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.
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.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
Ruizhi
R
Han
Long
L
Liu
Hyun-Dong
HD
Shin
Rachel R
RR
Chen
Jianghua
J
Li
Guocheng
G
Du
Jian
J
Chen
D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D001665:Binding Sites; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D060847:Metabolic Engineering / Q000379:methods*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
C484753:2-O-(beta-D-glucopyranosyl)ascorbic acid; D050505:Mutant Proteins; D011134:Polysaccharides; C008315:maltodextrin; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D001205:Ascorbic Acid