Mutagenesis of beta cyclodextrin clucanotransferase gene from Bacillus circulans A11 That affects the y-cyclodextrin production

Abstract

Cyclodextrins are cyclic oligosaccharides of 6, 7 and 8 glucose units, called α -, β- and γ -cyclodextrins (CDs), 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. CGTase consists of 5 domains, A, B, C, D and E; domain B divides domain A into subdomain A1 and A2. It has been known that natural CGTases produce a mixture of α -, β- and γ-CDs at different ratios making the separation of CDs time¬ consuming and costly. A CGTase that can produce one type of CD is desirable. The γ -CD, the largest of the three cyclodextrins, can trap larger molecules that cannot be trapped by α -and β -CDs. Although there is a demand for γ -CDs in various industries, the γ -CD is not widely used in practice due to low production of this substance. To engineer a CGTase that produces γ -CD, parts of the enzyme that involved in product specificity should be verified. By using the information from bioinformatics, the amino acid sequence comparison of the three CGTase types shows that they have moderate homology but γ -CGTase has 3 regions that are different from the other two CGTases. These are amino acid sequences at position 87-95, 141-152 and 532-536 (B. circulans A11 CGTase numbering). The first and second regions are at the substrate binding subsites -3 and -7, respectively, in the active site region. The third region is located in domain D. In this study, the three equivalent regions in β -CGTase from Bacillus circulans A11 are mutated in favor of the γ -CGTase sequence. The activities of the mutants are studied in order to determine how these different regions affect the production of γ -CD. The three regions in β -CGTase gene are mutated using USE mutagenesis method, which includes deletion and substitution of some amino acid • residues. The mutant plasmids, pNan1, 2 and 3 that have the mutation sites I (position 87-95), II (position 141-152) and III (position 532-536), respectively, are obtained . Then, the mutant plasmids containing the combinations of these 3 mutation regions are constructed. The dextrinizing and CD-forming activities of the mutant enzymes from these clones are studied. From the results, subsites -3 (mutation site I) plays an important role in the production of β -CD and subsite -7 (mutation site II) is involved in the proportion of γ -CD production. Surprisingly, domain D is involved in the activity and product specificity of the enzyme, which indicates that the other domains also contributed to the specificity as well not just the active site.