A Highly Active Alpha Amylase from Bacillus licheniformis: Directed Evolution, Enzyme Characterization and Structural Analysis

作者：Liu, Yihan; Fan, Shuai; Liu, Xiaoguang; Zhang, Zhimeng; Wang, Jianling; Wang, Zhengxiang^*; Lu, Fuping

来源：Journal of Microbiology and Biotechnology, 2014, 24(7): 898-904.

DOI：10.4014/jmb.1402.02004

• Direct evolution
• Bacillus licheniformis
• alpha-amylase
• characterization
• specific activity
• structure model

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 degrees 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.

• 出版日期2014-7
• 单位天津科技大学