Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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A Highly Active Alpha Amylase from Bacillus licheniformis: Directed Evolution, Enzyme Characterization and Structural Analysis

  • Liu, Yihan (Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education) ;
  • Fan, Shuai (Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education) ;
  • Liu, Xiaoguang (Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education) ;
  • Zhang, Zhimeng (Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education) ;
  • Wang, Jianling (The College of Biotechnology, Tianjin University of Science and Technology) ;
  • Wang, Zhengxiang (Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education) ;
  • Lu, Fuping (Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education)
  • Received : 2014.02.04
  • Accepted : 2014.04.10
  • Published : 2014.07.28
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Abstract

The stability of Bacillus licheniformis alpha-amylase (BLA) under acid condition was enhanced through direct evolution using the error-prone polymerase chain reaction. One beneficial mutation site, H281I, was obtained in BLA. The specific activity of H281I was 161/352 U/mg, which was 62.6/27.5% higher than that of the wild-type (WT) (99/276 U/mg) at pH 4.5/6.5 and $95^{\circ}C$. The pH optimum for H281I was decreased about 1 unit, whereas no significant changes of optimum temperature and thermostability were observed compared with the wild type (WT). The $k_{cat}/K_m$ value of H281I was 1.7-/1.4-fold higher at pH 4.5/6.5, respectively, than that of WT. The structure model analysis indicated that the H281I mutation altered the predicted interaction between the amino acid residues at 281 and 273, thus creating a conducive local environment for substrate binding, as reflected by its decreased $K_m$, and consequently increased the specific activity.

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