Mutagenesis of cyclodextrin clucanotransferase gene to determine the thermostability of the enzyme

Abstract

Cyclodextrins (CDs) are cyclic oligosaccharides of 6, 7 and 8 glucose units, linked by α-1, 4-glycosidic bonds, called α-, β- and γ-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 is the central catalytic domains while others perform accessory functions. The commercial production of CDs required that the starch be liquefied at high temperature before the CGTase reaction at much lower temperature. Thermostable CGTase would, therefore, be useful for efficient production of CDs. By using amino acid sequence comparison between the Bacillus circulans A11 CGTase and the thermostable CGTase,l four major different regions, I, II, III and IV were found at position 89-94, 265-271, 333-339, and 538-540 (B. circulans A11 CGTase numbering), respectively. The relevant regions I-III were located in domains A/B. In this study, these three regions in β-CGTase from Bacillus circulans A11 were mutated in favor of the thermostable CGTase sequences using the unique site elimination (USE) mutagenesis method. The mutant plasmids, pRS1, 2 and 3 that have the mutation region I, II, and III, respectively, were obtained. Then, the recombinant plasmids containing the various combinations of the 3 mutation regions were constructed. The dextrinizing activity, thermostability and CD-forming activity of the mutant enzymes from these clones were studied in order to determine whether these different regions affect the stability of CGTase. We found that all the three mutation regions gave rise to an increase in dextrinizing activity, a decrease in optimum temperature and no increase in thermostability. All CGTase mutants were active in CD-forming activity; all but one with altered product specificity.