title: Enhanced maltose production through mutagenesis of acceptor binding subsite +2 in Bacillus stearothermophilus maltogenic amylase.
abstract: Maltogenic amylases are used to decrease the maltotriose content of high maltose syrups. However, due to the interplay between the hydrolysis and transglycosylation activities of maltogenic amylases, the maltotriose contents of these syrups are still greater than that necessary for pure maltose preparation. In this study, the maltogenic amylase from Bacillus stearothermophilus was engineered to decrease its transglycosylation activity with the expectation that this would enhance maltose production. Site-directed mutagenesis was used to generate Trp 177 variants W177F, W177Y, W177L, W177N, and W177S. The transglycosylation activities of the mutant enzymes decreased as the hydrophilicity of the residue at position 177 increased. The mutant enzymes exhibited notable enhancements in maltose production, with a minimum of maltotriose contents of 0.2%, compared with 3.2% for the wild-type enzyme. Detailed characterization of the mutant enzymes suggests that the best of them, W177S, will deliver performance superior to that of the wild-type under industrial conditions.
authors.affiliation: 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: syc-1028@163.com.
authors.forename: Yecheng
authors.initials: Y
authors.lastname: Sun
authors.affiliation: 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: xgduan@jiangnan.edu.cn.
authors.forename: Xuguo
authors.initials: X
authors.lastname: Duan
authors.affiliation: 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: wangleijs@163.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: 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.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D020909:Acarbose / Q000378:metabolism; D000681:Amylases / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics*; D005947:Glucose / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006868:Hydrolysis; D008320:Maltose / Q000096:biosynthesis*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D014312:Trisaccharides / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D014312:Trisaccharides; C008317:maltotriose; D008320:Maltose; D013213:Starch; D000681:Amylases; D006026:Glycoside Hydrolases; C120909:glucan 1,4-alpha-maltohydrolase; D005947:Glucose; D020909:Acarbose
keywords: Bacillus stearothermophilus; High maltose syrup; Maltogenic amylase; Transglycosylation