Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
6490629 | Journal of Biotechnology | 2016 | 8 Pages |
Abstract
The thermostability of the maltohexaose-forming α-amylase from Bacillus stearothermophilus (AmyMH) without added Ca2+ was improved through structure-based rational design in this study. Through comparison of a homologous model structure of AmyMH with the crystal structure of the thermostable α-amylase from Bacillus licheniformis, Ser242, which located at the beginning of fourth α-helix of the central (β/α)8 barrel was selected for mutation to improve thermostability. In addition, an amide-containing side chain (Asn193) and a loop in domain B (ÎIG mutation), which have been proven to be important for thermostability in corresponding position of other α-amylases, were also investigated. Five mutants carrying the mutations ÎIG, N193F, S242A, ÎIG/N193F, and ÎIG/N193F/S242A were generated and their proteins characterized. The most thermostable mutant protein, ÎIG/N193F/S242A, exhibited a 26-fold improvement in half-life at 95 °C compared to the wild-type enzyme without added Ca2+. Mutant ÎIG/N193F/S242A also exhibited substantially better activity and stability in the presence of the chelator EDTA, demonstrating enhanced Ca2+ binding. These results suggest that mutant ÎIG/N193F/S242A has potential for use in the industrial liquefaction of starch.
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Authors
Zhu Li, Xuguo Duan, Jing Wu,