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Improving the Thermostability and Enhancing the Ca(2+) Binding of the Maltohexaose-Forming α-amylase From Bacillus Stearothermophilus

Improving the Thermostability and Enhancing the Ca(2+) Binding of the Maltohexaose-Forming α-amylase From Bacillus Stearothermophilus

Zhu Li, Xuguo Duan, Jing Wu

J Biotechnol. 2016 Mar 20;222:65-72.

PMID: 26869314

Abstract:

The thermostability of the maltohexaose-forming α-amylase from Bacillus stearothermophilus (AmyMH) without added Ca(2+) 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 (ΔIG 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 ΔIG, N193F, S242A, ΔIG/N193F, and ΔIG/N193F/S242A were generated and their proteins characterized. The most thermostable mutant protein, ΔIG/N193F/S242A, exhibited a 26-fold improvement in half-life at 95°C compared to the wild-type enzyme without added Ca(2+). Mutant ΔIG/N193F/S242A also exhibited substantially better activity and stability in the presence of the chelator EDTA, demonstrating enhanced Ca(2+) binding. These results suggest that mutant ΔIG/N193F/S242A has potential for use in the industrial liquefaction of starch.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP34620774	Maltohexaose		34620-77-4	Price

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