Mutants of glycoside hydrolases and uses thereof for synthesizing complex oligosaccharides and disaccharide intermediates

A method for preparing the building block corresponding to a disaccharide α-D-glucopyranosyl-(1-4)-N-acetyl-α-D-glucopyranosaminyl of formula (Ia):

• wherein said method comprises the step of reacting:
• A) a mutant of a glycoside hydrolase,
• wherein said wild type glycoside hydrolase has 450 to 850 amino acids and comprises eleven motifs defined by the following consensus motifs:
• (1) the amino acid sequence LGVNYLHLMPL (SEQ ID NO:
• 1), which is located in the β-strand 2 of said wild type glycoside hydrolase;
• (2) the amino acid sequence DGGYAV (SEQ ID NO:
• 2), which is located in the loop 2 of the (β/α)8-barrel of said wild type glycoside hydrolase;
• (3) the amino acid sequence DFVFNH (SEQ ID NO:
• 3) which is located in the β-strand 3 of said wild type glycoside hydrolase;
• (4) the amino acid sequence LREIFPDTAPGNF (SEQ ID NO:
• 4), which is located in the domain B of said wild type glycoside hydrolase;
• (5) the amino acid sequence FNSYQWDLN (SEQ ID NO:
• 5), which is located in the C-terminal part of the domain B of said wild type glycoside hydrolase;
• (6) the amino acid sequence ILRLDAVAFLWK (SEQ ID NO:
• 6), which is located in the β-strand 4 of said wild type glycoside hydrolase;
• (7) the amino acid sequence EAIV (SEQ ID NO:
• 7), which is located in the β-strand 5 of said wild type glycoside hydrolase;
• (8) the amino acid sequence YVRCHDDI (SEQ ID NO:
• 8), which is located in the β-strand 7 of said wild type glycoside hydrolase;
• (9) the amino acid sequence RISGTLASLAG (SEQ ID NO:
• 9), which is located in the domain B′ of said wild type glycoside hydrolase;
• (10) the amino acid sequence GIPLIYLGDE (SEQ ID NO:
• 10), which is located in the β-strand 8 of said wild type glycoside hydrolase;
• (11) the amino acid sequence RWVHRP (SEQ ID NO:
• 11), which is located in the loop 8 of the (β/α)8-barrel,
• and the sequence formed by said eleven motifs from said wild type glycoside hydrolase joined end-to-end from motif (1) to motif (11) has at least 65% sequence identity or at least 80% sequence similarity with the amino acid sequence SEQ ID NO:
• 12;
• wherein said mutant has one or two mutation(s) consisting of, when said mutant has only one mutation:
• the substitution of the amino acid residue at position 4 in said motif (4) with any amino acid selected from the group consisting of alanine (A), cysteine (C), glutamic acid (E), glycine (G), histidine (H), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), serine (S), threonine (T), valine (V) with the provisio that said wild type glycoside hydrolase does not contain a valine at this position, tryptophan (W) and tyrosine (Y), or
• the substitution of the amino acid residue at position 5 in said motif (4) with any amino acid selected from the group consisting of leucine (L), methionine (M) and valine (V), or
• the substitution of the amino acid residue at position 8 in said motif (6) with any amino acid selected from the group consisting of glutamic acid (E), phenylalanine (F), glycine (G), lysine (K), leucine (L), methionine (M), proline (P), glutamine (Q), arginine (R) and valine (V), or
• the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V) and tryptophan (W), or
• the substitution of the amino acid residue at position 4 in said motif (7) with any amino acid selected from the group consisting of alanine (A), cysteine (C), aspartic acid (D), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), asparagine (N), serine (S), threonine (T) and tyrosine (Y), or
• the substitution of the amino acid residue at position 7 in said motif (8) with any amino acid selected from the group consisting of alanine (A) and valine (V), or
• the substitution of the amino acid residue at position 1 in said motif (9) with any amino acid selected from the group consisting of alanine (A), cysteine (C), phenylalanine (F), glycine (G) with the provisio that said wild type glycoside hydrolase does not contain a glycine at this position, lysine (K), asparagine (N), glutamine (Q), serine (S) with the provisio that said wild type glycoside hydrolase does not contain a serine at this position, threonine (T) and tryptophan (W), or
• when said mutant has two mutations:
• the substitution of the amino acid residue at position 4 in said motif (4) with an alanine (A) and the substitution of the amino acid residue at position 9 in said motif (6) with a histidine (H), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a cysteine (C) and the substitution of the amino acid residue at position 5 in said motif (4) with a leucine (L), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a lysine (K) and the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of leucine (L) and tryptophan (W), or
• the substitution of the amino acid residues at positions 4 and 5 in said motif (4) respectively with a leucine (L), or
• the substitution of the amino acid residues at positions 4 and 5 in said motif (4) respectively with a methionine (M), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a proline (P) and the substitution of the amino acid residue at position 9 in said motif (6) with a cysteine (C), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a threonine (T) and the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of histidine (H) and lysine (K), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a valine (V) and the substitution of the amino acid residue at position 5 in said motif (4) with any amino acid selected from the group consisting of leucine (L) and methionine (M), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a valine (V) and the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of histidine (H), lysine (K), arginine (R), and valine (V), or
• the substitution of the amino acid residue at position 8 in said motif (6) with a histidine (H) and the substitution of the amino acid residue at position 9 in said motif (6) with a serine (S), or
• the substitution of the amino acid residue at position 8 in said motif (6) with a proline (P) and the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of cysteine (C), isoleucine (I) and leucine (L), or
• the substitution of the amino acid residue at position 8 in said motif (6) with a threonine (T) and the substitution of the amino acid residue at position 9 in said motif (6) with a histidine (H);
• B) with the acceptor of formula (IIa):
• wherein Y is selected from —O— and —S— and R is selected from the group consisting of:
• C1-C6 alkyl, C1-C6 alkenyl, aryl, allyl, —CO-alkyl (C1-C6), —CO-alkenyl (C1-C6), —CO-aryl,
• R′ designates a group selected from:
• acetyl, trichloroacetyl, trifluoroacetyl,
• and
• with a donor of formula (IIIa):
• wherein R1 represents a group selected from:

A mutant of a wild type glycoside hydrolase, wherein said wild type glycoside hydrolase has 450 to 850 amino acids and comprises eleven motifs defined by the following consensus motifs:

• (1) the amino acid sequence LGVNYLHLMPL (SEQ ID NO:
• 1), which is located in the β-strand 2 of said wild type glycoside hydrolase;
• (2) the amino acid sequence DGGYAV (SEQ ID NO:
• 2), which is located in the loop 2 of the (β/α)8-barrel of said wild type glycoside hydrolase;
• (3) the amino acid sequence DFVFNH (SEQ ID NO:
• 3) which is located in the β-strand 3 of said wild type glycoside hydrolase;
• (4) the amino acid sequence LREIFPDTAPGNF (SEQ ID NO:
• 4), which is located in the domain B of said wild type glycoside hydrolase;
• (5) the amino acid sequence FNSYQWDLN (SEQ ID NO:
• 5), which is located in the C-terminal part of the domain B of said wild type glycoside hydrolase;
• (6) the amino acid sequence ILRLDAVAFLWK (SEQ ID NO:
• 6), which is located in the β-strand 4 of said wild type glycoside hydrolase;
• (7) the amino acid sequence EAIV (SEQ ID NO:
• 7), which is located in the β-strand 5 of said wild type glycoside hydrolase;
• (8) the amino acid sequence YVRCHDDI (SEQ ID NO:
• 8), which is located in the β-strand 7 of said wild type glycoside hydrolase;
• (9) the amino acid sequence RISGTLASLAG (SEQ ID NO:
• 9), which is located in the domain B′ of said wild type glycoside hydrolase;
• (10) the amino acid sequence GIPLIYLGDE (SEQ ID NO:
• 10), which is located in the β-strand 8 of said wild type glycoside hydrolase;
• (11) the amino acid sequence RWVHRP (SEQ ID NO:
• 11), which is located in the loop 8 of the (β/α)8-barrel,
• and the sequence formed by said eleven motifs from said wild type glycoside hydrolase joined end-to-end from motif (1) to motif (11) has at least 65% sequence identity or at least 80% sequence similarity with the amino acid sequence SEQ ID NO:
• 12;
• wherein said mutant has one or two mutation(s) consisting of, when said mutant has only one mutation:
• the substitution of the amino acid residue at position 4 in said motif (4) with any amino acid selected from the group consisting of alanine (A), cysteine (C), glutamic acid (E), glycine (G), histidine (H), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine (Y), or
• the substitution of the amino acid residue at position 5 in said motif (4) with any amino acid selected from the group consisting of leucine (L), methionine (M) and valine (V), or
• the substitution of the amino acid residue at position 8 in said motif (6) with any amino acid selected from the group consisting of glutamic acid (E), phenylalanine (F), glycine (G), lysine (K), leucine (L), methionine (M), proline (P), glutamine (Q), arginine (R) and valine (V), or
• the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V) and tryptophan (W), or
• the substitution of the amino acid residue at position 4 in said motif (7) with any amino acid selected from the group consisting of alanine (A), cysteine (C), aspartic acid (D), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), asparagine (N), serine (S), threonine (T) and tyrosine (Y), or
• the substitution of the amino acid residue at position 7 in said motif (8) with a valine (V), or
• the substitution of the amino acid residue at position 1 in said motif 9 with any amino acid selected from the group consisting of alanine (A), cysteine (C), phenylalanine (F), glycine (G), lysine (K), asparagine (N), glutamine (Q), serine (S), threonine (T) and tryptophan (W), or
• when said mutant has two mutations:
• the substitution of the amino acid residue at position 4 in said motif (4) with an alanine (A) and the substitution of the amino acid residue at position 9 in said motif (6) with a histidine (H), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a cysteine (C) and the substitution of the amino acid residue at position 5 in said motif (4) with a leucine (L), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a lysine (K) and the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of leucine (L) and tryptophan (W), or
• the substitution of the amino acid residues at positions 4 and 5 in said motif (4) respectively with a leucine (L), or
• the substitution of the amino acid residues at positions 4 and 5 in said motif (4) respectively with a methionine (M), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a proline (P) and the substitution of the amino acid residue at position 9 in said motif (6) with a cysteine (C), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a threonine (T) and the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of histidine (H) and lysine (K), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a valine (V) and the substitution of the amino acid residue at position 5 in said motif (4) with any amino acid selected from the group consisting of leucine (L) and methionine (M), or
• the substitution of the amino acid residue at position 4 in said motif (4) with a valine (V) and the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of histidine (H), lysine (K), arginine (R), and valine (V), or
• the substitution of the amino acid residue at position 8 in said motif (6) with a histidine (H) and the substitution of the amino acid residue at position 9 in said motif (6) with a serine (S), or
• the substitution of the amino acid residue at position 8 in said motif (6) with a proline (P) and the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of cysteine (C), isoleucine (I) and leucine (L), or
• the substitution of the amino acid residue at position 8 in said motif (6) with a threonine (T) and the substitution of the amino acid residue at position 9 in said motif (6) with a histidine (H).

A method for determining whether a wild type protein is a wild type glycoside hydrolase, said method comprising the steps of:

• a) determining the amino acid sequence of said protein,
• b) identifying in the amino acid sequence of said protein, preferably from the N- to C-terminus, eleven motifs defined by the following consensus motifs:
• (SEQ ID NO:
• 1)
• (1)
• the amino acid sequence LGVNYLHLMPL;
• (SEQ ID NO:
• 2)
• (2)
• the amino acid sequence DGGYAV;
• (SEQ ID NO:
• 3)
• (3)
• the amino acid sequence DFVFNH;
• (SEQ ID NO:
• 4)
• (4)
• the amino acid sequence LREIFPDTAPGNF;
• (SEQ ID NO:
• 5)
• (5)
• the amino acid sequence FNSYQWDLN;
• (SEQ ID NO:
• 6)
• (6)
• the amino acid sequence ILRLDAVAFLWK;
• (SEQ ID NO:
• 7)
• (7)
• the amino acid sequence EAIV;
• (SEQ ID NO:
• 8)
• (8)
• the amino acid sequence YVRCHDDI;
• (SEQ ID NO:
• 9)
• (9)
• the amino acid sequence RISGTLASLAG
• (SEQ ID NO:
• 10)
• (10)
• the amino acid sequence GIPLIYLGDE;
• (SEQ ID NO:
• 11)
• (11)
• the amino acid sequence RWVHRP;
• determining the sequence identity percent or sequence similarity percent between the sequence formed by said eleven motifs joined end-to-end from motif (1) to motif (11) with the amino acid sequence SEQ ID NO:
• 12, and if the sequence identity percent is at least 65%, or if the sequence similarity percent is at least 80%, then the wild type protein is a wild type glycoside hydrolase.

A molecule selected from the group consisting of:

• Allyl α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-α-D-glucopyranoside (XX2),
• Allyl α-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-α-D-glucopyranoside (XX3),
• Allyl 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-α-D-glucopyranoside (XX4),
• Allyl α-D-glucopyranosyl-(1→4)-2-amino-2-deoxy-α-D-glucopyranoside (XX5),
• Allyl α-D-glucopyranosyl-(1→4)-2-amino-2-N,3-O-carbonyl-2-deoxy-α-D-glucopyranoside (XX6),
• Allyl 2,4,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-6-O-benzyl-2-benzylamino-2-N,3-O-carbonyl-2-deoxy-α-D-glucopyranoside (XX7),
• Allyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1-*4)-6-O-benzyl-2-benzylamino-2-N,3-O-carbonyl-2-deoxy-α-D-glucopyranoside (XX8),
• Allyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-6-O-benzyl-2-amino-2-N,3-O-carbonyl-2-deoxy-α-D-glucopyranoside (XX8A),
• Allyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-6-O-benzyl-2-benzylamino-2-deoxy-α-D-glucopyranoside (XX9),
• Allyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-6-O-benzyl-2-benzylacetamido-2-deoxy-α-D-glucopyranoside (XX10),
• Allyl 2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-α-L-rhamnopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-6-O-benzyl-2-benzylacetamido-2-deoxy-α-D-glucopyranoside (XX11),
• Allyl 2-O-acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→3)[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-6-O-benzyl-2-benzylacetamido-2-deoxy-α-D-glucopyranoside (XX12),
• Allyl α-D-glucopyranosyl-(1→4)-2-deoxy-2-trichloroacetamido-α-D-glucopyranoside (XX13),
• Allyl 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-3,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-α-D-glucopyranoside (XX14),
• 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-3,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-α-D-glucopyranose (XX15),
• 2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-3,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-α-D-glucopyranosyl trichloroacetimidate (XX16),
• Allyl 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-3,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-α-D-glucopyranosyl-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside (XX17),
• Allyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-α-D-glucopyranoside (XX24), and
• Allyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-2-acetamido-6-O-benzyl-2-deoxy-α-D-glucopyranoside (XX25).