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

J Biochem. 2019 Jun 1;165(6):497-503. doi: 10.1093/jb/mvy123.

Abstract

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.

Keywords: DMT-glycoside; GH19 chitinase; chitin oligosaccharide; glycosynthase.

MeSH terms

  • Biocatalysis*
  • Bryopsida / enzymology
  • Bryopsida / genetics
  • Carbohydrate Conformation
  • Chitin / chemistry
  • Chitin / metabolism*
  • Chitinases / genetics*
  • Chitinases / metabolism*
  • Glycosylation
  • Models, Molecular
  • Mutation*
  • Oligosaccharides / chemical synthesis*
  • Oligosaccharides / chemistry
  • Oligosaccharides / metabolism*

Substances

  • Oligosaccharides
  • Chitin
  • Chitinases