γ-Cyclodextrin (γ-CD), a cyclic oligosaccharide containing eight glucose units linked by α-1,4-glycosidic bonds, can be produced from starch using cyclodextrin glycosyltransferase (CGTase). Unfortunately, this enzymatic process produces mixtures of α-, β-, and γ-CD. In this study, amino acid residues in the subsite -3 (T47 and F91) and the central subsite (Y186) of Bacillus clarkii γ-CGTase were modified to improve the γ-CD production. The cyclization activities and product specificities of mutants T47H and F91W were similar to those of the wild-type. The cyclization activities of mutants F91N and F91L were significantly greater than those of the wild-type but their γ-CD product specificities were lower. Finally, the central subsite mutant Y186W displayed a γ-CD specificity (94.6%) significantly greater than that of the wild-type (77.1%). To maximize the γ-CD yield, the effects of added complexing agents were investigated. Among the cyclic complexing agents tested, low-boiling cyclododecanone was the smallest that precipitated with γ-CD. When cyclododecanone was used with Y186W, the total CD yield reached 72.6%, and 96.6% of the product was γ-CD. These results, which represent the highest γ-CD yield ever reported, may provide a way to improve large-scale γ-CD preparation and expand the uses of γ-CD in the future.
Keywords: complexing agent; cyclodextrin; cyclodextrin glycosyltransferase; enzymatic conversion; modification.