title: [Improving maltodextrin specificity by site-saturation engineering of subsite +1 in cyclodextrin glycosyltransferase from Paenibacillus macerans].
abstract: By engineering the subsite +1 of cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans, we improved its maltodextrin specificity for 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) synthesis. Specifically, we conducted site-saturation mutagenesis on Leu194, Ala230, and His233 in subsite +1 separately and gained 3 mutants L194N (leucine --> asparagine), A230D (alanine --> aspartic acid), and H233E (histidine --> glutamic acid) produced higher AA-2G yield than the wild-type and the other mutant CGTases. Therefore, the 3 mutants L194N, A230D, and H233E were further used to construct the double and triple mutations. Among the 7 obtained combinational mutants, the triple mutant L194N/A230D/H233E produced the highest AA-2G titer of 1.95 g/L, which was increased by 62.5% compared with that produced by the wild-type CGTase. Then, we modeled the reaction kinetics of all the mutants and found a substrate inhibition by high titer of L-AA for the mutants. The optimal temperature, pH, and reaction time of all the mutants were also determined. 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 three positions (194, 230 and 233) and the substrate sugars.
authors.forename: Qiaoyan
authors.initials: Q
authors.lastname: Xu
authors.forename: Ruizhi
authors.initials: R
authors.lastname: Han
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D001205:Ascorbic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology*; D011134:Polysaccharides / Q000737:chemistry*; D015202:Protein Engineering; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011134:Polysaccharides; C065378:ascorbic acid 2-O-glucoside; C008315:maltodextrin; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D001205:Ascorbic Acid