Acholetin biopolymers and methods for enzymatic synthesis
AssigneeThe University of British Columbia
InventorsStephen G. Withers, Spencer S.S. MacDonald
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Abstract
Provided herein are β-1,3-linked biopolymers (acholetin polysaccharides). Furthermore, provided herein are enzymatic methods and systems for producing β-1,3-linked oligosaccharides and polysaccharides using β-1,3-N-acetylglucosaminide phosphorylase (Acholetin phosphorylase (AchP)). The AchP was sourced from the genome of the cell wall-less Mollicute bacterium, Acholeplasma laidlawii and was found to synthesize β-1,3-linked N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) oligomers using the donor. α-N-acetylglucosamine 1-phosphate (GlcNAc1-P) or N-acetylgalactosamine 1-phosphate (GalNAc1-P).
Claims54
A polysaccharide, the polysaccharide comprising repeated monomers of N-acetyl-glucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) linked by glycosidic bonds having a β-configuration between the C1 position of the first GlcNAc or GalNAc ring and the C3 position of the adjacent GlcNAc or GalNAc ring, having the structure of Formula I
- wherein,
- n is an integer of 10 or greater;
- and
- X is selected from —OH;
- and
A method of making an oligosaccharide or a polysaccharide, the method comprising enzymatic synthesis with a glycoside phosphorylase (GP) that links monomeric GlcNAc or GalNAc via a β-1,3-glycosidic linkage, wherein the oligosaccharide or the polysaccharide comprises repeated monomers of GlcNAc or GalNAc linked by glycosidic bonds having a β-configuration between the C1 position of the first GlcNAc or GalNAc ring and the C3 position of the adjacent GlcNAc or GalNAc ring, having the structure of Formula I
- wherein,
- n is an integer between 2 and 9, to form the oligosaccharide;
- n is an integer of 10 or greater, to form the polysaccharide;
- and
- X is selected from —OH;
A method of making an oligosaccharide or a polysaccharide, the method comprising:
- (a) generating a GlcNAc-1-P or GalNAc-1-P, as a glycosyl donor, by reacting an N-acetylhexosamine-1-kinase (NahK) with GlcNAc or GalNAc and ATP;
- (b) initiating a reverse phosphorolysis reaction by mixing the GlcNAc-1-P or GalNAc-1-P precipitate from step (a) with a glycosyl acceptor with a glycoside phosphorylase;
- wherein the oligosaccharide or the polysaccharide comprises repeated monomers of GlcNAc or GalNAc linked by glycosidic bonds having a β-configuration between the C1 position of the first GlcNAc or GalNAc ring and the C3 position of the adjacent GlcNAc or GalNAc ring, having the structure of Formula II
- wherein,
- n is an integer between 2 and 9, to form the oligosaccharide;
- and
- n is an integer of 10 or greater, to form the polysaccharide.
A method of adding GlcNAc or GalNAc via a β-1,3 linkage to a GlcNAc or GalNAc at the non-reducing end of an oligosaccharide, a polysaccharide, a chitin or a chito-oligosaccharide, the method comprising reverse phosphorolysis with a glycoside phosphorylase.
A reaction composition, the reaction composition comprising at least:
- (a) a GlcNAc-1-P or GalNAc-1-P as a glycosyl donor;
- (b) GlcNAc or GalNAc as a glycosyl acceptor;
- and
- (c) a β-1,3-GlcNAc phosphorylase enzyme comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:
- 2, wherein the amino acid sequence has β-1,3-GlcNAc phosphorylase enzyme activity, and wherein the β-1,3-GlcNAc phosphorylase enzyme synthesizes a β-1,3-glycosidic linkages between the donor and acceptor.
Dependencies are inferred from the claim text.