title: Asp577 mutations enhance the catalytic efficiency of cyclodextrin glycosyltransferase from Bacillus circulans.
abstract: The amino acid residue Asp 577 is located in calcium-binding site III (CaIII) of the cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) from Bacillus circulans STB01. In the present study, the effects of replacing Asp577 with glycine, alanine, valine, leucine, and isoleucine on the catalytic efficiency of this CGTase were investigated. Two of these replacements, D577G and D577A, increased the β-cyclization activity of CGTase. Kinetic studies showed that the Km values of D577G and D577A were 36.1% and 18.0% lower and the kcat/Km values were 43.9% and 23.0% higher than those of the wild-type enzyme, respectively. These mutations increased both the affinity of CGTase for maltodextrin and the catalytic efficiency of the cyclization reaction. Furthermore, although D577G and D577A only slightly enhanced β-cyclodextrin production, compared with the wild-type enzyme, their higher β-cyclization activities resulted in a significant reduction in the amount of mutant protein required during the cyclodextrin production process. Thus, the two mutants are more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhancement of catalytic efficiency may be due to the smaller size of the glycine and alanine side chains, which may weaken the impact of this residue on CaIII.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China.
authors.forename: Zhaofeng
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors.forename: Min
authors.initials: M
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China. Electronic address: zhengbiaogu@jiangnan.edu.cn.
authors.forename: Zhengbiao
authors.initials: Z
authors.lastname: Gu
authors.affiliation: Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1065, USA.
authors.forename: Tod P
authors.initials: TP
authors.lastname: Holler
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors.forename: Li
authors.initials: L
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Hong
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China. Electronic address: caimingli@jiangnan.edu.cn.
authors.forename: Caiming
authors.initials: C
authors.lastname: Li
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis / Q000187:drug effects*; D003505:Cyclodextrins / Q000235:genetics; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics; D047392:beta-Cyclodextrins / Q000378:metabolism
chemical_list: D003505:Cyclodextrins; D050505:Mutant Proteins; D047392:beta-Cyclodextrins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase
keywords: Calcium-binding site; Cyclodextrin; Cyclodextrin glycosyltransferase