A highly active alpha amylase from Bacillus licheniformis: directed evolution, enzyme characterization and structural analysis.
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.
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.
Yihan
Y
Liu
Shuai
S
Fan
Xiaoguang
X
Liu
Zhimeng
Z
Zhang
Jianling
J
Wang
Zhengxiang
Z
Wang
Fuping
F
Lu
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli; D007700:Kinetics; D008958:Models, Molecular; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D000516:alpha-Amylases