Mutant endoglycoceramidases with enhanced synthetic activity

摘要

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.

主权项

1. A method of producing a glycolipid, the method comprising: contacting a donor substrate having an activated saccharide moiety and an acceptor substrate with a variant of a wild-type endoglycoceramidase in a reaction mixture, wherein the variant catalyzes the transfer of the saccharide moiety from the donor substrate to the acceptor substrate at a rate that exceeds hydrolysis of the glycolipid, wherein the wild-type endoglycoceramidase has a nucleophilic region comprising a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-(Glu/Asp)-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence, said variant comprises an amino acid substitution of the Glu residue within said sequence in any one of SEQ ID NOs:2, 5, 6, 8, 11, 12, 14, 17, 18, 20, 84, and 85 wherein the amino acid substitution is selected from Ser, Gly, and Ala and wherein the acceptor substrate is sphingosine or a sphingosine analog.