title: Improving maltodextrin specificity for enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid by site-saturation engineering of subsite-3 in cyclodextrin glycosyltransferase from Paenibacillus macerans.
abstract: In this work, the subsite-3 of cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was engineered to improve maltodextrin specificity for 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) synthesis. Specifically, the site-saturation mutagenesis of tyrosine 89, asparagine 94, aspartic acid 196, and aspartic acid 372 in subsite-3 was separately performed, and three mutants Y89F (tyrosine→phenylalanine), N94P (asparagine→proline), and D196Y (aspartic acid→tyrosine) produced higher AA-2G titer than the wild-type and the other mutants. Previously, we found the mutant K47L (lysine→leucine) also had a higher maltodextrin specificity. Therefore, the four mutants K47L, Y89F, N94P, and D196Y were further used to construct the double, triple, and quadruple mutations. Among the 11 combinational mutants, the quadruple mutant K47L/Y89F/N94P/D196Y produced the highest AA-2G titer of 2.23g/L, which was increased by 85.8% compared to that produced by the wild-type CGTase. The reaction kinetics of all the mutants were modeled, and the pH and thermal stabilities of all the mutants were analyzed. The structure modeling indicated that the enhanced maltodextrin specificity may be related with the changes of hydrogen bonding interactions between the side chain of residue at the four positions (47, 89, 94, and 196) and the substrate sugars.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Qiaoyan
authors.initials: Q
authors.lastname: Xu
authors.forename: Ruizhi
authors.initials: R
authors.lastname: Han
authors.forename: Hyun-dong
authors.initials: HD
authors.lastname: Shin
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Chen
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis; D001483:Base Sequence; D004795:Enzyme Stability / Q000235:genetics; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism*; D015202:Protein Engineering; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D011134:Polysaccharides; C065378:ascorbic acid 2-O-glucoside; C008315:maltodextrin; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D001205:Ascorbic Acid
keywords: 2-O-Glucopyranosyl-l-ascorbic acid (AA-2G); Cyclodextrin glycosyltransferase (CGTase); Maltodextrin; Site-saturation mutagenesis; l-Ascorbic acid (l-AA)