Glucan branching enzymes and their methods of use

Patent metadata

Field	Value
Publication number	US-2016265013-A1
Application number	US-201414780106-A
Country	US
Kind code	A1
Filing date	Mar 26, 2014
Priority date	Mar 26, 2013
Publication date	Sep 15, 2016
Grant date	—

Abstract

The present invention relates to the use of β-glucan branching enzymes in transglycosylation reactions.

First claim

1 . A method for transglycosylation of β-glucan oligo- and/or polysaccharides for making branched oligo- and/or polysaccharides, comprising
providing an isolated enzyme of bacterial origin having glycosyltransferase activity on β-glucan oligo- and/or polysaccharides, such that it cleaves a donor β-glucan oligo- or polysaccharide and transfers a part of said β-glucan oligo- or polysaccharide to internal glucose unit of an additional β-glucan oligo- or polysaccharide acting as acceptor,
bringing the enzyme in contact with a substrate comprising beta-glucan oligo- or -polysaccharides with chains comprising five or more glucose units,
forming a covalently linked branch comprising three or more glucose units from a donor β-glucan oligo- or polysaccharide, on an acceptor oligo- or polysaccharide.

2 . The method of claim 1 wherein said isolated enzyme of bacterial origin catalyses a transglycosylation reaction such that it releases two glucose units from the reducing end of said donor β-glucan oligo- or polysaccharide and transfers the remaining non-reducing end to an internal glucose unit of said additional β-glucan oligo- or polysaccharide thus forming a covalently linked branch comprising three or more glucose units on said acceptor polysaccharide.

3 . The method of claim 1 wherein said isolated enzyme transfers said part of said donor β-glucan oligo- or polysaccharide to the third glucose residue from the reducing end of said acceptor β-glucan oligo- or polysaccharide.

4 . The method of claim 1 wherein said isolated enzyme of bacterial origin has preferential transferase activity over hydrolase activity in the presence of reactive acceptor β-glucan oligo- or polysaccharide.

5 . The method of claim 1 wherein a covalently linked branch is formed on another covalently linked branch in an acceptor β-glucan oligo- or polysaccharide thus forming a product oligo- or polysaccharide with branch on branch.

6 . The method of claim 1 wherein said branch is covalently linked through a (β1→6) linkage between two glucose units.

7 . The method of claim 6 wherein the donor and/or the acceptor oligo- or polysaccharide β-glucan comprises any oligo- or polysaccharide from the group consisting of yeast β-glucan, laminarin, lichenan, curdlan and barley β-glucan.

8 . The method of claim 1 wherein the sequence of said isolated enzyme is from an isolated enzyme of bacterial origin and belonging to glycosyl hydrolase family GH17.

9 . The method of claim 8 wherein the sequence of said isolated enzyme is from a bacterial strain from the taxonomic family of Bradyrhizobiaceae.

10 . The method of claim 8 wherein the sequence of said isolated enzyme is from a bacterial strain from the taxonomic genus of Bradyrhizobium.

11 . The method of claim 8 wherein the sequence of said isolated enzyme is from a bacterial origin comprises bacterial strains belonging to the species Bradyrhizobium japonicum or Bradyrhizobium diazoefficiens.

12 . The method of claim 8 wherein said isolated enzyme comprises an amino acid sequence with more than 75% sequence identity to SEQ ID NO: 1, identified as sequence no. 1 in FIG. 1 .

13 . A β-glucan polysaccharide product produced by the method of claim 1 .

14 . The β-glucan polysaccharide of claim 13 having a branched structure, composed predominantly of (β1→3) linkages with (β1→6)-side chain branches.

15 . A β-glucan polysaccharide of claim 13 composed predominantly of (β1→3) linkages with multiple (β1→6)-side chain branches comprising at least one branch covalently linked to another branch.

16 . The method of claim 1 wherein the produced branched oligo- and/or polysaccharides have biological activity.

17 . The method of claim 16 where the biological activity comprises immune activity such as anti-tumoral, anti-bacterial, anti-viral or anti-fungal activity.

18 . The method of claim 16 for the production of bio-active β-glucan molecules for cosmetic purposes.

19 . The method of claim 17 for the production of bio-active β-glucan molecules for medical/pharmaceutical use.

20 . The method of 19 for the production of bio-active β-glucan molecules as constituents of drugs.

Assignees

• Matis Ohf
• Univ Groningen

Inventors

• Jonsson Wheat Jon Oskar
• Hreggvidsson Gudmundur Oli
• Fridjonsson Olafur Hedinn
• Dobruchowska Justyna M
• Kamerling Johannis P

Classifications

• C08B37/0024

  beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof · CPC title

• C12P19/04

  Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds · CPC title

• C12P19/18 Primary

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title