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
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Ascomycota / enzymology*
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Ascomycota / genetics
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Ascomycota / metabolism
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Azides / chemical synthesis
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Azides / chemistry
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Carbohydrate Sequence
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Catalysis
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Chromatography, High Pressure Liquid
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Disaccharides / chemical synthesis
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Disaccharides / chemistry
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Disaccharides / metabolism
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Glycoside Hydrolases / chemistry
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Glycoside Hydrolases / genetics*
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Glycoside Hydrolases / metabolism*
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Glycosides / biosynthesis
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Glycosides / chemistry
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Lactose / analogs & derivatives
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Lactose / chemistry
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Mutagenesis, Site-Directed
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Oligosaccharides / chemistry
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Oligosaccharides / metabolism
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Stereoisomerism
Substances
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Azides
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Disaccharides
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Glycosides
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O-allyl N(1)-acetyl-2(II)-azidochitobioside
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Oligosaccharides
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lactosyl fluoride
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Glycoside Hydrolases
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Lactose