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.
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School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, 106 91, Stockholm, Sweden.
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CERMAV-FR-CNRS 2607, BP 53, F-38041 Grenoble, France.
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.
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Centre de Recherche sur les Macromolécules Végétales (CERMAV-CNRS), BP53, 38041 Grenoble Cedex 9, France.
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Department of Chemistry, Columbia University, New York, New York 10027, USA.
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