Iterative Saturation Mutagenesis of-6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-O-D-Glucopyranosyl-L-Ascorbic Acid Synthesis

作者：Han, Ruizhi; Liu, Long^*; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua; Du, Guocheng; Chen, Jian

来源：Applied and Environmental Microbiology, 2013, 79(24): 7562-7568.

DOI：10.1128/AEM.02918-13

• L-ASCORBIC-ACID
• CIRCULANS STRAIN 251
• 2-O-ALPHA-D-GLUCOPYRANOSYL-L-ASCORBIC ACID
• ENZYMATIC TRANSFORMATION
• DIRECTED EVOLUTION
• GLUCANOTRANSFERASE
• TRANSGLUCOSYLATION
• 2-GLUCOSIDE
• AMYLASE
• STARCH

2-O-D-Glucopyranosyl-L-ascorbic acid (AA-2G), a stable L-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to -7). In this study, iterative saturation mutagenesis (ISM) was performed on the -6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites-Y167, G179, G180, and N193-was first performed and revealed that four mutants-Y167S, G179R, N193R, and G180R-produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the -6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.

• 出版日期2013-12
• 单位江南大学