Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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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
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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.

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