Polypeptide having the ability to form connections of glucosyl units in alpha-1,3 on an acceptor

Patent metadata

Field	Value
Publication number	US-2020323899-A1
Application number	US-202016915559-A
Country	US
Kind code	A1
Filing date	Jun 29, 2020
Priority date	Jun 17, 2013
Publication date	Oct 15, 2020
Grant date	—

Abstract

Polypeptides having the ability to specifically form connections of glucosyl units in alpha 1,3 on an acceptor having at least one hydroxyl moiety are presented. The polypeptides include i) the pattern I of sequence SEQ ID NO: 1, ii) the pattern II of sequence SEQ ID NO: 2, iii) the pattern III of sequence SEQ ID NO: 3, and iv) the pattern IV of sequence SEQ ID NO: 4, or derivates from one or several of said patterns, wherein the polypeptide furthermore has an aspartic residue (D) at position 5 of the pattern II (SEQ ID NO: 2), a glutamic acid residue (E) at position 6 of the pattern III (SEQ ID NO: 3) an an aspartic acid residue (D) at position 6 of the pattern IV (SEQ ID NO: 4). Methods for producing acceptors connected to glucosyl units in alpha 1,3 using the polypeptides are also provided.

First claim

1 . A recombinant polypeptide comprising an amino acid sequence that is at least 99 percent identical to SEQ ID NO: 12, and wherein said polypeptide maintains an aspartic acid residue (D) at position 5 of the pattern II (SEQ ID NO: 2), a glutamic acid residue (E) at position 6 of the pattern III (SEQ ID NO: 3), and an aspartic acid residue (D) at position 6 of the pattern IV (SEQ ID NO: 4).

2 . An expression vector comprising a nucleic acid encoding the recombinant polypeptide of claim 1 .

3 . A host cell comprising the vector of claim 2 .

4 . A composition comprising the recombinant polypeptide of claim 1 .

5 . A composition comprising the expression vector of claim 2 .

6 . A composition comprising the host cell of claim 3 .

7 . A method of producing a recombinant polypeptide from a cell, said method comprising culturing the host cell of claim 3 so that the polynucleotide encoding the recombinant polypeptide is expressed and the recombinant polypeptide is secreted.

8 . A method for producing acceptors connected to glucosyl units in alpha 1,3 comprising a rate of connections of such glucosyl units in alpha 1,3 between 1 and 50%, said method comprising the steps of:
(i) mixing in a reaction medium: an isolated polypeptide having the ability to specifically form connections of glucosyl units in alpha-1,3 on an acceptor having at least one hydroxyl moiety, said polypeptide having a sequence identity of at least 99% with SEQ ID NO: 12, and wherein said polypeptide maintains an aspartic acid residue (D) at position 5 of the pattern II (SEQ ID NO: 2), a glutamic acid residue (E) at position 6 of the pattern III (SEQ ID NO: 3), and an aspartic acid residue (D) at position 6 of the pattern IV (SEQ ID NO: 4); a substrate of said polypeptide and; an acceptor comprising at least one hydroxyl moiety; and (ii) incubating said mixture obtained in step (i) so as to obtain connection of glucosyl units in alpha-1,3 on said acceptor,
wherein the rate of connections of such glucosyl units in alpha 1,3 is controlled by varying the ratio between the substrate concentration and the acceptor concentration.

9 . The method of claim 6 , wherein the substrate is selected from the group consisting of α-D-glucopyranosyl fluoride, p-nitrophenyl α-D-glucopyranoside, α-D-glucopyranosyl, α-L-sorofuranoside, lactulosucrose, and sucrose.

10 . The method of claim 6 , wherein the acceptor comprising at least one hydroxyl moiety is selected from the group of polysaccharides comprising glucans, dextrans branched in α-1,2, alternans, mutans, reuterans, starch, amylopectin, amylose, glycogen, and pullulan.

11 . The method of claim 6 , wherein the acceptor connected to glucosyl units in alpha-1,3 is a bulking agent, a thickener, an emulsifier, a texturing agent and/or a stabilizer.

Assignees

• Institut National De Rech Pour Lagriculture Lalimentation Et Lenvironnement
• Institut Nat Des Sciences Appliquees De Toulouse
• Centre Nat Rech Scient

Inventors

• Remaud-Simeon Magali
• Vuillemin Marlene
• Moulis Claire
• Monsan Pierre
• Morel Sandrine

Classifications

• C12P19/18

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

• C08L5/02

  Dextran; Derivatives thereof · CPC title

• A61K31/715 Primary

  Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters · CPC title

• C12P19/10

  Pullulan · CPC title

• C12Y204/01

  Hexosyltransferases (2.4.1) · CPC title