Construction of chimeric cyclodextrin glucanotransferases by homologous recombination and study of their activites / Anusak Keadsin = การสร้างไซโคลเดกซ์ทรินกลูคาโนทรานเฟอเรสไคเมอริกโดยอาศัยโฮโมโลกัสรีคอมบิเนชันและศึกษาการทำงานของเอนไซม์ไคเมอริก / อนุศักดิ์ เกิดสิน
Cyclodextrins are cyclic oligosaccharides of 6, 7 and 8 glucose units, called alpha-, beta-, and g-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. CGTase is an enzyme consisting 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 alpha-, beta- and gamma-CDs with different ratios and the separation of CDs is time-consuming and employs expensive organic solvents. This study is aimed to determine the essential part of enzyme that may be involved in different ratio of CDs production by using in vivo homologous recombination between alpha-CGTase from Paenibacillus macerans and beta-CGTase from Bacillus circulans A11. Two plasmids, containing a direct repeat of either the beta- and alpha-CGTase genes (pVR321) or alpha- and beta- CGTase genes (pVR388), were used to construct the chimeric CGTase genes. After appropriate restriction enzyme digestion, each linearized plasmid was transformed into a recombination-proficient host. The resulting chimeric clones were characterized. Two series of chimeric plasmids were obtained. For chimeric CGTases from pVR321 series, we found that recombination within the domains C and D decreased the production of alpha-CD significantly and increased the production of beta- and gamma-CDs. Recombination within the 3-half of subdomain A2 had little effect on the production of alpha- and beta-CDs, while the proportion of gamma-CD was slightly increased. The chimeric CGTases in this series still produce beta-CD as a major product. For the chimeras in pVR388 series, several of them showed neither dextrinizing nor cyclization activities. Interestingly, two chimeras in this series, pVR402 and 403 whose N-terminal regions about 100 amino acid residues (N-terminal half of subdomain A1) derived from a-CGTase, produced beta-CD as major product. Replacing the subdomain A2, domains C, D and E in pVR402 with the wild type alpha-CGTase sequence results in a chimeric plasmid pVR404, whose CGTase gene is equivalent to alpha-CGTase containing the C-terminal half of subdomain A1 and domain B of the beta-CGTase sequence. The pVR404 produced alpha-CD as major product. By comparing the results from pVR404 with those of pVR402, 403 and pVR321 series, the subdomain A2 region most likely contains the determinant for CGTase product specificity. Nevertheless, the existence of the beta-CGTase sequence in this region renders the CGTase produced more beta- and less alpha-CDs. In summary, it can be seen that although the A and B domains are considered to be important for product specificity, the other domains also contributed to this specificity as well.
