Enzymatic Synthesis Of2-O-α-D-glucopyranosyl-L-Ascorbic Acid By Molecular Engineering Of Cyclodextrin Glycosyltransferase From Paenibacillus Macerans

Vitamin C (VC), acting as its reduced form L-ascorbic acid (L-AA), is a water-solublevitamin. It’s widely used in the pharmaceutical and cosmetic industries as well as apreservative with its excellent antioxidant activity. However, due to its extremely instablechemical structure, the application of VC has been greatly limited. To solve this problem,2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), a kind of VC glycosyl derivative, isconsidered to be the best with its high non-reducibility, excellent antioxidant ability andeffortless release of L-AA and glucose.AA-2G is synthesized by specific glycosyltransferases transglycosylation, transferingglucosides from glycosyl donors to C-2of VC. Among all the glycosyltransferases, CGTase(EC2.4.1.19) from Paenibacillus macerans is the superior one due to its broader substratesources and higher product specificity for AA-2G. As for glycosyl donors, cyclodextrins arenot suitable because of the high cost and low transformation efficiency. Maltodextrin, with itslow cost and high solubility in aqueous solution, has a great potential in large-scaleproduction of AA-2G..In this work, the subsites-3and+1of CGTase was determined by structure simulationand references. Using the recombinant plasmid pET20b(+)/cgt expressed in E. coli BL21(DE3) as the template, site-saturation engineering of amino acid residues around the twosubsites were seperately performed to improve the maltodextrin specificity for AA-2Gsynthesis. Among all the obtained mutants, the double-subsite combinational mutantK47L/Y89F/N94P/D196Y/L194N/A230D/H233E produced the highest AA-2G titer of2.37g·L-1, which was increased by97.4%compared with that produced by the wild-type CGTase.The pH and thermal stabilities of all the mutants were analyzed and the reaction kineticswere also modeled. The optimum reaction temperature of mutants is36oC，which is the samewith the wild-type. The optimum pH of mutants fluctuates in the range of5.5to7. Initialanalysis showed that amino acids of different polarities introduced by the mutants mightchange pKa of ionizing group at the active site of the enzyme. It was also found that mutantsmaintained good thermal stability and acid tolerance at40oC and pH5.5, respectively. Thehighest yield of AA-2G produced by the wild-type and mutant CGTases were all obtained at24h of the reaction. The structure modeling indicated that the enhanced maltodextrinspecificity may be related to the changes of hydrogen bonding interactions between the sidechain of residue and the substrate sugars.