Mutant endoglycoceramidases with enhanced synthetic activity

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, 4, 6, 8, 10, 12, 14, 16-20, and wherein the amino acid substitution is selected from Ser, Gly, and Ala, and wherein the acceptor substrate is a sphingosine or a sphingosine analog.

A method of producing GM1, the method comprising:

• contacting a fluorinated GM1 sugar donor substrate 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 fluorinated GM1 donor substrate to the acceptor substrate at a rate that exceeds hydrolysis of the GM1, wherein said wild-type endoglycoceramidase has the amino acid sequence of SEQ ID NO:
• 2 and said variant has a serine substitution of the Glu residue at position 354 of SEQ ID NO:
• 2, and wherein the acceptor substrate is a sphingosine or a sphingosine analog.