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http://dx.doi.org/10.4014/jmb.1402.02004

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)
Publication Information
Journal of Microbiology and Biotechnology / v.24, no.7, 2014 , pp. 898-904 More about this Journal
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.
Keywords
Direct evolution; Bacillus licheniformis; alpha-amylase; characterization; specific activity; structure model;
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