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J Microbiol Biotechnol. 2014 Jul;24(7):898-904.

A highly active alpha amylase from Bacillus licheniformis: directed evolution, enzyme characterization and structural analysis.

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Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin 300457, P. R. China , Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300457, P. R. China, National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, P. R. China.


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°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 kcat/Km 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 Km, and consequently increased the specific activity.

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