title: Improving the thermostability and enhancing the Ca(2+) binding of the maltohexaose-forming α-amylase from Bacillus stearothermophilus.
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.
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors: Zhu
authors: Z
authors: Li
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors: Xuguo
authors: X
authors: Duan
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Electronic address: jingwu@jiangnan.edu.cn.
authors: Jing
authors: J
authors: Wu
mesh_terms: D004492:Edetic Acid; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; C016549:maltohexaose; D004492:Edetic Acid; D000516:alpha-Amylases
keywords: Alpha-amylase; Ca(2+) Binding; Site-directed mutagenesis; Thermostability