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

Inactive Publication Date: 2016-05-19
INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about a polypeptide that can create connections between glucosyl units in a specific way. This polypeptide has a preference for creating those connections in a certain position on an acceptor molecule. The invention also includes a method for using a specific polynucleotide to produce this polypeptide. The technical effect of this invention is the ability to create specific connections between glucosyl units, which can be useful in various applications such as drug discovery and materials science.

Problems solved by technology

Furthermore, the synthesis of polysaccharides connected with controlled rates of glucosyl units linked in alpha-1,3 has never been described, and no such connected product existed hitherto on the market.

Method used

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

Examples

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Effect test

example 1

Screening of New Enzymes in L. citreum NRRL B-742

[0137]After sequencing of the genome of the strain L. citreum NRRL B-742, a gene proved particularly original. Indeed the corresponding putative protein was found to have a sequence having a maximum of only 54% identity with the putative glycoside hydrolase Leuconostoc fallax KCTC 3537 whose sequence is available in the database, and referenced by the NCBI under number ZP_08312597. Now, any other protein sequence with significant identity could be identified.

[0138]This gene encodes a putative transglucosylase of 1888 amino acids, having the characteristic catalytic triad DED and the 4 conserved regions usually described in transglucosylases of family 70. The schematic representation of the protein is shown in FIG. 1 (based on the alignment of protein sequences with GTF180) with 5 domains: i) domain V (403-446 and 1356 to 1800), ii) domain IV (446-586 and 1284-1356), iii) domain A (catalytic) (636-899, 1052-1191 and 1231-1270), iv) dom...

example 2

Production of a New Enzyme in E. coli

[0141]The gene encoding this enzyme has been cloned into several vectors (pET 53, 55, 49 and 60) commercially available from NOVAGEN or INVITROGEN, and expressed in different various of E. coli (TOP10, BL21AI, BL21 DE3 Star, Arctic Express DE3).

[0142]This cloning resulted in a consistent production of the protein. This production has helped initiate the experiments of biochemical characterisation to clarify the catalytic properties of the identified enzyme.

[0143]Simultaneously, a truncated form of the signal peptide and C-terminal, APS AC-1313 SEQ ID no 12 and SEQ ID no 13 for the nucleic and protein sequences, respectively) of the protein has been cloned and expressed in the strain of E. coli BL21 DE3 star, allowing again a significant expression of the protein; which expression has proved almost twice higher than that of the wild-type protein.

example 3

Reaction of a Polypeptide According to the Invention with Sucrose, a Dextran-Type Acceptor and the Enzyme Object of the Invention and Analysis of the Products of this Reaction

[0144]To characterise the functional properties of this putative transglucosylase, the enzyme was first implemented on sucrose alone, a natural substrate of enzymes of the GH70 family.

[0145]Unexpectedly, the chromatographic analyses (HPAEC-PAD, HPSEC) showed that the enzyme alone is only capable of hydrolysing the substrate in equimolar amounts of glucose and fructose. Now and from sucrose alone, this enzyme showed no ability to produce polymers of glucosyl units, as well as its truncated form.

[0146]It is interesting to note that to date, bioinformatic analyses on the primary structure of the GH of the family 70 would not predict this feature (structural determinants governing the ability—or not—of a transglucosylase polymerising are not yet known).

[0147]While nothing presaged that this protein had still an act...

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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.

Description

[0001]The present international application claims priority of application FR 13 / 01402 filed on 17 Jun. 2013, which is incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention relates to an isolated polypeptide having the ability to form connections of glucosyl units in alpha 1,3 on an acceptor, a polynucleotide encoding said polypeptide, its use in a production process of acceptors connected to glucosyl units in alpha 1,3, said acceptors connected to glucosyl units in alpha 1,3 and the use thereof.PRIOR ART[0003]Glucosyltransferases are enzymes capable of catalysing the synthesis of glucose polymers from an inexpensive substrate, such as sucrose, alone or in the presence of an acceptor of glucosyl units comprising at least one hydroxyl moiety. Within these acceptor molecules, the glucosyl units are coupled by glycosidic linkages of variable nature (α-1,6, α-1,4, α-1,2 or α-1,3).[0004]The transglucosylases (or glucan saccharases) belonging to the family 70 ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/715C12P19/04C12P19/18C12N9/10
CPCA61K31/715C12N9/1051C12Y204/01C12P19/18C12P19/04C08B37/0021C12P19/08C12Y204/01005C08L5/02C12P19/10
Inventor REMAUD-SIMEON, MAGALIVUILLEMIN, MARLENEMOULIS, CLAIREMONSAN, PIERREMOREL, SANDRINE
Owner INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE
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