Unexpected regioselectivity of Humicola insolens Cel7B glycosynthase mutants

Sophie Blanchard; Sylvie Armand; Pedro Couthino; Shamkant Patkar; Jesper Vind; Eric Samain; Hugues Driguez; Sylvain Cottaz

doi:10.1016/j.carres.2006.12.017

Unexpected regioselectivity of Humicola insolens Cel7B glycosynthase mutants

Carbohydr Res. 2007 Apr 9;342(5):710-6. doi: 10.1016/j.carres.2006.12.017. Epub 2006 Dec 22.

Authors

Sophie Blanchard¹, Sylvie Armand, Pedro Couthino, Shamkant Patkar, Jesper Vind, Eric Samain, Hugues Driguez, Sylvain Cottaz

Affiliation

¹ CERMAV-FR-CNRS 2607, BP 53, F-38041 Grenoble, France.

PMID: 17224137
DOI: 10.1016/j.carres.2006.12.017

Abstract

Four Humicola insolens Cel7B glycoside hydrolase mutants have been evaluated for the coupling of lactosyl fluoride on O-allyl N(I)-acetyl-2(II)-azido-beta-chitobioside. Double mutants Cel7B E197A H209A and Cel7B E197A H209G preferentially catalyze the formation of a beta-(1-->4) linkage between the two disaccharides, while single mutant Cel7B E197A and triple mutant Cel7B E197A H209A A211T produce predominantly the beta-(1-->3)-linked tetrasaccharide. This result constitutes the first report of the modulation of the regioselectivity through site-directed mutagenesis for an endoglycosynthase.

MeSH terms

Ascomycota / enzymology*
Ascomycota / genetics
Ascomycota / metabolism
Azides / chemical synthesis
Azides / chemistry
Carbohydrate Sequence
Catalysis
Chromatography, High Pressure Liquid
Disaccharides / chemical synthesis
Disaccharides / chemistry
Disaccharides / metabolism
Glycoside Hydrolases / chemistry
Glycoside Hydrolases / genetics*
Glycoside Hydrolases / metabolism*
Glycosides / biosynthesis
Glycosides / chemistry
Lactose / analogs & derivatives
Lactose / chemistry
Mutagenesis, Site-Directed
Oligosaccharides / chemistry
Oligosaccharides / metabolism
Stereoisomerism

Substances

Azides
Disaccharides
Glycosides
O-allyl N(1)-acetyl-2(II)-azidochitobioside
Oligosaccharides
lactosyl fluoride
Glycoside Hydrolases
Lactose