EP3271469A1 - Production de synthons glycosyles par voie enzymatique - Google Patents

Production de synthons glycosyles par voie enzymatique

Info

Publication number
EP3271469A1
EP3271469A1 EP16711230.9A EP16711230A EP3271469A1 EP 3271469 A1 EP3271469 A1 EP 3271469A1 EP 16711230 A EP16711230 A EP 16711230A EP 3271469 A1 EP3271469 A1 EP 3271469A1
Authority
EP
European Patent Office
Prior art keywords
sequence
amino acid
seq
identity
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP16711230.9A
Other languages
German (de)
English (en)
French (fr)
Inventor
Isabelle Andre
Stéphane GRELIER
David GUIEYSSE
Alvaro LAFRAYA
Pierre Monsan
Claire Moulis
Frédéric PERUCH
Magali Remaud-Simeon
Marlène VUILLEMIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Institut National des Sciences Appliquees de Toulouse
Universite de Bordeaux
Institut Polytechnique de Bordeaux
Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut National des Sciences Appliquees de Toulouse
Institut National de la Recherche Agronomique INRA
Universite de Bordeaux
Institut Polytechnique de Bordeaux
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Institut National des Sciences Appliquees de Toulouse, Institut National de la Recherche Agronomique INRA, Universite de Bordeaux, Institut Polytechnique de Bordeaux filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP3271469A1 publication Critical patent/EP3271469A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
    • C08F20/10—Esters
    • C08F20/26—Esters containing oxygen in addition to the carboxy oxygen
    • C08F20/28—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
    • C08F20/52—Amides or imides
    • C08F20/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F20/58—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-acryloylmorpholine
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/58—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound through only acyclic carbon atoms to a non-saccharide heterocyclic ring, e.g. bleomycin, phleomycin

Definitions

  • the present invention relates to the field of enzymatic glycosylation of hydroxylated synthons to obtain glycosylated monomers.
  • the present invention also relates to novel chemo-enzymatic glyco (co) polymer synthesis pathways based on chemical polymerization or on a chemical coupling reaction of glycosylated monomers obtained enzymatically.
  • Glyco (co) polymers have attracted increasing interest in recent years because of their wide potential in many biotechnology and industrial sectors.
  • carbohydrate units in synthetic macromolecules can give polymers new physicochemical properties, for example to increase their solubilities, modify their hydrophobic characteristics, and thus open the way to new industrial applications.
  • these glyco (co) polymers are of great interest in the manufacture of biomaterials for the repair of lesions, tissue engineering (Cho et al., Biomaterials, 2006 Feb; 27 (4): 576- 85. Epub 2005 Aug 8), the vectorization of active principles (Ahmes and Narain, Biomaterials 201 1 Aug, 32 (22): 5279-90), or to modify and make a hydrophobic surface biocompatible.
  • glyco (co) polymers are of particular interest in the field of biological diagnosis (Abraham et al., Biomaterials, 2005 Aug; 26 (23): 4767-78), as a support for covalent coupling of biomolecules, or as multivalent architectures favoring recognition processes with certain proteins (Spain et al., Polym Chem, 2011,2, 60-68, Vasquez-Dorbatt et al., Chembiochem 2012 Nov 26; 13 (17): 2478-87 ).
  • hybrid glycopolymers differing from natural polysaccharides, and formed of a synthetic part, of (meth) acrylate, (meth) acrylamide, styrene, norbornenyl, vinyl acetate, peptide, .. and a carbohydrate moiety.
  • the reactive functions carried by the polymer must be removed from the polymer backbone in order to increase the reactivity while decreasing the steric hindrance generated by the grafting of the pendant saccharide sequences.
  • glycomonomers In general, the synthesis of glycomonomers is often very painful, requiring time-consuming steps of protection and deprotection as well as the use of metal catalysts or toxic solvents. In addition, the diversity of saccharide structures accessible by these routes is still limited. Finally, in some cases, Mixtures of undesirable structures are obtained and make it difficult to control the polymerization reaction and subsequently the structure and properties of the polymers.
  • HEMA glucosylated 2- (hydroxy) ethyl methacrylate
  • MEGlc 2-methacryloyloxyethyl- ⁇ -D-glucopyranoside
  • Kitazawa Korean et al., Chem Lett, 1990, 19, No. 9, 1733-1736
  • glucose donor such as methylglucoside
  • phosphomolybdic acid as a catalyst
  • 2,4-dinitrochlorobenzene as inhibitor
  • Nakaya Nakaya et al., Makromol Chem., Rapid Commun, 1993, 14, 77-83 synthesized another glucopolymer in 1993 by reacting HEMA with 2,3-bromide. 4,6-tetra-O-acetyl- ⁇ -D-glucopyranose in the presence of silver oxide or mercury cyanide, according to the method of Helferich (Helferich and Weis, Chem., Ber., 1956, 89, 314-321) .
  • the enzymatic processes are very favorably positioned because of the specificity and selectivity of the action of the enzymes to circumvent the difficulties of chemical synthesis and to propose an "eco-compatible" alternative, decreasing not only the use harmful products (metal catalysts, organic solvents) but also the costs and production times of the glycosylated monomers.
  • Kobayashi described the use of ⁇ -galactosidase with 4-nitrophenyl N-acetyl- ⁇ -Dglucosaminide and lactose as substrates for p-Nitrophenyl N-acetyl- ⁇ -lactosaminide (Kobayashi et al., J Carbohydr Chem, 1994, 13, 753-766). This enzymatic step is followed by a reduction of the nitro function in amine function to then hook an acrylate function.
  • lipases were used for the esterification of various sugars with (meth) acrylates, but with limited yields and selectivities (Albertin et al, Macromolecules, 2004, 37 (20), pp 7530-7537 Miura et al., Polym Poli Part A-Polym Chem, 2004, 42, 4598-4606, Park et al., J Biomed Mater Res A. 2004 Dec 1; 71 (3): 497-507; Kulshrestha et al, ACS; Symp Ser, 2005, 900, pp. 327-342, Tsukamoto et al., J Chem Technol Biotechnol, 2008, 83, 1486-1492).
  • the present invention provides a method of manufacturing a glycosylated synthon, or monomer, comprising at least one step of bringing at least one glycan-saccharase into contact with at least one hydroxylated synthon and at least one sucrose, wherein :
  • R1 represents a hydrogen atom or a C1-C3 alkyl
  • R 2 represents a C 1 -C 20 alkylene group; or a group (C 2 H 4 O) n , where n is an integer of 1 to 10;
  • Xi represents - (O) -, - (NH) -, - (S) - or - (NR ' 2 (OH)) -, preferably - (O) -, - (NH) -, or - (NR' 2 (OH)) -, with R '2 is an alkylene group Ci-C 2 o; or a group - (C 2 H 4 O) m -, with m being an integer of 1 to 10;
  • R 3 represents a covalent bond; alkylene Ci-C 2 o; or a group (C 2 H 4 O) n , where n is an integer of 1 to 10;
  • R 4 represents a covalent bond; alkylene Ci-C 2 o; or a group (C 2 H 4 O) n , where n is an integer of 1 to 10;
  • R 5 represents a covalent bond; alkylene Ci-C 2 o; or a group (C 2 H 4 O) m , with m being an integer of 1 to 10;
  • n an integer from 1 to 20;
  • X 2 represents - (O) -, - (NH) - or - (S) -;
  • R O represents hydrogen or an alkyl group Ci-C 2 o
  • R 7 represents a C 1 -C 20 alkylene group
  • the hydroxylated synthon is chosen from the group consisting of:
  • R 1 represents a hydrogen atom or a C 1 -C 3 alkyl
  • R 2 represents a C 1 -C 20 alkylene group; or a group (C 2 H 4 O) n , where n is an integer of 1 to 10;
  • Xi represents - (O) -, - (NH) -, - (S) - or - (NR ' 2 (OH)) -, preferably - (O) -, - (NH) -, or - (NR' 2 (OH)) -, with R '2 is an alkylene group Ci-C 2 o; or a group - (C 2 H 4 O) m -, with m being an integer of 1 to 10; (ii) styrenic synthons of formula (II):
  • R 3 represents a covalent bond; a C 1 -C 20 alkylene group; or a group (C 2 H 4 O) N , where n is an integer of 1 to 10;
  • R 4 represents a covalent bond; a C 1 -C 20 alkylene group; or a group (C 2 H 4 O) N , where n is an integer of 1 to 10;
  • R 5 represents a covalent bond; a C 1 -C 20 alkylene group; or a group (C 2 H 4 O) M , with m being an integer between 1 and 10;
  • n an integer from 1 to 20;
  • X 2 represents - (O) -, - (NH) - or - (S) -;
  • R O represents hydrogen or an alkyl group C 1 -C 2 0;
  • the glycan-sucrase is chosen from the group comprising:
  • a sequence having at least 80% identity with SEQ ID NO: 1 mutated once at any of the positions R226, 1228, F229, A289, F290, 1330, V331, D394 and R446 which can be chosen from:
  • R226Xi said sequence having an amino acid X1 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W and Y.
  • a sequence having at least 80% identity with SEQ ID NO: 3 ASNP I228X 2
  • said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, L, M, N, P, Q, R, S, T, V, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 4 (ASNP F229X 3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A, C, D, E, G , H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;
  • sequence having an amino acid X 4 represents an amino acid selected from the group consisting of C, D, E, F , G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;
  • SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W and Y;
  • sequence having an amino acid X 7 represents an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 9 (ASNP D394X "), said sequence having an amino acid Xs representing an amino acid selected from the group consisting of A, C, E, F, G , H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and
  • R.446X 9 said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, Q, S , T, V, W and Y.
  • a sequence having at least 80% identity with SEQ ID NO: 1 mutated once at any of the R226, 1228, F229, A289, F290, 1330, V331, D394 and R446 positions may be chosen from: a sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of C, H, K, M, N, Q, S, T and V;
  • sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of H, L, T, V, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 4 (ASNP F229X3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of C, D, E, G, H, I, K, M, N, P, Q, V, W and Y, in particular of C, D, E, G, I, K, M, N, P, V, W and Y, and more preferably of M and Y;
  • sequence having an amino acid X 4 represents an amino acid selected from the group consisting of C, D, E, F , M, N, P, Q, S, T, V and W and more particularly selected from the group consisting of F, M, N, P, Q, S and T;
  • SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, P, Q, S, T, V and W, more preferably A, C, D, H, I, K, L, M, Q, S, T, V and W, in particular A, C, I, L, V, S, T and W;
  • SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, M, N, Q, S, V and Y, in particular A and C, more preferably A;
  • sequence having an amino acid X 7 represents an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, N, Q, R, S, T, W and Y, more preferably C, D, E, F, G, N, R, S, T, W and Y , in particular E, T and W;
  • sequence having at least 80% identity with SEQ ID NO: 9 (ASNP D394X "), said sequence having an amino acid Xs representing an amino acid selected from the group consisting of A, E, F, G, H I, K and L, especially A and E; a sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R.446X9), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, G, K, L, M, N, and S, especially A, N, and M.
  • the hydroxylated synthon used is more particularly chosen from the group consisting of HEMA, NHAM, HEAA, VP, VBA, HMNCA, AHMCL, MVL, BME. , allyl alcohol and NNHEA.
  • HEMA high-tension polymer
  • NHAM high-tension polymer
  • HEAA high-tension polymer
  • allyl alcohol e.g., ethylene glycol
  • NNHEA e.g., ethylene glycol
  • BME e.g., ethylene glycol
  • a hydroxylated synthon according to the invention implemented in an enzymatic glycosylation process of the invention is glucosylated or fructosylated at the end of this process, preferably glucosylated.
  • the subject of the invention is also a process for the manufacture of a glyco (co) polymer comprising the polymerization of at least 2 monomers obtained, independently, at the end of the enzymatic glycosylation process according to the invention.
  • the method of manufacturing a glyco (co) polymer according to the invention comprises, in order, the following steps:
  • the method of manufacturing a (co) polymer glyco according to this embodiment may furthermore comprise, independently:
  • the subject of the invention is also a process for producing a glyco (co) polymer, preferably in block form, comprising the coupling of at least 2 monomers obtained, independently, at the end of the enzymatic glycosylation process according to US Pat. invention, preferably monomers obtained from the synthons of formula (V) and / or (VI) according to the invention.
  • alkyl a linear or branched saturated hydrocarbon aliphatic group comprising from 1 to 20, especially from 1 to 10, preferably from 1 to 6 carbon atoms;
  • alkylene a linear or branched divalent alkylene group comprising from 1 to
  • glycoside units are known to those skilled in the art.
  • a glycoside unit according to the invention is chosen from one or more glucose (s), one or more fructose (s) or a mixture of glucose. (s) and fructose (s).
  • the inventors have developed an enzymatic method for the glycosylation of hydroxylated synthons using specific glucan-saccharases identified by the applicant, capable of effecting such glycosylation, in particular glucosylation or fructosylation.
  • glycosylated synthons, or monomers are advantageously used in a chemical process for preparing glyco (co) polymers of controlled structures and functionalities.
  • the inventors have developed a method for the chemo-enzymatic synthesis of glyco (co) polymers based on chemical polymerization or a coupling reaction of glycosylated monomers obtained enzymatically.
  • the methods according to the present invention advantageously make it possible to better control the degrees of glycosylation of the synthons and the structures as well as their distribution.
  • these methods advantageously provide access to a greater diversity of macro-molecular architectures for different fields of application, such as biomaterials, implant equipment, tissue engineering, biological diagnosis and principles delivery. assets.
  • the present invention relates in the first place to a process for manufacturing a glycosylated synthon, or monomer, comprising at least one step of placing at least one glycan-saccharase of the invention in contact with at least one hydroxylated synthon according to the invention. invention and at least one sucrose.
  • the enzymes of the invention are advantageously able to glycosylate synthons at their hydroxyl function.
  • the enzymes according to the invention are capable of glucosylating or fructosylating the synthons of the invention.
  • glycansucrase is chosen from the group consisting of glycoside hydrolases belonging to families 13, 68 and 70 of glycoside hydrolases (GH13, GH68 and GH70).
  • glycoside hydrolases belonging to the 13 family are naturally occurring amylosaccharases produced by the bacteria of the Deinococcus, Neisseria or Alteromonas genera.
  • glycoside hydrolases belonging to the 70 family are in turn glucan saccharases naturally produced by lactic acid bacteria of the genera Leuconostoc, Lactobacillus, Streptococcus or Weissela sp.
  • Bacillus subtilis fructosyltransferase is also used in a glycosylation process according to the invention and belongs to the 68 family of glycoside hydrolases.
  • the enzymes of the invention are all capable of transferring glucose or fructose from sucrose to the hydroxylated synthons of the invention.
  • the nucleotide sequence of the wild-type form of the ASNp (Neisseria polysaccharea amylosucrose) (GH13 family) gene is GenBank LA11781.1, while its polypeptide sequence is Uniprot Q9ZEU2 (SEQ ID NO: 1).
  • the nucleotide sequence of the wild form of the DSR-S enzyme (derived from the strain Leuconostoc mesenteroides B-512F) is GenBank reference 109598.
  • the nucleotide sequence of the wild form of the DSR-E enzyme (from Leuconostoc mesenteroides strain NRRL B-1299) is GenBank AJ430204.1 and reference Uniprot Q8G9Q2.
  • the enzyme GBD-CD2 (sequence SEQ ID NO: 13) is a truncated form of the aforementioned DSR-E enzyme as described in Brison et al., J. Biol. Chem., 2012, 287, 7915-24.
  • an enzyme according to the invention can be synthesized by conventional methods of synthetic chemistry, or homogeneous chemical syntheses in solution or in solid phase.
  • those skilled in the art can use the polypeptide synthesis techniques in solution described by HOUBEN WEIL (1974, In the method of Organizational Chemistry, E. Wunsh ed., Volume 15-1 and 15-11, Thieme , Stuttgart.).
  • An enzyme according to the invention may also be chemically synthesized in the liquid or solid phase by successive couplings of the different amino acid residues (from the N-terminal end to the C-terminal end in the liquid phase, or from the C-terminus towards the N-terminus in solid phase).
  • an enzyme according to the invention can be synthesized by genetic recombination, for example according to a production method comprising the following steps:
  • step (b) transfecting a host cell with the recombinant vector obtained in step (a);
  • step b) culturing the host cell transfected in step b) in a suitable culture medium
  • glycan-saccharase used in a process of the invention are chosen from a group comprising:
  • the mutation of a sequence having at least 80% identity with SEQ ID NO: 1 in position R226, 1228, F229, A289, F290, 1330, V331, D394 or R446 is a mutation. by substitution.
  • a sequence having at least 80% identity with SEQ ID NO: 1 when mutated once at position R226, it is chosen from a sequence having at least 80% identity with the SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid X1 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W and Y.
  • X 1 preferably represents an amino acid selected from the group consisting of C, H, K, M, N, Q, S, T and V.
  • a sequence having at least 80% identity with SEQ ID NO: 1 when mutated once at position 1228, it is chosen from a sequence having at least 80% identity with the SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, L, M, N, P , Q, R, S, T, V, W and Y.
  • X 2 preferably represents an amino acid selected from the group consisting of H, L, T, V, W and Y.
  • a sequence having at least 80% identity with SEQ ID NO: 1 when mutated once at position F229, it is chosen from a sequence having at least 80% identity with the SEQ ID NO: 4 (ASNP F229X3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, N, P , Q, R, S, T, V, W and Y.
  • X 3 preferably represents an amino acid selected from the group consisting of C, D, E, G, H, I, K, M, N, P, Q, V, W and Y, in particular of C, D, E, G, I, K, M, N, P, V, W and Y, and more preferably of M and Y.
  • a sequence having at least 80% identity with SEQ ID NO: 1 when mutated once at position A289, it is chosen from a sequence having at least 80% identity with the SEQ ID NO: 5 (ASNP A289X 4), said sequence having an X amino acid 4 representing an amino acid selected from the group consisting of C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.
  • X 4 preferably represents an amino acid selected from the group consisting of C, D, E, F, M, NP, Q, S, T, V and W, and more particularly chosen from the group consisting of of F, M, N, P, Q, S and T.
  • a sequence having at least 80% identity with SEQ ID NO: 1 when mutated once at position F290, it is chosen from a sequence having at least 80% identity with the sequence SEQ ID NO: 6 (ASNP F29OX 5 ), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.
  • X 5 preferably represents an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, P, Q, S, T, V and W, more preferably A, C, D, H, I, K, L, M, Q, S, T, V and W, in particular A, C, I, L, V, S, T and W.
  • a sequence having at least 80% identity with SEQ ID NO: 1 when mutated once at position 1330, it is chosen from a sequence having at least 80% identity with the sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, M, N , P, Q, R, S, T, V, W and Y.
  • X 6 preferably represents an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, M, N, Q, S, V and Y, in particular A and C, more preferably A.
  • a sequence having at least 80% identity with SEQ ID NO: 1 when mutated once at position V331, it is chosen from a sequence having at least 80% identity with the SEQ ID NO: 8 (ASNP V331X 7), said sequence having an amino X 7 acid representing an amino acid selected from the group consisting of a, C, D, E, F, G, H, I, K, L, M , N, P, Q, R, S, T, W and Y.
  • X 7 preferably represents an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, N, Q, R, S, T, W and Y, more preferably C, D, E, F, G, N, R, S, T, W and Y, in particular E, T and W;
  • a sequence having at least 80% identity with SEQ ID NO: 1 when mutated once at position D394, it is chosen from a sequence having at least 80% identity with the SEQ ID NO: 9 (ASNP D394Xs), said sequence having an amino acid Xs representing an amino acid selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, P , Q, R, S, T, V, W and Y.
  • X 8 preferably represents an amino acid selected from the group consisting of A, E, F, G, H, I, K and L, particularly A and E.
  • a sequence having at least 80% identity with SEQ ID NO: 1 when mutated once at position R446, it is chosen from a sequence having at least 80% identity with the SEQ ID NO: 10 (ASNP R446X9), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N , Q, S, T, V, W and Y.
  • X 9 preferably represents an amino acid selected from the group consisting of A, C, G, K, L, M, N, and S, especially A, N, and M.
  • a sequence having at least 80% identity with SEQ ID NO: 1 mutated once at any one of the positions R226, 1228, F229, A289, F290, 1330, V331, D394 and R446 is selected from any one of SEQ ID NO: 2 to 10 defined above.
  • a sequence having at least 80% identity with SEQ ID NO: 1 mutated once at any of the positions R226, 1228, F229, A289, F290, 1330, V331, D394 and R446 is selected from:
  • R226Xi said sequence having an amino acid X1 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W and Y.
  • sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, L, M, N, P, Q, R, S, T, V, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 4 (ASNP F229X3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;
  • sequence having an amino acid X 4 represents an amino acid selected from the group consisting of C, D, E, F , G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;
  • sequence having at least 80% identity with the sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W and Y; - a sequence having at least 80% identity with SEQ ID NO: 8 (ASNP V331X 7), said sequence having an amino acid X 7 represents an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 9 (ASNP D394Xs), said sequence having an amino acid Xs representing an amino acid selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and
  • sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R446X9), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W and Y.
  • a sequence having at least 80% identity with SEQ ID NO: 1 mutated once at any of the positions R226, 1228, F229, A289, F290, 1330, V331, D394 and R446 is selected from:
  • sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of C, H, K, M, N, Q, S, T and V;
  • sequence having at least 80% identity with SEQ ID NO: 3 (ASNP H28X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of H, L, T, V, W and Y;
  • F229X3 said X3 amino acid sequence being an amino acid selected from the group consisting of C, D, E, G, H, I, K, M, N, P, Q, V, W and Y, especially C, D, E, G, I, K, M, N, P, V, W and Y, and more preferably M and Y;
  • sequence having an amino acid X 4 represents an amino acid selected from the group consisting of C, D, E, F , M, NP, Q, S, T, V and W, and more particularly selected from the group consisting of F, M, N, P, Q, S and T;
  • sequence having at least 80% identity with the sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, P, Q, S, T, V and W, more preferably A, C, D, H, I, K, L, M, Q, S, T, V and W, in particular A, C, I, L, V, S, T and W; a sequence having at least 80% identity with the sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, M, N, Q, S, V and Y, in particular A and C, more preferably A;
  • V331X 7 said sequence having an amino X 7 acid representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, N, Q, R, S, T, W and Y, more preferably C, D, E, G, F, N, R, S, T, W and Y, in particular E, T and W;
  • sequence having at least 80% identity with SEQ ID NO: 9 (ASNP D394Xs), said sequence having an amino acid Xs representing an amino acid selected from the group consisting of A, E, F, G, H, I, K and L, in particular A and E;
  • sequence having an amino acid X9 represents an amino acid selected from the group consisting of A, C, G, K , L, M, N, and S, especially A, N, and M.
  • all the enzymes possessing one of these peptide sequences have a capacity that is statistically greater or even much greater than that of the wild-type enzyme to glucosylate the hydroxylated synthons of the invention.
  • hydroxylated synthon used in a process of the invention is HEMA
  • certain enzymes advantageously make it possible to obtain only mono-glucosylated HEMA, such as, for example, a sequence enzyme having at least 80 % identity with SEQ ID NO: 13 (ANi 23 -GBD-CD2).
  • hydroxylated synthon used in a process of the invention is HEMA
  • certain enzymes advantageously make it possible to obtain only mono-glucosylated HEMA and di-glucosylated HEMA, such as, for example, selected enzymes in the group:
  • FH9X3 said sequence having an amino acid X3 representing W; a sequence having at least 80% identity with the sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing A;
  • hydroxylated synthon used in a process of the invention is HEMA
  • certain enzymes advantageously make it possible to obtain a mixture of mono-glucosylated, di-glucosylated and triglucosylated HEMA, such as, for example the enzymes selected in the group:
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, I, K, L, M and V;
  • enzymes selected from the group:
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing A;
  • hydroxylated synthon used in a process of the invention is NHAM
  • certain enzymes advantageously make it possible to obtain only mono-glucosylated NHAMs, such as, for example, the enzymes chosen from the group:
  • certain enzymes advantageously make it possible to obtain only mono-glucosyl NHAMs and di-glucosylated NHAMs, such as, for example, selected enzymes in the group:
  • sequence having at least 80% identity with the sequence SEQ ID NO: 5 (ASNP A289X 4), said sequence having an amino acid X 4 represents an amino acid selected from the group consisting of N, P and Q;
  • SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing N;
  • hydroxylated synthon used in a process of the invention is NHAM
  • certain enzymes advantageously make it possible to obtain a mixture of mono-glucosylated, di-glucosylated and tri-glucosylated NHAMs, such as, for example the enzymes selected in the group:
  • sequence having at least 80% identity with SEQ ID NO: 8 (ASNP V331X 7), said sequence having an amino acid X 7 represents an amino acid selected from the group consisting of C, D and E;
  • hydroxylated synthon used in a method of the invention is NNHEA
  • certain enzymes advantageously make it possible to obtain only mono-glucosyl NNHEAs, such as, for example, the enzymes chosen from the group:
  • NNHEA hydroxylated synthon used in a process of the invention
  • other enzymes also make it possible to advantageously obtain only mono-glucosyl NNHEAs, namely:
  • SEQ ID NO: 5 (ASNP A289X 4), said sequence having an amino acid X 4 Q represents; a sequence having at least 80% identity with the sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of C, L and V representative; and
  • the present invention also encompasses sequences having an amino acid sequence of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of amino acid identity with one of SEQ ID NO: 1 to 18 such as previously defined and a biological activity of the same nature.
  • the "percentage of identity" between two nucleic acid or amino acid sequences is determined by comparing the two optimally aligned sequences, through a comparison window.
  • the part of the nucleotide sequence in the comparison window may thus comprise additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or deletions) so as to obtain a optimal alignment between the two sequences.
  • the percent identity is calculated by determining the number of positions at which an identical nucleotide base (or identical amino acid) is observed for the two compared sequences, and then dividing the number of positions at which there is identity between the two nucleic bases. (or between the two amino acids) by the total number of positions in the comparison window, then multiplying the result by one hundred in order to obtain the percentage of nucleotide (or amino acid) identity of the two sequences between them.
  • the optimal alignment of the sequences for the comparison can be performed in a computer manner using known algorithms.
  • the present invention also relates to sequences whose amino acid sequence has 100% amino acid identity with amino acids 225 to 450 of SEQ ID NO: 2 to 10 and at least 80%, 81%. 82% 83% 84% 85% 86% 87% 88% 89% 90% 91% 92% 93% 94% 95% 96% 97% 98 %, 99% or 100% of amino acid identity with the rest of the sequences SEQ ID NO: 2 to 10 as defined above, and a biological activity of the same kind.
  • the enzymes according to the invention make it possible to produce glycosylated or monomeric synthons which can be mono- or poly-glycosylated.
  • hydroxylated synthons specifically used in an enzymatic process for the manufacture of monomers of the invention are chosen from the compounds of formulas (I) to (VI).
  • the hydroxylated synthons of the invention may be compounds of formula (I): (i) (meth) acrylate / (meth) acrylamide synthons of formula (I):
  • R 1 represents a hydrogen atom or a C 1 -C 3 alkyl
  • R 2 represents a C 1 -C 20 alkylene group; or a group (C 2 H 4 O) n , where n is an integer of 1 to 10;
  • Xi represents - (O) -, - (NH) -, - (S) - or - (NR ' 2 (OH)) -, preferably - (O) -, - (NH) -, or - (NR' 2 (OH)) -, with R ' 2 representing a C 1 -C 20 alkylene group; or a group - (C 2 H 4 O) m -, with m being an integer of 1 to 10;
  • R 1 represents a hydrogen atom. According to another embodiment, R 1 represents a C 1 -C 3 alkyl, preferably a methyl.
  • R 2 is alkylene C 1 -C 20, especially C 1 -C 10, in particular Ci-C 5. More particularly, R 2 may be selected from the group consisting of methylene, ethylene, propylene, butylene and pentylene, and is preferably methylene or ethylene.
  • X 1 represents - (O) -, - (NH) - or - (NR ' 2 (OH)), preferably - (O) - or - (NH) -.
  • R '2 is alkylene C 1 -C 20, especially C 1 -C 10, in particular Ci-C 5. More particularly, R ' 2 may be selected from the group consisting of methylene, ethylene, propylene, butylene and pentylene, and is preferably methylene or ethylene.
  • a synthon of formula (I) of the invention is such that:
  • R 1 represents a hydrogen atom or a C 1 -C 3 alkyl, preferably a hydrogen or a methyl
  • R 2 represents an alkyl group C 1 -C 20, preferably a C 10 -C alkylene, in particular C 1 -C 5, more particularly a methylene or ethylene;
  • Xi represents - (O) -, - (NH) - or - (NR ' 2 (OH)) -, preferably - (O) - or - (NH) -.
  • a synthon of formula (I) of the invention is such that:
  • Xi represents - (O) -
  • R 1 represents a hydrogen atom or a C 1 -C 3 alkyl, preferably a C 1 -C 3 alkyl, in particular a methyl;
  • R 2 represents a C 1 -C 20 alkyl group, preferably a C 1 -C 10 , in particular C 1 -C 5 , alkylene, more particularly a methylene or an ethylene, preferentially an ethylene.
  • Such a synthon of formula (I) may in particular be a 2- (hydroxy) ethyl methacrylate (HEMA):
  • a synthon of formula (I) of the invention is such that:
  • Xi represents - (NH) -
  • R 1 represents a hydrogen atom or a C 1 -C 3 alkyl, preferably a hydrogen or a methyl
  • R 2 represents a C 1 -C 20 alkyl group, preferably a C 1 -C 10 , in particular C 1 -C 5 , alkylene, more particularly a methylene or an ethylene.
  • Such a synthon of formula (I) may in particular be an N- (hydroxy) methyl acrylamide (NHAM) or an N- (hydroxy) ethyl acrylamide (HEAA):
  • a synthon of formula (I) of the invention is such that:
  • Xi is - (NR '2 (OH)) -, wherein R' 2 represents an alkylene group Ci-C 2 o, in particular C 1 -C 10, in particular to C 5, more particularly a methylene, an ethylene, a propylene, a butylene or a pentylene, preferably a methylene or an ethylene, more preferably an ethylene;
  • Ri represents a hydrogen atom
  • R 2 represents an alkyl group Ci-C 2 o, preferably Cl- Cio alkylene, in particular C 1 -C 5, more particularly a methylene or ethylene, in particular ethylene.
  • Such a synthon of formula (I) may in particular be an N, N-bis (2-hydroxyethyl) acrylamide (NNHEA):
  • a synthon of the invention of formula (I) is chosen from 2- (hydroxy) ethylmethacrylate (HEMA), N- (hydroxy) methyl acrylamide (NHAM), N- (hydroxy) ethylacrylamide (HEAA) and N, N-bis (2-hydroxyethyl) acrylamide (NNHEA), preferably from HEMA, NHAM and HEAA.
  • the hydroxylated synthons of the invention may be compounds of formula (II):
  • R 3 represents a covalent bond; a C1-C20 alkylene group; or a group (C 2 H 4 0) n, with n being an integer between 1 and 10.
  • R 3 represents a covalent bond or an alkylene group
  • Such an alkylene group may in particular be C1-C10, especially C1-C5. More particularly, R 3 may be selected from the group consisting of methylene, ethylene, propylene, butylene and pentylene, and is preferably methylene or ethylene.
  • R 3 represents a covalent bond, a methylene or an ethylene.
  • a synthon of formula (II) can in particular be a 4-vinylphenol (VP) or a 4-vinyl benzyl alcohol (VBA):
  • hydroxylated synthons of the invention may be compounds of formula (III):
  • R 4 represents a covalent bond; a C1-C20 alkylene group; or a group (C 2 H 4 0) n, with n being an integer between 1 and 10.
  • R4 represents a C1-C20 alkylene, in particular C1-C10, especially C1-C5, alkylene group.
  • R4 may be selected from the group consisting of methylene, ethylene, propylene, butylene and pentylene, and is preferably methylene or ethylene, more particularly methylene.
  • a synthon of formula (III) may in particular be a 4- (hydroxy) methyl oxazolidine-2,5-dione (HMNCA):
  • hydroxylated synthons of the invention may be compounds of formula (IV):
  • R 5 represents a covalent bond; a C1-C20 alkylene group; or a group (C 2 H 4 0) m, with m being an integer between 1 and 10;
  • n an integer from 1 to 20;
  • X 2 represents - (O) -, - (NH) - or - (S) -;
  • R O represents hydrogen, alkyl C1 -C20.
  • X 2 represents - (O) -.
  • n is between 1 and 10, in particular between 1 and
  • n is in particular chosen from 1, 2, 3, 4 and 5, and preferably represents the value 1 or 2.
  • R 5 represents a covalent bond or a C 1 -C 10 alkylene group, in particular a C 1 -C 5 alkylene group.
  • the alkylene group may be selected from the group consisting of methylene, ethylene, propylene, butylene and pentylene, preferably methylene or ethylene, more particularly methylene. .
  • R 5 represents a covalent bond or methylene.
  • R 5 represents a hydrogen or a C 1 -C 10 alkyl group, in particular a C 1 -C 5 alkyl group.
  • the alkyl group can be chosen from the group consisting of a methyl, an ethyl, a propyl, a butyl and a pentyl, preferably a methyl or an ethyl, more particularly a methyl .
  • Re represents hydrogen or methyl
  • a compound of formula (IV) is such that:
  • R 5 represents a C 1 -C 10 alkylene group, especially a C 1 -C 5 alkylene group, and is in particular a methylene;
  • n represents an integer from 1 to 5; and preferably represents the value 1 or 2, in particular the value 2;
  • X 2 represents - (O) -
  • R D represents hydrogen
  • Such a synthon of formula (IV) may in particular - (hydroxy) methyl caprolactone (AHMCL):
  • a compound of formula (IV) is such that:
  • R5 represents a covalent bond
  • n represents an integer from 1 to 5; and preferably represents the value 1 or 2, in particular the value 1;
  • X 2 represents - (O) -
  • R represents a C 1 -C 10 alkyl group, in particular a C 1 -C 5 alkyl group, and is in particular methyl.
  • Such a synthon of formula (IV) may in particular be a ( ⁇ ) - mevalonolactone
  • a synthon of the invention of formula (IV) is chosen from ⁇ - (hydroxy) methyl caprolactone (AHMCL) and i-mevalonolactone (MVL).
  • hydroxylated synthons of the invention may be compounds of formula (V):
  • R 7 represents a C 1 -C 20 alkylene group.
  • R 7 represents a C 1 -C 10 alkylene group, especially a C 1 -C 5 alkylene group. More particularly, R 7 may be selected from the group consisting of methylene, ethylene, propylene, butylene and pentylene, preferably methylene or ethylene, more particularly ethylene.
  • Such a synthon of formula (V) may in particular be a 2-mercaptoethanol (BME):
  • hydroxylated synthons of the invention may be compounds of formula (VI):
  • Rs is alkylene C 1 -C 10, especially C 1 -C 5. More particularly, R 8 may be selected from the group consisting of methylene, an ethylene, a propylene, a butylene and a pentylene, preferably a methylene n ethylene, more particularly a methylene.
  • a synthon of the invention may be selected from the group consisting of 2 (hydroxy) ethylmethacrylate (HEMA), N- (hydroxy) methyl acrylamide (NHAM), N- (hydroxy) ethylacrylamide (HEAA ), 4-vinylphenol (VP),
  • VBA 4-vinyl benzyl alcohol
  • HNCA 4- (hydroxy) methyl oxazolidine-2,5-dione
  • HMCL ⁇ - (hydroxy) methyl caprolactone
  • MVL ⁇ - mevalonolactone
  • NHEA N, N-bis (2-hydroxyethyl) acrylamide
  • a synthon according to the invention is in particular chosen from the group consisting of
  • HEMA hydroxyethylmethacrylate
  • NHAM N- (hydroxy) methyl acrylamide
  • HEAA N- (hydroxy) ethylacrylamide
  • VBA 4-vinyl benzyl alcohol
  • the enzymatic glycosylation of the hydroxylated and in particular monohydroxylated synthons of the invention is in the presence of sucrose.
  • glycosylated synthons and “monomers” are used interchangeably to designate the glycosylated synthons obtained from the enzymatic process according to the invention for the glycosylation of hydroxylated synthons.
  • the synthons of the invention are more particularly glucosylated or fructosylated during the enzymatic glycosylation process according to the invention.
  • the process according to the invention for manufacturing a glycosylated synthon, or monomer, of the invention may in particular be implemented under the conditions set forth in Example 1, point 1.3 below.
  • the monomers of the invention may be mono- or multi-glycosylated, as illustrated in the examples of the present application.
  • the enzyme can advantageously be inactivated. This inactivation may, for illustrative purposes, be carried out by thermal inactivation, for example at a temperature greater than 60 ° C., in particular greater than 80 ° C., preferably greater than 90 ° C.
  • the reaction medium containing the glycosylated synthons according to the invention is concentrated by lyophilization, in particular in anticipation of a subsequent purification step.
  • reaction medium containing the glycosylated synthons according to the invention is not concentrated by freeze-drying.
  • the glycosylated synthons according to the invention are purified after their preparation.
  • This purification step when it is carried out, can take place after a step of inactivation of the enzyme used and / or after a lyophilization step.
  • a purification step occurs after a step of inactivation of the enzyme, in the absence of a lyophilization step.
  • This purification step may advantageously also comprise a liquid / liquid extraction step in order to eliminate the residual synthon.
  • the glucan-saccharase used can advantageously be chosen from the group comprising:
  • sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of A, C, N, P, S and T, preferably C;
  • sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of P, T and V, preferably T;
  • F229X 3 said sequence having an amino acid X3 representing W; - a sequence having at least 80% identity with the sequence SEQ ID NO: 5 (ASNP A289X 4), said sequence having an amino acid X 4 is H, I, K, L, M and W, preferably H and L;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, H, I, K, L, M, N, P, Q, V and W, preferably A, C, I, K, L, M, P and V, in particular A, C, I, K, L, M and V, and in particular A, C, I, L and V;
  • sequence having an amino acid X 7 represents an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, M, especially C, D, E, G, H, and especially E;
  • sequence having at least 80% identity with SEQ ID NO: 9 (ASNP D394X 8), said sequence having an amino acid representing Xs A, C, E, G, H, I, L, M and N, in particular A;
  • sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R446X9), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M and N, preferably C, G, K, L and N, especially N;
  • SEQ ID NO: 17 ASR-C-del-bis
  • SEQ ID NO: 18 Bacillus substrilis fructosyltransferase
  • the glucansucrase used in a method of the invention may be chosen from the group comprising:
  • sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of C, E, L, M, N, P, Q, R, T, V and Y;
  • F229X3 said X3 amino acid sequence being an amino acid selected from the group consisting of A, C, I, L, M, N, P, Q, V, W and Y, preferably M;
  • sequence having an amino acid X 4 represents an amino acid selected from the group consisting of C, D, E, F G, H, I, L, M, N, P, Q, S, T and V, preferably N, P, Q, S, and V, especially N, P and Q;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, G, L, M, N, P, Q, V, W and Y;
  • SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, M, N and V, preferably N;
  • sequence having at least 80% identity with SEQ ID NO: 8 (ASNP V331X 7), said sequence having an amino acid X 7 represents an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T and Y, preferably C, D, E, N, and T, especially E; a sequence having at least 80% identity with SEQ ID NO: 9 (ASNP D394Xs), said sequence having an amino acid Xs representing an amino acid selected from the group consisting of C, E, G and N, preferably E;
  • sequence having an amino acid X9 represents an amino acid selected from the group consisting of A, C, F, G, K L, M, N, Q, S, T and Y;
  • SEQ ID NO: 18 Bacillus subtilis fructosyltransferase
  • a process for producing a glycosylated synthon, or monomer comprises at least one step of placing at least one glycan-saccharase with at least 2-mercaptoethanol (BME) as a synthon hydroxyl and at least one sucrose, wherein the glycan-sucrase is selected from the group consisting of:
  • sequence having at least 80% identity with SEQ ID NO: 1 (ASNP WT); a sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of A, E, F, H, L, M, N, Q, R, V, W and Y, preferably L, V, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 4 (ASNP F229X 3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A, C, I, L, M , P, Q, R, V, W and Y, preferably C, M, P, Q, V, W and Y, in particular C, M, P, V and Y, especially Y;
  • sequence having an amino acid X 4 represents an amino acid selected from the group consisting of C, D, E, F , G, H, I, M, N, P, Q, R, S, T, V and W, preferably C, D, E, F, M, N, P, Q, S, T, V and W , and especially F, M, N, P, Q, S and T;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably A, C, D, E, G, I, M, P, Q, S, T, V and W, especially D, Q, S, T and W, and especially S, T and W;
  • sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, N, Q, S, T, V and W, preferably Q and V;
  • V331X 7 said sequence having an amino X 7 acid representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, N, Q, R, S, T, W and Y, preferably A, C, D, E, F, G, H, I, N, Q, R, S, T, W and Y, in particular C, D, E, F, N, R, S, T, W and Y, and especially E, T, and W;
  • said Xs amino acid sequence being an amino acid selected from the group consisting of A, E and S, preferably E;
  • sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R.446X 9 ), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, Q, S and T, preferably S;
  • a process for producing a glycosylated synthon, or monomer comprises at least one step of placing at least one glycan-saccharase with at least one propen-1-ol (allylic alcohol) at least one as hydroxylated synthon and at least one sucrose, wherein the glycan-sucrase is selected from the group consisting of:
  • R226Xi said sequence having an amino acid X1 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V and Y;
  • sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of D, E, F, G, H, P, Q, R, S, T, V, W and Y, preferably H;
  • sequence having at least 80% identity with SEQ ID NO: 4 (ASNP F229X3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of C, D, E, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably D, E, G, H, I, K and M, in particular D, E, G, I, K and M;
  • SEQ ID NO: 5 (ASNP A289X 4), said sequence having an X amino acid 4 representing an amino acid selected from the group consisting of C, D, E, F, G, H, I, K, L, M, P , Q, R, S, T, V, W and Y, preferably C, D, E, F, G, H, I, K, L, MW and Y;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably C, D, E, G, H, I, K, L, M and W, in particular C and H; a sequence having at least 80% identity with the sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W and Y, preferably C, D, E, F, G, H, K, L, M, S and Y, especially C;
  • V331X 7 said sequence having an amino X 7 acid representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W and Y, preferably C, D, E, F, G, H, I, K, L, N and W;
  • sequence having at least 80% identity with SEQ ID NO: 9 (ASNP D394Xs), said sequence having an amino acid Xs representing an amino acid selected from the group consisting of A, C, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably E, F, G, H, I, K and L, in particular E;
  • sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R.446X9), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, Q, S, T, V, W and Y, preferably M;
  • SEQ ID NO: 18 Bacillus subtilis fructosyltransferase
  • a process for manufacturing a glycosylated synthon, or monomer comprises at least one step of placing at least one glycan-saccharase with at least one VBA as a hydroxylated synthon and at least one sucrose, wherein the glycan-sucrase is selected from the group consisting of
  • WT a sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of H, K, M, N, Q, S, T, V and Y, in particular H, K, M, N, Q, S, T and V, preferably H, K, M, N, Q, T and V;
  • I228X 2 said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of V;
  • sequence having an amino acid X 4 represents an amino acid selected from the group consisting of M, N, P, Q S, T and V, preferably M, N, P, Q, S and T, and especially P, Q and S;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of V and W;
  • V331X 7 said X 7 amino acid sequence being an amino acid selected from the group consisting of C, D, G, S, T and Y, preferably C, G, S, T and Y;
  • a process for producing a glycosylated synthon, or monomer comprises at least one step of placing at least one glycan-saccharase with at least one NNHEA as a hydroxylated synthon and at least one sucrose, wherein the glycan-sucrase is selected from the group consisting of
  • sequence having at least 80% identity with SEQ ID NO: 1 (ASNP WT), a sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of A, D, E, I, K, L, M, N, Q, S, T, V, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of A, C, E, F, H, L, M, N, P, Q, S, T, V, W and Y, preferably V;
  • sequence having at least 80% identity with SEQ ID NO: 4 (ASNP F229X3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of C, I, L, M, N, Q, R, V, W and Y, preferably M;
  • SEQ ID NO: 5 (ASNP A289X 4), said sequence having an X amino acid 4 representing an amino acid selected from the group consisting of C, D, E, F, G, H, I, K, L, M, N , P, Q, R, S, T, V and W, preferably C, G, M, N, Q, S, T and V, in particular Q;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably A, C, G, I, K, L, M, Q, S, T, V and W, especially C, L, V and W;
  • sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of C, K, M, Q, T, V and Y, preferably V;
  • sequence having an amino acid X 7 represents an amino acid selected from the group consisting of C, D, E, F, G, H, I, K, L, M, N, R, S, T, W and Y, preferably D, E, G, N, S, T and Y;
  • sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R446X9), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of C, F, G, K, L, M, N, Q, S, T and Y;
  • a sequence having at least 80% identity with SEQ ID NO: 12 may also be used in a glycosylated synthon production method according to the invention when NNHEA is the hydroxylated synthon to be glycosylated, especially to glucosylate.
  • a process for producing a glycosylated synthon, or monomer comprises at least one step of bringing at least one glycan-sucrase into contact with at least HEAA as a hydroxylated synthon and at least one sucrose, wherein the glycan-sucrase is selected from the group consisting of
  • SEQ ID NO: 18 Bacillus subtilis fructosyltransferase
  • the present invention more preferably relates to a process for manufacturing a glycosylated synthon, or monomer, comprising at least one step of placing at least one glycan-saccharase in contact with at least one hydroxylated synthon and at least one sucrose, in which :
  • glycosylated synthon is HEMA and the glycansucrase used is selected from the group consisting of:
  • sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of A, C, N, P, S and T, preferably C;
  • a sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of P, T and V, preferably T;
  • sequence having at least 80% identity with the sequence SEQ ID NO: 5 (ASNP A289X 4), said sequence having an amino acid X 4 is H, I, K, L, M and W, preferably H and L;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, H, I, K, L, M, N, P, Q, V and W, preferably A, C, I, K, L, M, P and V, in particular A, C, I, K, L, M and V, and in particular A, C, I, L and V;
  • sequence having an amino acid X 7 represents an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, M, especially C, D, E, G, H, and especially E;
  • sequence having at least 80% identity with SEQ ID NO: 9 (ASNP D394X 8), said sequence having an amino acid representing Xs A, C, E, G, H, I, L, M and N, in particular A;
  • sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R.446X 9 ), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, D, E , F, G, H, I, K, L, M and N, preferably C, G, K, L and N, especially N;
  • GBD-CD2 a sequence having at least 80% identity with SEQ ID NO: 14
  • the hydroxylated synthon is NHAM and the glucan-saccharase used in a method of the invention is chosen from the group comprising:
  • sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of C, E, L, M, N, P, Q, R, T, V and Y;
  • sequence having at least 80% identity with SEQ ID NO: 4 (ASNP F229X3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A, C, I, L, M, N, P, Q, V, W and Y, preferably M;
  • sequence having an amino acid X 4 represents an amino acid selected from the group consisting of C, D, E, F G, H, I, L, M, N, P, Q, S, T and V, preferably N, P, Q, S, and V, especially N, P and Q;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, G, L, M, N, P, Q, V, W and Y;
  • sequence having at least 80% identity with the sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, M, N and V, preferably N; - a sequence having at least 80% identity with SEQ ID NO: 8 (ASNP V331X 7), said sequence having an amino acid X 7 represents an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T and Y, preferably C, D, E, N, and T, especially E;
  • D394Xs said Xs amino acid sequence being an amino acid selected from the group consisting of C, E, G and N, preferably E;
  • sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R446X9), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, F, G, K, L, M, N, Q, S, T and Y;
  • the glycosylated synthon is 2-mercaptoethanol (BME) and the glycan-sucrase is selected from the group consisting of:
  • sequence having at least 80% identity with SEQ ID NO: 1 (ASNP WT); a sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of A, E, F, H, L, M, N, Q, R, V, W and Y, preferably L, V, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 4 (ASNP F229X 3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A, C, I, L, M , P, Q, R, V, W and Y, preferably C, M, P, Q, V, W and Y, in particular C, M, P, V and Y, especially Y;
  • sequence having an amino acid X 4 represents an amino acid selected from the group consisting of C, D, E, F , G, H, I, M, N, P, Q, R, S, T, V and W, preferably C, D, E, F, M, N, P, Q, S, T, V and W , and especially F, M, N, P, Q, S and T;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably A, C, D, E, G, I, M, P, Q, S, T, V and W, especially D, Q, S, T and W, and especially S, T and W;
  • sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, N, Q, S, T, V and W, preferably Q and V;
  • V331X 7 said sequence having an amino X 7 acid representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, N, Q, R, S, T, W and Y, preferably A, C, D, E, F, G, H, I, N, Q, R, S, T, W and Y, in particular C, D, E, F, N, R, S, T, W and Y, and especially E, T, and W;
  • said Xs amino acid sequence being an amino acid selected from the group consisting of A, E and S, preferably E;
  • sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R.446X 9 ), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, Q, S and T, preferably S;
  • the hydroxylated synthon is propen-1-ol (allyl alcohol) and the glycan-sucrase is selected from the group consisting of:
  • sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V and Y;
  • sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of D, E, F, G, H, P, Q, R, S, T, V, W and Y, preferably H;
  • sequence having at least 80% identity with SEQ ID NO: 4 (ASNP F229X3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of C, D, E, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably D, E, G, H, I, K and M, in particular D, E, G, I, K and M;
  • sequence having an amino acid X 4 represents an amino acid selected from the group consisting of C, D, E, F , G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably C, D, E, F, G, H, I, K, L, MW and Y;
  • SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably C, D, E, G, H, I, K, L, M and W, especially C and H;
  • sequence having at least 80% identity with the sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W and Y, preferably C, D, E, F, G, H, K, L, M, S and Y, especially C; - a sequence having at least 80% identity with SEQ ID NO: 8 (ASNP V331X 7), said sequence having an amino acid X 7 represents an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W and Y, preferably C, D, E, F, G, H, I, K, L, N and W;
  • D394Xs said Xs amino acid sequence being an amino acid selected from the group consisting of A, C, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably E, F, G, H, I, K and L, in particular E;
  • sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R.446X9), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, Q, S, T, V, W and Y, preferably M;
  • the glycosylated synthon is VBA and the glycan-sucrase is selected from the group consisting of:
  • sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of H, K, M, N, Q, S, T, V and Y, in particular H, K, M, N, Q, S, T and V, preferably H, K, M, N, Q, T and V;
  • sequence having an amino acid X 2 representing an amino acid selected from the group consisting of V; - a sequence having at least 80% identity with the sequence SEQ ID NO: 5 (ASNP A289X 4), said sequence having an amino acid X 4 represents an amino acid selected from the group consisting of M, N, P, Q S, T and V, preferably M, N, P, Q, S and T, and especially P, Q and S;
  • SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of V and W;
  • sequence having an amino acid X 7 represents an amino acid selected from the group consisting of C, D, G, S, T and Y, preferably C, G, S, T and Y;
  • glycosylated synthon is NNHEA and the glycan-sucrase is selected from the group consisting of:
  • sequence having at least 80% identity with SEQ ID NO: 2 (ASNP R226Xi), said sequence having an amino acid Xi representing an amino acid selected from the group consisting of A, D, E, I, K, L, M, N, Q, S, T, V, W and Y;
  • sequence having at least 80% identity with SEQ ID NO: 3 (ASNP I228X 2 ), said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of A, C, E, F, H, L, M, N, P, Q, S, T, V, W and Y, preferably V;
  • sequence having at least 80% identity with SEQ ID NO: 4 (ASNP F229X3), said sequence having an amino acid X3 representing an amino acid selected from the group consisting of C, I, L, M, N, Q, R, V, W and Y, preferably M;
  • SEQ ID NO: 5 (ASNP A289X 4), said sequence having an amino acid X 4 representing a amino acid selected from the group consisting of C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V and W, preferably C, G, M, N, Q, S, T and V, especially Q;
  • sequence SEQ ID NO: 6 (ASNP F290Xs), said sequence having an amino acid X 5 representing an amino acid selected from the group consisting of A, C, D, E, G, H, I, K, L, M, P, Q, R, S, T, V, W and Y, preferably A, C, G, I, K, L, M, Q, S, T, V and W, especially C, L, V and W;
  • sequence SEQ ID NO: 7 (ASNP ⁇ 330 ⁇ ), said sequence having an amino acid X 6 representing an amino acid selected from the group consisting of C, K, M, Q, T, V and Y, preferably V;
  • V331X 7 said sequence having an amino X 7 acid representing an amino acid selected from the group consisting of C, D, E, F, G, H, I, K, L, M, N, R, S, T, W and Y, preferably D, E, G, N, S, T and Y;
  • sequence having at least 80% identity with SEQ ID NO: 9 (ASNP D394Xs), said sequence having an amino acid Xs representing an amino acid selected from the group consisting of E, G, R and S;
  • sequence having at least 80% identity with SEQ ID NO: 10 (ASNP R446X9), said sequence having an amino acid X9 representing an amino acid selected from the group consisting of C, F, G, K, L, M, N, Q, S, T and Y;
  • glycosylated synthon is HEAA and the glycan-sucrase is selected from the group consisting of:
  • the monomers of the invention are polymerizable and / or can be coupled by coupling reaction, and can be implemented in a process for producing a glyco (co) polymer according to the invention, comprising the polymerization and / or a coupling reaction of at least two of these monomers.
  • a chemo-enzymatic process according to the invention is advantageous with regard to many aspects, such as in particular the very short reaction time allowing, within a few hours, in particular in the space of 24 hours, to pass hydroxylated synthons at glyco (co) polymers of interest.
  • the polymerization and the coupling reaction according to the invention also make it possible to access original molecular architectures, such as comb or block (co) polymers, which can be evaluated for the development of new hydrophilic balance materials. hydrophobic modulable.
  • Such a method can in particular be implemented under the conditions set forth in Example 6 below.
  • a method of manufacturing by polymerization of a glyco (co) polymer according to the invention comprises, in order, the following steps:
  • such a polymerization process may further comprise, independently:
  • step c ' after any one of the steps c), in which the monomer chain obtained at the end of the preceding step is polymerized with at least one non-glycosylated synthon.
  • a library comprising 8 wild-type enzymes (belonging to the GH70 families and 13 glycoside hydrolases) and 171 single mutants (positions 226, 228, 229, 289, 290, 330, 331, 394 and 446) constructed from the amylosucrase of Neisseria polysaccharea (ASNp) (GH13 family) were tested for their ability to glucosylate F HEMA, and NHAM.
  • ASNp Neisseria polysaccharea
  • Table 1 illustrates a number of the glucansucrases tested in the examples of the present text and specifies: column 1: the organism from which the enzyme originates; column 2: the various wild-type enzymes tested as well as the mutated positions of the active site of these wild-type enzymes in the mutated glucan-saccharases also tested; column 3: the bibliographic references in which these enzymes, both in wild and mutated forms, have been described in the state of the art.
  • the set of Escherichia coli strains, overexpressing hetero-glucan saccharases of the wild-type GH13 and GH70 families or mutants thereof (Table 1), are stored in 96-well microplate format to facilitate future steps of synthon glucosylation screening. hydroxylated.
  • precultures are in turn used to seed the "deep-well" microplates, each well containing 1 ml per well of self-inducing medium ZYM5052 containing in particular 0.2% (w / v) -lactose, 0.05% (w / v) D-glucose, 0.5% (w / v) glycerol and 0.05% (w / v) L-arabinose (Studier et al., Protein Expr Purif 2005 May, 41 (1): 207-34.).
  • the cell suspension is centrifuged for 20 min at 3000 g at 4 ° C.
  • Cell pellets are resuspended in 96-well deep-well microplates, with 300 ⁇ l of phosphate buffered saline (24 mM sodium / potassium phosphate and 274 mM NaCl) containing 0.5 g / L lysozyme and 5 mg / L Bovine pancreatic RNAse.
  • microplates are then stored overnight at -80 ° C. After thawing, the microplates are vigorously stirred and then centrifuged for 20 min at 3000 g at 4 ° C.
  • the centrifuged supernatants containing the recombinant enzymes are transferred into deep-well 96-well microplates to carry out the acceptor reactions.
  • the enzymatic activity is evaluated in microplate format end point after 30 minutes of incubation of 146 mM final sucrose by assaying reducing sugars by 3,5-dinitrosalicylic acid (DNS).
  • DNS 3,5-dinitrosalicylic acid
  • the absorbance is read at 540 nm.
  • the acceptor reactions are carried out in deep-well microplates in a volume of 300 ⁇ l, in the presence of 73 mM sucrose, 73 mM acceptor ( HEMA, NHAM, or other ...) in final concentrations and 150 of centrifuged cell lysate.
  • microplates are incubated at 30 ° C. and 700 rpm.
  • the enzymes are denatured at 95 ° C for 10 minutes.
  • microplates are stored at -20 ° C for rapid analysis of glucosylation by HPLC / MS.
  • the acceptor reactions are carried out in tubes in a volume of 1 ml, in the presence of 146 mM sucrose, 438 mM acceptor (HEMA or NHAM) in final concentrations of 1 U / mL. enzymatic activity.
  • the tubes are incubated at 30 ° C. and shaken at 500 rpm.
  • the enzymes are denatured at 95 ° C for 10 minutes, the reaction media are centrifuged, filtered, diluted and analyzed by LC / MS.
  • a first short HPLC analysis on a hypercarb column (6 minutes) is carried out to identify the presence or absence of glucosylation products.
  • the second Hypercarb or Amino HPLC analysis (30 minutes) allows separation and identification of all components of the reaction mixture.
  • the Dionex HPLC system is coupled to a mass spectrometer, single quadrupole MSQPlus, Thermo S cientific.
  • the amount of glycosylation products (in g / L) was estimated based on the response coefficient of the acceptor in UV detection.
  • Example 2 Determination of Glucosylation Efficiencies of HEMA and NHAM by the Enzymes of Example 1
  • Tables 3 and 4 show the conversion rate of sucrose and the three main glucosylation products obtained in the presence, respectively, of HEMA and NHAM in g / L, characterized by MS and respectively corresponding to the mono-glucosyl acceptor (Acceptor -Glcl), di-glucosylated (Acceptor-Glc2) and tri-glucosylated (Acceptor-Glc3). Products in smaller amounts are also detected, corresponding to tetra-, penta-, hexa- and heptaglucosylated forms.
  • the structure of the di- and tri-glucosylated products may differ depending on the specificity of the enzyme and the structure of the products can not be determined without ambiguity only by NMR analysis.
  • ASR synthesizes a significantly higher level of glucosylated HEMA (21 g / L). With ASR, the products formed are distributed in equivalent proportions between the mono-, di- and tri-glucosylated forms of HEMA.
  • GBD-CD2 enzyme leads only to mono-glucosyl HEMA.
  • this enzyme advantageously makes it possible to obtain only mono-glucosyl HEMA.
  • Glucosylated NHAM with levels of the order of 2 g / L.
  • HEMA and NHAM were then assayed as an acceptor reaction using Neisseria polysaccharea amylosucrase (ASNP) and its library of mono-mutants identified in Table 1.
  • ASNP Neisseria polysaccharea amylosucrase
  • glucosylation reactions carried out in the presence of acceptor were analyzed initially using a short HPLC method (6 min) in order to quickly identify the enzymes effective in glucosylation of the acceptor.
  • the ASNp wild-type enzyme as well as 69 mono-mutated enzymes were found to be able to glucosylate HEMA, as shown in Table 5.
  • 25 mutants appeared to glucosylate HEMA more efficiently than the parental wild-type enzyme, namely R226C, I228T, F229W, A289H, A289L, F290A, F290C, F290I, F290K, F290L, F290M, F290P, F290V, I330A, V331C.
  • the wild-type ASNp enzyme as well as 103 mono-mutated enzymes were found to be able to glucosylate NHAM, as shown in Table 6.
  • 13 mutants appeared to glucosylate NHAM at least as efficiently as the parental wild-type enzyme, namely F229M, A289N, A289P, A289Q, A289S, A289V, I330N, V331C, V331D, V331E, V331N, V331T and D394E.
  • Example 3 Following the screening, a number of mutants tested in Example 3 were analyzed more finely by LC-MS (detection UV 21 0 for HEMA, detection UV215 for NHAM), according to the protocol indicated in the example 1 to analyze the glucosylation products obtained.
  • HEMA was glucosylated by the enzymes described in Table 7, resulting from the screening presented in Example 3. HEMA mono-, di- and / or triglucosyl compounds were thus obtained.
  • Example 5 Gram-scale production of glucosylation products of HEMA and NHAM using the selected enzymes
  • the reactions are carried out in a final volume of 500 ml, in the presence of 143 mM sucrose, 438 mM acceptors and 1 U / ml of enzyme. After total consumption of the sucrose, the enzyme is deactivated at 95 ° C. and the reaction medium is concentrated by lyophilization for a subsequent purification step.
  • Purification aims to eliminate residual sugars.
  • the purification is carried out by flash chromatography on a C18 Reveleris column of 80 g to 120 g (Alltech, Epernon, France) using a water / acetonitrile gradient.
  • ATRP Atom Transfer Radical Polymerization
  • RAFT Reversible Addition Fragmentation Transfer
  • a typical ATRP polymerization proceeds as follows.
  • the solution is then degassed by bubbling argon and the bipyridine (ligand, 15.7 mg, 100 ⁇ ., 2 eq.) And CuIBr (catalyst, 7.1 mg, 50 ⁇ ., 1 eq.) Are added in this order.
  • 20 ml of a degassed solution of the glucosylated monomer-HEMA mixture are added (0.78 g, 2.0 mmol, 40 eq.).
  • the polymerization then takes place for 8 hours at room temperature.
  • the reaction is stopped by air oxidation of the CuII Cu (the solution changes from brown to blue).
  • the solution is then passed through a silica column to get rid of the Cuil.
  • the reaction medium is then lyophilized to recover the polymer in the form of a white powder.
  • the polymerization yield is generally greater than 90%.
  • Example 7 Implementation of the glucosylation method on 2-propenol
  • 60 mutants appeared to glucosylate allyl alcohol at least as efficiently as the parental wild-type enzyme, namely I228H, F229D, F229E, F229G, F229H, F229I, F229K, F229M, A289C, A289D, A289E, A289F, A289G, A289H, A289I, A289K, A289L, A289M, A289W, A289Y, F290C, F290D, F290E, F290G, F290H, F290I, F290K, F290L, F290M, F290W, I330C, I330D, I330E, I330F, I330G, I330H, I330K, I330L, I330M, I330S, I330Y, V331C, V331D, V331E, V331F, V331G, V331H, V331I, V331K, V331
  • Wild ASNp and 89 mono-mutants are also shown to be able to glucosylate BME, as shown in Table 13.
  • 59 mutants appeared to glucosylate BME at least as efficiently as the parental wild-type enzyme, namely:
  • VBA was assayed as an acceptor reaction using Neisseria polysaccharea amylosucrase (ASNp), its mono mutant library identified in Table 1, as described in Example 3, and also in a-l, 2-BrS and ASR.
  • ASNp Neisseria polysaccharea amylosucrase
  • VBA can be glucosylated by wild-type ASNp as well as by 26 of its mono-mutants tested, namely: R226H, R226K, R226M, R226N, R226Q, R226S, R226T, R226V, R226Y, I228V, A289M, A289N, A289P, A289Q, A289S, A289T, A289V, F290V, F290W, V331C, V331D, V331G, V331S, V331T, V331Y and R446A.
  • Example 9 Determination of the glucosylation efficiencies of N, N-bis (2-hydroxyethyl) acrylamide (NNHEA) by N. polysaccharea amylosucrase (ASNp), its mutants and certain enzymes of Example 1
  • NNHEA was assayed as an acceptor reaction using Neisseria polysaccharea amylosucrase (ASNp), its mono-mutant library identified in Table 1, as described in Example 3, and also in a-1, 2-BrS, a-1,3-BrS and ASR.
  • ASNp Neisseria polysaccharea amylosucrase
  • the results obtained for NNHEA with the enzymes al, 2-BrS, ⁇ 1, 3-BrS and ASR are shown in Table 16, whereas the results obtained for NNHEA with ASNp and its mono-mutants are summarized in Table 1.
  • Table 17 Glucosylation results of NNHEA
  • ⁇ -1,2-BrS, ⁇ -1,3-BrS and ASR enzymes can glucosylate NNHEA and lead only to the production of mono-glucosyl NNHEA. Traces of di-glucosylated NNHEA have also been detected.
  • traces of a compound is meant in the sense of the invention that this compound is present in a sample in an amount sufficient to be detected by a measurement method such as that implemented in the present examples, but in a quantity too weak to be measured.
  • the value of 0 given in the tables of this document means that the compound concerned is either absent or present in such a small quantity that it is not detectable by a measurement method such as that implemented in the present examples.
  • NNHEA can be glucosylated by wild-type ASNp as well as by 113 of its mono-mutants tested, as shown in Table 17.
  • the HEAA was fructosylated, 1, 4, 5, 6 and even 7 times. Since these acceptors have only one reactive hydroxyl group, only one type of each fructosylation level of these molecules is obtained.
  • sucrose consumption of the fructosyltransferase implemented and the production rates (in g / L) of the tested fructosylated acceptors were measured.
  • Example 9 The glucosylation products obtained in Example 9 with some of the enzymes tested were analyzed more finely by LC-MS (UV204 detection for NNHEA), according to the protocol indicated in Example 1.
  • NNHEA mono-glucosylated by the enzymes tested, in particular by wild-type ASNp and its mutants A289Q, F290C, F290L, and F290V. Traces of di-glucosylated NNHEA were also detected with the monomers F290C, F290L and F290V.
  • Example 12 Determination of Glucosylation Efficacy of HEAA by Certain Enzymes of Example 1
  • acceptor reactions were performed with a panel of 3 wild Glycansucrases, namely: ASR, ⁇ -1,3-BrS and Bacillus subtilis fructosyltransferase shown in Table I.
  • reaction medium is analyzed by HPLC-MS on a Hypercarb column (30 min) to identify the glucosylation products.
  • Table 19 shows the conversion rate of sucrose and the total glucosylation of each of these enzymes.
  • ASR provides mono-glycosylated HEAA, di-glucosylated HEAA, and tri-glucosylated HEAA.
  • ⁇ -1,3-BrS and the D394E mono-mutant of wild-type ASNp only make it possible to obtain mono-glucosylated HEAAs. Only traces of di-glucosylated HEAA are observed with these two enzymes.
  • GBD-CD2 23.7 2.168 2.168 0 0
  • DSR-OK> 95 0.149 0.149 0 0 a-1.2-BrS> 95 1.886 1.781 0.054 0.050 a-1.3-BrS> 95 2.078 1.918 0.160 0
  • SEQ ID NO: 8 series: (Proteins mutated sequences of glucan-saccharase ASNp (Amylosucrase Neisseria polysaccharea) V331X 7

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Macromonomer-Based Addition Polymer (AREA)
  • Polymerisation Methods In General (AREA)
EP16711230.9A 2015-03-19 2016-03-17 Production de synthons glycosyles par voie enzymatique Pending EP3271469A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR1552254A FR3033800B1 (fr) 2015-03-19 2015-03-19 Production de synthons glycosyles par voie enzymatique
PCT/EP2016/055843 WO2016146764A1 (fr) 2015-03-19 2016-03-17 Production de synthons glycosyles par voie enzymatique

Publications (1)

Publication Number Publication Date
EP3271469A1 true EP3271469A1 (fr) 2018-01-24

Family

ID=53794304

Family Applications (1)

Application Number Title Priority Date Filing Date
EP16711230.9A Pending EP3271469A1 (fr) 2015-03-19 2016-03-17 Production de synthons glycosyles par voie enzymatique

Country Status (6)

Country Link
US (1) US10696995B2 (pt)
EP (1) EP3271469A1 (pt)
BR (1) BR112017019907A2 (pt)
CA (1) CA2979123A1 (pt)
FR (1) FR3033800B1 (pt)
WO (1) WO2016146764A1 (pt)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3870616B1 (en) 2018-10-25 2023-10-11 DuPont Industrial Biosciences USA, LLC Alpha-1,3-glucan graft copolymers
CN111100893A (zh) * 2020-01-08 2020-05-05 西安岳达生物科技股份有限公司 一种采用酶催化法制备糖苷的方法
WO2022003305A1 (fr) * 2020-07-02 2022-01-06 Institut National Des Sciences Appliquees De Toulouse Procédé de preparation de polyglucosides d'alkyle et polyglucosides d'alkyle obtenus selon le procédé

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19716731A1 (de) * 1997-04-14 1998-10-15 Biopract Gmbh Verfahren zur enzymatischen Herstellung von Fructosylverbindungen
EP2100966A1 (en) 2008-03-12 2009-09-16 Institut Pasteur Mutants of glycoside hydrolases and uses thereof for synthesizing complex oligosaccharides and disaccharide intermediates
EP2100965A1 (en) 2008-03-12 2009-09-16 Institut Pasteur Mutants of glycoside hydrolases and uses thereof for synthesizing complex oligosaccharides and disaccharide intermediates
US8486664B2 (en) 2010-07-29 2013-07-16 Basf Se Enzymatic production of an ethylenically unsaturated glycoside using polysaccharides
US8580538B2 (en) 2010-07-29 2013-11-12 Basf Se Enzymatic production of an ethylenically unsaturated glycoside

Also Published As

Publication number Publication date
WO2016146764A1 (fr) 2016-09-22
CA2979123A1 (fr) 2016-09-22
FR3033800A1 (fr) 2016-09-23
BR112017019907A2 (pt) 2018-06-12
US20180258456A1 (en) 2018-09-13
FR3033800B1 (fr) 2020-10-23
US10696995B2 (en) 2020-06-30

Similar Documents

Publication Publication Date Title
CA2378562C (fr) Procede de production d'oligosaccharides
EP3122890A1 (fr) Nouveaux flavonoïdes o-alpha-glucosylés sur le cycle b, procédé d'obtention et utilisations
EP3271469A1 (fr) Production de synthons glycosyles par voie enzymatique
EP3011019B1 (fr) Polypeptide ayant la capacite de former des branchements d'unites glucosyle en alpha-1,3 sur un accepteur
FR2897069A1 (fr) Construction de nouveaux variants de l'enzyme dextrane-saccharase dsr-s par ingienerie moleculaire.
EP1109916A1 (en) Nucleic acid molecules encoding an amylosucrase
US20230227489A1 (en) Glycolipopeptide biosurfactants
EP2501734B1 (fr) Polymères comprenant une majorité de monomères amphiphiles destinés au piégeage et à la manipulation de protéines membranaires
EP3174978B1 (fr) Proteine a activite dextrane-saccharase et applications
FR2822163A1 (fr) Molecules d'acides nucleiques codant une dextrane-saccharase catalysant la synthese de dextrane portant des ramifications de type alpha-1,2 osidiques
Ambikapathy et al. The role of proline in osmoregulation in Phytophthora nicotianae
WO2007144475A1 (fr) Procede de production de lipase, cellule de yarrowia lipolytica transformee apte a produire ladite lipase et leurs applications
Klingel et al. Enzymatic synthesis and characterization of mono-, oligo-, and polyglucosylated conjugates of caffeic acid and Gallic acid
WO1996034971A1 (fr) Procede de production de vanilline par bioconversion de precurseurs benzeniques
WO2023222986A1 (fr) Procédé de préparation de glycolipides, glycolipides et leurs utilisations
US20080134363A1 (en) Identification and characterization of a novel alpha-amylase from maize endosperm
FR3092339A1 (fr) Cellule, notamment levure, resistante au phloroglucinol
JPH09271390A (ja) β−１，３−グルカナーゼ反応残物からのオリゴ糖の製造方法
NO20011669L (no) Polyglukan og polyglukanderivater som kan oppnås fra amylosukrase ved biokatalytisk fremstilling i n¶rv¶r av biogenesubstanser
FR2822162A1 (fr) Molecules d'acides nucleiques codant une dextrane-saccharase calatalysant la synthese de dextrane portant des ramifications de type alpha-1,2 osidiques

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20171019

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

RIN1 Information on inventor provided before grant (corrected)

Inventor name: MOULIS, CLAIRE

Inventor name: VUILLEMIN, MARLENE

Inventor name: LAFRAYA, ALVARO

Inventor name: ANDRE, ISABELLE

Inventor name: PERUCH, FREDERIC

Inventor name: MONSAN, PIERRE

Inventor name: REMAUD-SIMEON, MAGALI

Inventor name: GUIEYSSE, DAVID

Inventor name: GRELIER, STEPHANE

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20200224

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: INSTITUT NATIONAL DES SCIENCES APPLIQUEES DE TOULOUSE (INSAT)

Owner name: INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULTURE, L'ALIMENTATION ET L'ENVIRONNEMENT

Owner name: INSTITUT POLYTECHNIQUE DE BORDEAUX

Owner name: UNIVERSITE DE BORDEAUX

Owner name: CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS