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
View PDF0 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0071]The advantage of the invention lies in that the polypeptide as described above is responsible for the formation of connecting glucosyl units in the alpha-1,3 position of an acceptor.
[0072]Preferably, the polypeptide according to the invention has the ability to form connections of glucosyl units in alpha 1,3 on an acceptor at a rate between 1 and 50%, preferably between 5% and 40%, and more preferably still between 10 and 40%.
[0073]Even more preferably, the polypeptide according to the invention has the ability to form connections of glucosyl units in alpha 1,3 on an acceptor at a maximum rate of 50%.
[0074]Another object of the invention concerns an isolated polynucleotide encoding a polypeptide as defined above, a fragment or a derivative thereof.
[0075]According to the invention, said polynucleotide is a DNA or RNA molecule.
[0076]By “polynucleotide” is meant broadly a DNA molecule such as for instance a cDNA (complementary DNA) or genomic or synthetic DNA, or an RNA molecule, such as a messenger RNA or synthetic RNA, as well as analogues of DNA or RNA containing non-natural nucleotide analogues, non-natural internucleotide linkages, or both. Preferably, said polynucleotide is a DNA molecule. The polynucleotides may have any topological conformation, such as linear or circular.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Experimental program
Comparison scheme
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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
pHaaaaaaaaaa
concentrationaaaaaaaaaa
Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap