Polypeptide having the capacity to form alpha-1,3 glucosyl unit branchings on an acceptor

Patent metadata

Field	Value
Publication number	US-2018104274-A1
Application number	US-201715712173-A
Country	US
Kind code	A1
Filing date	Sep 22, 2017
Priority date	Jun 17, 2013
Publication date	Apr 19, 2018
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) and 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 method for producing acceptors connected to glucosyl units in alpha 1,3 comprising a rate of connections of said glucosyl units in alpha 1,3 between 1% and 50%, the 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 comprising:
(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 a derivative of one or more of said domains having at least 80% identity with them, wherein said derivative 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.

2 . The method according to claim 1 , wherein the ratio between the substrate concentration and the acceptor concentration is greater than or equal to 1 so as to obtain a connection rate of between 35% to 50%.

3 . The method according to claim 1 , wherein the ratio between the substrate concentration and the acceptor concentration varies between 0.5 and 1 so as to obtain a connection rate of 20% to 35%.

4 . The method according to claim 1 , wherein the ratio between the substrate concentration and the acceptor concentration is lees than 0.5 so as to obtain a connection rate of lower than 20%.

5 . The method according to claim 1 , wherein said acceptor connected to glucosyl units in alpha-1,3 is selected from the group consisting of polysaccharides, glucans, α-glucans, s dextran, dextrans branched in α-1,2, alternans, mutans, reuterans, starch, amylopectin, amylose, glycogen and pullulan.

6 . The method according to claim 1 , 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.

7 . An acceptor connected to glucosyl units in alpha-1,3 obtained by the method according to claim 1 .

8 . An acceptor connected to glucosyl units in alpha-1,3 obtained by the method according to claim 2 .

Assignees

• Inst Nat De La Rech Agronomique Inra
• Institut Nat Des Sciences Appliquees De Toulouse
• Centre Nat Rech Scient

Inventors

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

Classifications

• C12P19/04

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

• C12Y204/01005

  Dextransucrase (2.4.1.5) · CPC title

• C12P19/10

  Pullulan · CPC title

• 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