Biotechnology Bulletin ›› 2013, Vol. 0 ›› Issue (10): 153-159.

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Saturation Mutagenesis and the Enzyme Properties of the Alpha-Amylase from Geobacillus sp.

Xue Bei1,Pei Jianxin2, Wang Zhibin3, Luo Zhang1,Wei Yutuo4,   

  1. （1. Department of Food Science，Tibet Agricultural and Animal Husbandry College，Linzhi 860000 ；2. National Engineering Research Center for Non-food Biorefinery，Guangxi Academy of Science，Nanning 530007 ；3.Chengdu Rongsheng Pharmaceuticals Co.，Ltd，Chengdu 610041 ；4. Key Laboratory of Microbial and Plant Genetic Engineering of Ministry of Education， Guangxi University，Nanning 530005）
  • Received:2013-03-21 Revised:2013-10-15 Online:2013-10-14 Published:2013-10-15

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Abstract:

In this study, manipulation of half rational design was performed. Firstly, the dimensional structure of AMY（alpha-amylase of Geobacillus sp. GXS1）was built via homology modeling by using a close-related（33% homology in sequence）maltogenic amylase（BSMA） from B. stearothermophilus as a template. Secondly, three amino acid positions at the active site of AMY（Asp192, Glu221 and Asp289）were selected by structural superposition onto AMY combined with energy calculation by the computer-aided design software ProSa2003. These three putative amino acids for the active site of AMY, Asp192, Glu221 and Asp289, were subjected to saturation mutagenesis using degenerate primers. Four mutants（Asp192Ala, Glu221Asn, Glu221Leu and Asp289Leu）were obtained from the saturation mutant library, and studied the enzymatic properties of mutation enzyme simply.

Key words: Geobacillus, Alpha-amylase, Characterization, Site-saturation mutagenesis