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

Patent metadata

Field	Value
Publication number	US-2016136199-A1
Application number	US-201414898991-A
Country	US
Kind code	A1
Filing date	Jun 17, 2014
Priority date	Jun 17, 2013
Publication date	May 19, 2016
Grant date	—

Abstract

An isolated polypeptide having the ability to specifically form connections of glucosyl units in alpha 1,3 on an acceptor including at least one hydroxyl moiety. The polypeptide includes i) the pattern I of sequence SEQ ID NO: 1, ii) the pattern II of sequence SEQ ID NO: 2, iii) the pattern II of sequence SEQ ID NO: 3, iv) the pattern IV of sequence SEQ ID NO: 4 or derivates from one or several of said patterns. The polypeptide furthermore has the aspartic residue (D) in position 5 of the pattern II (SEQ ID NO: 2), the glutamic acid residue (E) at position 6 of the pattern III (SEQ ID NO: 3) and the aspartic acid residue (D) in position 6 of the pattern IV (SEQ ID NO: 4); and its uses.

First claim

1 . An isolated polypeptide having the ability to form specifically connections of glucosyl units in alpha-1,3 on an acceptor comprising at least one hydroxyl moiety and characterised in that said polypeptide comprises:
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 iv) the pattern IV of sequence SEQ ID no 4 or derivatives of one or more of said domains having at least 80% identity with them; wherein said polypeptide furthermore has the aspartic residue (D) in position 5 of the pattern II (SEQ ID no 2), the glutamic acid residue (E) at position 6 of the pattern III (SEQ ID no 3) and the aspartic acid residue (D) in position 6 of the pattern IV (SEQ ID no 4).

2 . The isolated polypeptide according to claim 1 , characterised in that said polypeptide comprises the sequence SEQ ID no 6, an orthologue, a derivative, or a fragment thereof.

3 . The polypeptide according to claim 1 , characterised in that said polypeptide comprises or consists of the sequence SEQ ID no 9, an orthologue, a derivative, or a fragment thereof.

4 . The polypeptide according to claim 1 , characterised in that said orthologue is selected from the group comprising the sequences SEQ ID no 15 and SEQ ID no 17.

5 . An isolated polynucleotide encoding a polypeptide according to claim 1 , preferably said polynucleotide is defined by the sequence SEQ ID no 10.

6 . An expression vector comprising a polynucleotide as defined in claim 5 .

7 . A transformed host cell including an expression vector as defined in claim 6 .

8 . A composition comprising at least one polypeptide as defined in claim 1 , a polynucleotide encoding said polypeptide, a vector comprising said polynucleotide or a transformed host cell including said vector.

9 . A method of producing a polypeptide as defined in any one of claim 1 , said method including the steps of:
a) inserting a polynucleotide encoding said polypeptide or a vector comprising said polynucleotide into a host cell; b) culturing said cell obtained in step a); and c) extracting the polypeptide from the culture obtained in step b).

10 . A process 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 a polypeptide as defined in claim 1 , 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 the connection of glucosyl units in alpha-1,3 on said acceptor.

11 . The method according to claim 10 , characterised in that the concentrations of substrate and of acceptor are adjusted so to get a connection rate between 35 and 50%.

12 . The method according to claim 10 , characterised in that the concentrations of substrate and of acceptor are adjusted so to get a connection rate of 20 to 35%.

13 . The method according to claim 10 , characterised in that the concentrations of substrate and of acceptor are adjusted so to get a connection rate lower than 20%.

14 . The method according to claim 1 , characterised in that said acceptor connected to glucosyl units in alpha-1,3 is selected from the group of polysaccharides, preferably glucans, including α-glucans such as dextran, dextrans branched in α-1,2, alternans, mutans, reuterans, starch, amylopectin, amylose, glycogen and pullulan.

15 . The method according to claim 10 , characterised in that the substrate is selected from the group comprising α-D-glucopyranosyl fluoride, p-nitrophenyl α-D-glucopyranoside, α-D-glucopyranosyl, α-L-sorofuranoside, lactulosucrose and sucrose.

16 . An acceptor connected to glucosyl units in alpha-1,3 likely to be obtained by the method according to claim 11 .

17 . A method for the production of acceptors connected to glucosyl units in alpha-1,3, comprising:
i) mixing in a reaction medium: a polypeptide as defined in claim 1 , of a polynucleotide encoding said polypeptide, of a vector comprising said polynucleotide, of a host cell including said vector and/or a composition comprising said polypeptide, said polynucleotide, said vector, or said host cell, 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 the connection of glucosyl units in alpha-1,3 on said acceptor.

18 . A composition comprising an acceptor connected to glucosyl units in alpha-1,3 as defined in claim 16 as a thickener, emulsifier and/or stabiliser, wherein said composition is a formulation selected from the group consisting of food industrial, cosmetic, agrochemical, petrochemical and pharmaceutical formulations.

19 . A method of providing a prebiotic effect comprising administering to a subject in need there of an effective amount of an acceptor connected to glucosyl units in alpha-1,3 as defined in claim 16 as an agent with prebiotic effect.

Assignees

• Agronomique Inst Nat Rech
• Inst Nat Sciences Appliq
• 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

• C12Y204/01

  Hexosyltransferases (2.4.1) · CPC title

• C12P19/04

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

• C08B37/0021

  Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran · CPC title