Mutations at calcium binding site III in cyclodextrin glycosyltransferase improve β-cyclodextrin specificity.
Cyclodextrin glycosyltransferases (CGTases, EC 2.4.1.19) are industrially important enzymes that produce cyclodextrins from starch by intramolecular transglycosylation. In this study, the effects of amino acid residue at position 315 in calcium binding site III (CaIII) on product specificity of CGTase were investigated by replacing Ala315 in the CGTase from Bacillus circulans STB01 with arginine, aspartic acid, threonine, leucine and valine. The cgt gene, which encodes this enzyme, was expressed in B. subtilis WB600 alongside site-directed mutants A315R, A315D, A315T, A315L and A315V. The results showed that CaIII plays an important role in cyclodextrin product specificity. Replacement of Ala315 by charged amino acid residues enhanced β-cyclodextrin specificity, compared with the wild-type CGTase. Mutations A315R and A315D resulted in an approximately 10% increase in β-cyclodextrin activity. Furthermore, under conditions resembling the industrial production processes, the mutants A315R and A315D displayed obvious increases in the production of β-cyclodextrin, indicating they were much more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhancement of β-cyclodextrin specificity for the mutants might be due to the stability of CaIII by charged amino acid substitutions.
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Xiaofeng
X
Ban
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.
Zhengbiao
Z
Gu
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Caiming
C
Li
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Min
M
Huang
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Li
L
Cheng
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.
Yan
Y
Hong
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: zfli@jiangnan.edu.cn.
Zhaofeng
Z
Li
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D003500:Cyclization; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D013213:Starch / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D047392:beta-Cyclodextrins / Q000737:chemistry / Q000378:metabolism*
D047392:beta-Cyclodextrins; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D002118:Calcium
Calcium binding site; Cyclodextrin glycosyltransferase; Product specificity; Residue 315; β-Cyclodextrin