/minute, and did not show any significant differences among them. The optimum pH of D328E ${alpha}$-amylase was shifted to more acidic pH. Also, the thermostability of H235Q ${alpha}$-amylase was increased compared to the wild type ${alpha}$-amylase.

Keywords

References

  1. Microbial Enzymes and Biotechnology Forgaty, W. M.
  2. EMBO J. v.6 Buisson, G.;Duee. E.;Haser, R.;Payan, F.
  3. J. Biochem. v.98 Ihara, H.;Sasaki, T.;Tsuboi, A.;Yamugato, H.;Tsukagoshi, N.;Udaka, S.
  4. Adv. Appl. Microbiol. v.24 Ingle, M. B.;Ericken, R. J.
  5. Protein engineering v.3 Holm, L.;Koivula, A. K.;Lehtovaara, P. M.;Hemminki, A.;Knowles, J. K. C.
  6. J. Biochem. v.95 Matsuura, Y.;Kusunoki, M.;Harada, W.;Kakudo, M.
  7. J. Biochem. v.92 Kita, Y.;Sakaguchi, M.;Nitta, Y.;Watanabe, T.
  8. I. Mol. Biol. (MOSC) v.22 no.5 Smirnova, N. A.;Sorokin, A. V.;Iomantas, V. Yu.;Abalakina, E. G.;Kozlov, Yu.;Stepanov, A. I.;Smirnova, N. A.;Sorokin, A. V.;Laptev, D. A.;Veiko, V. P.;Kozlov, Yu.
  9. Gene. v.19 Palva, I.
  10. Arch. Biochem. Biophysics v.155 Saito, N.
  11. Proc. Natl. Acad. Sci. v.74 Sanger, F. O.;Nicklem, S.;Coulson, A. R.
  12. J. Mol. Biol. v.53 Mandel, M.;Higa, A.
  13. Molecular Cloning Maniatis, T.;Fritsch, E. F.;Sambrook, J.
  14. Biotechnology v.55 Rehm, H. J.;Reed, G
  15. J. Biochem. v.41 Fuwa, H.
  16. Agric. Biol. Chem. v.43 Normura, D.
  17. Proc. Natl. Acad. Sci. USA v.80 Mielenze, J. R.
  18. FEMS microbial Lett. v.21 Joyet, P.;Guerineau, M.;Heslot, H.
  19. Mol. Gen. Genet. v.186 Cornelis, P.;Digneffe, C.;Willemot, K.
  20. J. Bacteriol. v.162 Ulmanen, I.;Lundstrom, K.;Lettovaara, P.;Sarvas, P.;Ruohonen, M.;Palva, I.
  21. J. Biochem. v.98 Yuuki, T.;Nomura, T.;Tezuka, H.;Tsuboi, A.;Yamagata, H.;Tsukagoshi, N.;Udaka, S.
  22. J. Biochem. v.84 Takahashi, T.;Tsuchida, Y.;Irie, M.
  23. Nucl. Acids Res. v.13 Taylor, J. W.;Ott, J.;Eckstein, F.
  24. Methods in enzymology v.I Bernflet, P.