title: Engineering of isoamylase: improvement of protein stability and catalytic efficiency through semi-rational design.
abstract: Isoamylase catalyzes the hydrolysis of α-1,6-glycosidic linkages in glycogen, amylopectin and α/β-limit dextrins. A semi-rational design strategy was performed to improve catalytic properties of isoamylase from Bacillus lentus. Three residues in vicinity of the essential residues, Arg505, Asn513, and Gly608, were chosen as the mutation sites and were substituted by Ala, Pro, Glu, and Lys, respectively. Thermal stability of the mutant R505P and acidic stability of the mutant R505E were enhanced. The k cat /K m values of the mutant G608V have been promoted by 49%, and the specific activity increased by 33%. This work provides an effective strategy for improving the catalytic activity and stability of isoamylase, and the results obtained here may be useful for the improvement of catalytic properties of other α/β barrel enzymes.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. liyouran@jiangnan.edu.cn.|National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China. liyouran@jiangnan.edu.cn.
authors.forename: Youran
authors.initials: Y
authors.lastname: Li
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. zhangl@jiangnan.edu.cn.|National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China. zhangl@jiangnan.edu.cn.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. bioding_jnu@yahoo.com.|National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China. bioding_jnu@yahoo.com.
authors.forename: Zhongyang
authors.initials: Z
authors.lastname: Ding
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. 327984744@qq.com.|National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China. 327984744@qq.com.
authors.forename: Zhenghua
authors.initials: Z
authors.lastname: Gu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. gyshi@jiangnan.edu.cn.|National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, People's Republic of China. gyshi@jiangnan.edu.cn.
authors.forename: Guiyang
authors.initials: G
authors.lastname: Shi
mesh_terms: D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D055162:Biocatalysis; D007517:Isoamylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D055550:Protein Stability
chemical_list: D007517:Isoamylase
keywords: Bacillus lentus; Catalytic efficiency; Isoamylase; Semi-rational design; Structure model