A novel method for chemo-enzymatic synthesis of chitin oligosaccharide catalyzed by the mutant of inverting family GH19 chitinase using 4,6-dimethoxy-1,3,5-triazin-2-yl α-chitobioside as a glycosyl donor

: A novel method for the chemo-enzymatic synthesis of chitin oligosaccharide catalyzed by mutants of BcChi-A, an inverting family GH19 chitinase from Bryum coronatum, has been developed using 4,6-dimethoxy-1,3,5-triazin-2-yl α-chitobioside [DMT-α-(GlcNAc)2)] as a donor substrate. Based on the glycosynthase derived from BcChi-A, Glu70, which acts as a catalytic base, and Ser102, which fixes a nucleophilic water molecule, were changed to generate several single and double mutants of BcChi-A, which were employed in synthetic reactions. Among the double mutants tested, E70G/S102G, E70G/S102C and E70G/S102A were found to successfully synthesize chitotetraose [(GlcNAc)4] from DMT-α-(GlcNAc)2 and (GlcNAc)2; however, the single mutants, E70G, S102G, S102C and S102A, did not. Among the mutants, E70G/S102A showed the highest synthetic activity. This is the first report of a glycosynthase that employs a dimethoxytriazine-type glycoside as a donor substrate.