Mutagenesis of cyclodextrin glucanotransferase gene from Bacillus circulans A11 to change the product specificity of the enzyme

Abstract

Cyclodextrins (CDs) are cyclic oligosaccharides of 6, 7 and 8 glucose units, linked by [alpha]-1,4-glycosidic bonds, called [alpha]-, [beta]- and [gamma]-cyclodextrins, respectively. CDs are the products of enzymatic conversion of starch and related substrates by cyclodextrin glucanotransferases (CGTases), and are useful carrier molecules for several applications in industries. The CGTase consists of 5 domains, A, B, C, D and E. Domains A/B are the central catalytic domain while others perform accessory functions. CGTases that predominantly produce one type of cyclodextrin are of great importance for industry because the costly and time-consuming separation of different cyclodextrins can be avoided. Amino acid sequence comparison of subdomain A2 between the Bacillus circulans A11 [beta]-CGTase and the [alpha]- and [gamma]- CGTases, four different regions I, II, III and IV were found at position 239-250, 262-271, 284-320 and 333-342 (B. circulans A11 CGTase numbering), respectively. In addition, S184, E264 and E268 were also selected to study the mode of substrate entrance. The four regions as well as S184, E264 and E268 in [beta]-CGTase from Bacillus circulans A11 were mutated using the unique site elimination (USE) mutagenesis method. The dextrinizing activity and CD-forming activity of the mutant enzymes were assayed. It was found that region I mutation had no influence on product specificity, regions II and III favored [beta]-CD production, and region IV showed little affect on [beta]-CD production. The S184K mutation slightly increased the proportion of [alpha]-CD with the expense of [gamma]-CD.