Mutant endoglycoceramidases with enhanced synthetic activity

Details Mutant endoglycoceramidases with enhanced synthetic activity Mutant endoglycoceramidases with enhanced synthetic activity

C - Chemistry, Metallurgy

12

N

C12N 9/24 (2006.01)

Patent

CA 2567581

The present invention relates to a novel endoglycoceramidase whose hydrolytic activity has been substantially reduced or eliminated, such that the enzyme is useful for synthesis of glycolipids from a monosaccharide or oligosaccharide and a ceramide. More specifically, the endoglycoceramidase is a mutant version of a naturally occurring endoglycoceramidase, preferably comprising a mutation within the active site or the nucleophilic site of the enzyme and more preferably comprising a substitution mutation of the Glu residue within the active site or the nucleophilic site. Also disclosed are a method for generating the mutant endoglycoceramidase and a method for enzymatically synthesizing glycolipids using this mutant enzyme.

La présente invention se rapporte à une nouvelle endoglycocéramidase dont l'activité hydrolytique a été sensiblement réduite ou éliminée, de façon que l'enzyme puisse servir à synthétiser des glycolipides à partir d'un monosaccharide ou d'un oligosaccharide et d'un céramide. Plus précisément, l'endoglycocéramidase selon l'invention est une version mutante d'une endoglycocéramidase d'origine naturelle, qui contient de préférence une mutation dans le site actif ou le site nucléophile de l'enzyme, et qui contient idéalement une mutation de substitution du résidu Glu dans son site actif ou son site nucléophile. L'invention a également trait à un procédé de génération de l'endoglycocéramidase mutante selon l'invention, et à un procédé de synthèse enzymatique de glycolipides faisant appel à ladite enzyme mutante.

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