School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
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.
Int J Biol Macromol. 2015 May;76:224-9. doi: 10.1016/j.ijbiomac.2015.02.036. Epub 2015 Mar 4.
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.
环糊精糖基转移酶（CGTase，EC 2.4.1.19）是一种工业上重要的酶，它通过分子内转糖基作用将淀粉转化为环糊精。在这项研究中，通过用精氨酸、天冬氨酸、苏氨酸、亮氨酸和缬氨酸替换环状芽孢杆菌 STB01 来源的 CGTase 中钙结合位点 III（CaIII）上第 315 位的氨基酸残基，研究了 CaIII 上氨基酸残基对 CGTase 产物特异性的影响。该酶的 cgt 基因与定点突变体 A315R、A315D、A315T、A315L 和 A315V 一起在枯草芽孢杆菌 WB600 中表达。结果表明，CaIII 对环糊精产物特异性起着重要作用。与野生型 CGTase 相比，带电荷氨基酸残基取代 Ala315 增强了β-环糊精特异性。突变体 A315R 和 A315D 使β-环糊精活性增加了约 10%。此外，在类似于工业生产过程的条件下，突变体 A315R 和 A315D 显示出β-环糊精产量的明显增加，表明它们比野生型酶更适合β-环糊精的工业生产。突变体对β-环糊精特异性的增强可能归因于带电荷氨基酸取代对 CaIII 的稳定性。
