Engineering ofBacillus amyloliquefaciensα‐Amylase for Improved Catalytic Efficiency by Error‐Prone PCR

Abstract

Bacillus amyloliquefaciens α‐amylase (BAA) is one of well‐known midrange thermostability amylases that are widely used in food and washing processes. However, the improvement of its catalytic properties by molecular modification is still lagging. To improve the activity of α‐amylase BAA, mutants BAA28 and BAA294 were constructed via error‐prone PCR and purified by column chromatography in the present study. The catalytic efficiencies (Kcat/Km) of BAA28 and BAA294 were 2.42 mL mg−1 s −1 and 2.73 mL mg−1 s−1, which were 43% and 61% higher than that of the wild‐type BAA, respectively. Their specific activities were also increased by 40% and 62%, respectively, with no apparent changes of optimum temperature and pH. Homology modeling and molecular docking analysis suggest that the reduced steric hindrance was an important factor that enhanced catalytic efficiencies and specific activities of the variants. These results may deepen our understanding of the mechanisms underlying the effects of each mutation on the catalytic efficiency of BAA and facilitate the construction of potent BAA mutants.This article is protected by copyright. All rights reserved