EP4176072A1 - Process for preparing alkyl polyglucosides, and alkyl polyglucosides obtained according to the process - Google Patents

Process for preparing alkyl polyglucosides, and alkyl polyglucosides obtained according to the process

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Publication number
EP4176072A1
EP4176072A1 EP21748921.0A EP21748921A EP4176072A1 EP 4176072 A1 EP4176072 A1 EP 4176072A1 EP 21748921 A EP21748921 A EP 21748921A EP 4176072 A1 EP4176072 A1 EP 4176072A1
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EP
European Patent Office
Prior art keywords
seq
alkyl
formula
family
glc
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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
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EP21748921.0A
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German (de)
French (fr)
Inventor
Etienne Severac
Claire Moulis
Davis GUIEYSSE
Sandrine Morel
Magali Remaud-Simeon
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
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 Recherche pour lAgriculture lAlimentation et lEnvironnement
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Publication of EP4176072A1 publication Critical patent/EP4176072A1/en
Pending legal-status Critical Current

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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
    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01005—Dextransucrase (2.4.1.5)

Definitions

  • the present invention relates to the field of the enzymatic preparation of alkyl polyglucosides.
  • the present invention also relates to the alkyl polyglucosides which can be obtained by said process.
  • Surfactants have been used for a very long time in a large number of fields such as pharmaceuticals, cosmetics, hygiene and cleaning products or industrial formulations due to their various properties: foaming, emulsifying, dispersing, detergents, etc.
  • Surfactants are structurally made up of a hydrophobic part and a hydrophilic part, the latter of which may be positively or negatively charged or even be uncharged.
  • Alkyl polyglucosides are glucolipidic surfactants whose polar head consists of glucosyl residues and a more or less long carbon chain typically having 1 to 18 carbon atoms.
  • APGs exhibit interesting surface properties, biodegradability and harmlessness to the skin and to the mucous membranes, which gives them a certain interest for numerous industrial applications.
  • APGs are conventionally synthesized via chemical routes.
  • the most common procedures for synthesizing APGs include the Fischer, Koenig-Knorr, or Schmidt method.
  • the Fischer method is the simplest to implement and is conventionally used industrially when the selectivity of synthesis is not a desired criterion.
  • the Fischer reaction can be carried out in one or two stages.
  • the carbohydrate is dissolved in excess alcohol in the presence of an acid catalyst and at elevated temperature.
  • the glucosyl units constituting APG can be present as a or b anomers, and pyranoside and furanoside isomers.
  • the average degree of polymerization of the carbohydrate part of commercial APGs currently remains below 2 (Ulvenlund et al., 2016) , and is typically between 1, 3 and 1, 6.
  • APGs whose carbohydrate portion is particularly long are required and requested by industrial players.
  • the enzymes most studied in this perspective are enzymes belonging to the family of glycoside hydrolases and more particularly those of the family of b-glycosidases.
  • the latter are enzymes without co-factors which naturally hydrolyze type b osidic bonds present in polysaccharides to produce mono- or oligosaccharides.
  • these enzymes are able to use a glycosyl donor and catalyze the transfer of the glycosyl to the free hydroxyl of an acceptor molecule.
  • alkyl-polyglucosides catalyzed by b-glycosidases generally retains a bond of configuration b between the sugars and the alkyl part (we will speak of b-alkyl-polyglucosides).
  • This synthesis is possible either by hydrolysis reversal (where the glucosyl donor can be cellulose or b-glucans), or by transglycosylation (where the glycosyl donors are natural polysaccharides or activated carbohydrates such as methyl ⁇ - D-glucopyranoside, rNR-bD-glucopyranoside or other aryl-glucosides).
  • Alkyl polyglucosides have been produced from other families of enzymes.
  • Dahiya et al. Successfully produced 1-O-hexyl-aD-mono, di- and tri- a-glucopyranosides from hexanol or octanol and sucrose using a strain of Microbacterium paraoxydans exhibiting amylosucrase-type membrane transglucosylation activity (Dahiya et al., 2015).
  • An alternative route is to use commercial APGs having a carbohydrate part of moderate size (typically degree of polymerization less than 3) and to lengthen the carbohydrate part enzymatically.
  • CGTases cyclodextrin glucano-transferases
  • CGTases belong to the glycoside hydrolase families GH 13 and GH57 and are specific for the formation of ⁇ -1, 4 glucosidic bonds. These enzymes catalyze four types of reactions from starch or amylose: cyclization, coupling, disproportionation and hydrolysis. In the case where a non-carbohydrate molecule carrying one or more glucosyl residues is introduced into the medium, the CGTases are capable of directing their activity towards a coupling and / or disproportionation reaction on this acceptor.
  • the APGs used could only be lengthened by 2 to 3 glucosyl units by the Bacillus stearothermophilus CGTase from dextrins of 10 to 15 glucosyl units.
  • Paul et al used a glucanotransferase of the GH57 family to extend dodecanol maltoside from starch.
  • APGs carrying 24 to 26 glucosyl units (C12G24 to C12G26) were identified by MS analysis (Paul, et al., 2015).
  • Glucan sucrases (GS) of the GH 13 and GH70 families can also be used for such glucosylation reactions.
  • glucansucrases catalyze the synthesis of glucans generally having very high molar masses and varied structures due to the presence of different types of osidic bonds (a-1, 2, a-1, 3, a -1, 4 and / or a- 1, 6) as well as their location in the polymer.
  • Sucrose isomers as well as glucose are also produced from sucrose but generally in much smaller quantities than the polymer.
  • glucansucrases of the GH13 and GH70 families have been used in acceptor reactions to extend the carbohydrate portion of short alkyl monoglucosides (1 to 8 carbon atoms) from sucrose.
  • Figure 1 illustrates the diagram of the enzymatic elongation reaction of the glucosidic part of an alkyl monoglucoside using ⁇ -transglucosylases of the GH70 family active on sucrose.
  • FIG. 2A shows the elongation profiles for the C8G1 substrate obtained with the ⁇ -transglucosylases of the GH70 family which use sucrose as a glucosyl donor.
  • Branching enzymes are used in reaction to 1 U.mL 1
  • Figure 3 compares the profile of the C8G1 elongation reaction catalyzed by GS-C (SEQ ID NO: 6) in the presence of 200 gL 1 of sucrose and 200 gL 1 of ⁇ -cyclodextrins and cyclodextrin glucanotransferases (CGTase) from Bacillus macerans (enzyme not in accordance with the invention) in the presence of 200 gL 1 of ⁇ -cyclodextrins (substrate not in accordance with the invention).
  • CTTase cyclodextrin glucanotransferases
  • Figure 4 compares the profiles of the C8G1 elongation reaction catalyzed by DSR-M D1 glucansucrase alone (SEQ ID NO: 1) and catalyzed by DSR-M D1 glucansucrase and branching enzymes.
  • BRS-A SEQ ID NO: 17
  • BRS-B D1 SEQ ID NO: 18
  • FIG. 5 shows the lengthening profile of alkyl glucosides of increasing sizes obtained with the glucansucrase DSR-M D1 (SEQ ID NO: 1) with initial sucrose concentrations of 200 gL 1 .
  • Inset A: [C8G1] 30mM;
  • Inset B: [C10G1] 10mM; Box C: Triton CG110 (Dow Chemicals, USA) at 15 gL 1 ;
  • the aim of the present invention is to overcome the drawbacks of the prior art and to provide a process for preparing an alkyl polyglucoside by enzymatic catalysis, said process being economical because using sucrose or one of its analogues as substrate and making it possible to obtain a great diversity of alkyl polyglucosides in terms of the size and structure of their glucosidic part.
  • the process of the invention makes it possible to obtain an alkyl polyglucoside with an adjustable number of glucosyl units of 2 to 200 glucosyl units.
  • the process also makes it possible to modulate the linear or branched structure of the carbohydrate part of the alkyl polyglucoside obtained, as well as the nature of the alpha-1, 2 osidic bonds; alpha-1, 3; alpha-1, 4 or alpha-1, 6 linking the glucosyl residues within the carbohydrate part.
  • the process of the invention makes it possible to prepare polyalkylglucosides having an alkyl group of large size, for example greater than 8 carbon atoms.
  • Another aim of the invention is to provide alkyl polyglucosides of great diversity in terms of size and structure of the glucosidic part, and in particular alkyl polyglucosides in which the size of the alkyl group is large, for example greater. with 8 carbon atoms.
  • the first subject of the present invention is a process for the preparation of an alkyl polyglucoside of formula (I)
  • R represents a linear or branched, saturated or unsaturated alkyl group comprising between 8 and 20 carbon atoms
  • [Glc] m - [Glc] n represents a linear or branched carbohydrate part comprising n + m glucosyl units, n + m being between 3 and 200; said process comprising at least one step i) of elongation of the glucosidic chain of an alkyl glucoside of formula (II)
  • R is as defined in formula (I),
  • [Glcj n represents a carbohydrate part comprising n glucosyl units n being between 1 and 15. said step comprising bringing said alkyl glucoside of formula (II) into contact with at least one ⁇ -transglucosylase of the GH70 family in the presence of sucrose or a sucrose analogue.
  • the method makes it possible to control the size, the branched or unbranched structure and the nature of the osidic bonds within the carbohydrate part of the alkyl polyglucoside obtained, and this even when the alkyl group of the alkyl glucoside used as substrate is large.
  • the ⁇ -transglucosylases of the GH70 family are water-soluble enzymes which naturally catalyze hydrophilic substances, and whose activity is a priori strongly limited in the presence of fat.
  • ⁇ -transglucosylases of the GH70 family are capable of accepting as substrate a C8-C20 alkyl glucosyl, that is to say a poorly soluble compound comprising a hydrophobic part unfavorable to the action of this type of enzyme, and to effectively catalyze the elongation of its carbohydrate part.
  • carbohydrate portion denotes a linear or branched polymer consisting of glucosyl units linked together by osidic bonds.
  • glycosidic bond or "glucosidic bond” or “osidic bond” can be used interchangeably and denote the covalent bond which links a glucosyl to another adjacent glucosyl.
  • the carbohydrate part [Glc] m - [Glc] n of the alkyl polyglucoside of formula (I) comprises two carbohydrate fractions [Glc] m and [Glc] n.
  • the carbohydrate fraction [Glc] n corresponds to the n glucosyl units of the carbohydrate part of the alkyl glucoside of formula (II) used as substrate in the process of the invention.
  • the carbohydrate fraction [Glc] m corresponds to the m glucosyl units added to the carbohydrate part [Glc] n of the alkyl glucoside of formula (II) during the glucosidic elongation step of the process of the invention.
  • n + m that is to say the number of glucosyl units in the carbohydrate part [Glc] m - [Glc] n of the alkyl polyglucoside of formula (I) is advantageously between 3 and 150, preferably between 3 and 100, more preferably between 3 and 30, more preferably between 3 and 25.
  • n + m is between 5 and 50, and can in particular be between 5 and 40, in particular still between 5 and 30, in particular between 5 and 25.
  • n + m is between 7 and 200, preferably between 8 and 200, preferably between 9 and 200, preferably between 10 and 200.
  • m that is to say say the number of glucosyl units in the carbohydrate fraction [Glc] m of the carbohydrate part [Glc] m - [Glc] n of the alkyl polyglucoside of formula (I), or in other words the number of glucosyl units added to the carbohydrate part [Glc] n of the alkyl glucoside of formula (II) during the implementation of the process of the invention is advantageously greater than 3.
  • m is between 3 and 150, preferably between 3 and 100, more preferably between 3 and 30.
  • n that is to say the number of glucosyl units in the carbohydrate fraction [Glc] n of the carbohydrate part [Glc] m - [Glc] n of the alkyl polyglucoside of formula (I), or in other words the number of glucosyl units of the carbohydrate part [Glc] n of the alkyl glucoside of formula (II), is advantageously between 1 and 8, preferably between 1 and 5, more preferably between 1 and 3.
  • n is equal to 1 or 2.
  • a bond denotes a covalent bond which links the carbon atom 1 of a glucosyl unit in its a configuration
  • b bond denotes a covalent bond which links carbon atom 1 of a glucosyl unit in its b configuration
  • the glucosyl unit adjacent to the alkyl group is linked to the alkyl group through an a bond or through a b bond, preferably through a b bond.
  • the glucosyl unit adjacent to the alkyl group is in the a or b configuration, preferably in the b configuration.
  • the other glucosyl units of the carbohydrate fraction [Glc] n of the alkyl polyglucoside of formula (I) or of the carbohydrate part [Glc] n alkyl glucoside of formula (II) are linked to each other by a and / or b, preferably by glucosidic bonds a.
  • ⁇ -1,3 bond refers to the covalent bond that links the carbon 1 of one glucosyl unit in its ⁇ configuration and the 3 carbon of another adjacent glucosyl unit.
  • ⁇ -1, 2 bond refers to the covalent bond that links the carbon 1 of one glucosyl unit in its ⁇ configuration and the 2 carbon of another adjacent glucosyl unit.
  • alpha-1,6 bond refers to the covalent bond that links the carbon 1 of one glucosyl unit in its alpha configuration and the 6 carbon of another adjacent glucosyl unit.
  • the alpha-glucosyl units are preferably alpha-D-glucosyl units.
  • glycosidic bonds of an alkyl polyglucoside can be carried out by any method known to those skilled in the art.
  • glucosidic bonds can be analyzed by nuclear magnetic resonance (NMR).
  • Step i) can be carried out with an alkyl glucoside of formula (II) essentially consisting of alkyl monoglucoside molecules, of alkyl diglucloside molecules, or of a mixture thereof, said mixture having advantageously an average degree of polymerization between 1 and 2, preferably between 1.3 and 1.6.
  • the mixture consists essentially of molecules of alkyl monoglucoside and of molecules of alkyl diglucloside, i.e.
  • alkyl monoglucoside and of alkyl diglucloside consists of at least 90% by weight of molecules of alkyl monoglucoside and of alkyl diglucloside, preferably at least 95% by weight of alkyl monoglucoside and alkyl diglucloside molecules, more preferably at least 97% by weight of alkyl monoglucoside and diglucloside molecules alkyl.
  • the remainder can advantageously consist of molecules of alkyl oligoglucosides, i.e. alkyl glucosides, the glucoside of which comprises 3 to 8 glucosyl units, preferably 3 to 5 glucosyl units.
  • the alkyl polyglucoside of formula (I) consists essentially of a mixture of alkyl polyglucoside molecules of formula (I), said mixture advantageously exhibiting an average degree of polymerization of between 3 and 25 , preferably between 5 and 25, preferably between 6 and 25, preferably between 7 and 25, more preferably between 8 and 25.
  • the mixture consists essentially of alkyl polyglucoside molecules of formula (I), i.e. it preferably consists of at least 90% by weight of said alkyl polyglucoside molecules, preferably at least 95% by weight of said polyalkylglucoside molecules, more preferably at least 97% by weight of said polyalkylglucoside molecules.
  • the “average degree of polymerization” accounts for the average distribution of the number of glucosyl units per molecule of alkyl polyglucoside of formula (I) or of alkyl glucoside of formula (II) within a mixture of polyalkyl glucoside molecules of formula (I) or of a mixture of alkyl glucoside molecules of formula (II).
  • this average degree of polymerization is determined from measurements made by nuclear magnetic resonance (NMR).
  • the average degree of polymerization is determined by calculating the ratio (average molar mass of the mixture of alkyl polyglucoside molecules of formula (I) - molar mass of the chain corresponding alkyl): (molar mass of a glucosyl unit).
  • the average degree of polymerization is determined by calculating the ratio (average molar mass of the mixture of alkyl glucoside molecules of formula (II) - molar mass of the corresponding alkyl chain): (molar mass of a glucosyl unit).
  • the alkyl group preferably comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 carbon atoms and at most 20 carbon atoms.
  • the alkyl group R advantageously comprises between 8 and 16 carbon atoms, more preferably comprises 8 and 12 carbon atoms.
  • the alkyl group R comprises between 9 and 20 carbon atoms, preferably comprises between 9 and 16 carbon atoms, more preferably comprises between 9 and 12 carbon atoms.
  • the alkyl group R comprises between 10 and 20 carbon atoms, preferably comprises between 10 and 16 carbon atoms, more preferably comprises between 10 and 12 carbon atoms.
  • the alkyl group R comprises between 13 and 20 carbon atoms, preferably comprises between 16 and 20 carbon atoms.
  • ⁇ -transglucosylases of the GH70 family are capable of catalyzing the elongation of the glucosidic part of an alkyl glucoside of formula (II) having an alkyl group R comprising between 8 and 20 atoms. carbon, that is to say an acceptor substrate having an alkyl pole much more hydrophobic than alkyl glucosides having an alkyl group such as a methyl, ethyl, propyl or butyl group.
  • the process of the invention is particularly advantageous in that it makes it possible to obtain an alkyl polyglucoside of formula (I) having an alkyl group R of between 8 and 20 carbon atoms and a carbohydrate part [Glc ] m - [Glc] n having an n + m as defined above.
  • R represents an alkyl group comprising from 8 to 12 carbon atoms
  • n + m is between 7 and 200, preferably between 8 and 200, preferably between 9 and 200, more preferably between 10 and 200.
  • n + m can advantageously be between 7 and 50, preferably between 8 and 50, preferably between 9 and 50, and more preferably between 10 and 50.
  • R represents an alkyl group comprising from 12 to 20 carbon atoms
  • n + m is between 3 and 200, preferably between 4 and 200, preferably between 5 and 200, and more preferably between 10 and 200.
  • n + m can advantageously be between 3 and 50, preferably between 4 and 50, preferably between 5 and 50, and more preferably between 10 and 50.
  • n + m is between 7 and 200, preferably between 8 and 200, preferably between 9 and 200, more preferably between 10 and 200 and may in particular be between 7 and 50, preferably between 8 and 50, preferably between 9 and 50, and more preferably between 10 and 50; and when R represents an alkyl group comprising from 12 to 20 carbon atoms, n + m is between 3 and 200, preferably between 4 and 200, preferably between 5 and 200, and more preferably between 10 and 200 and may in particular be between 3 and 50, preferably between 4 and 50, preferably between 5 and 50, and more preferably between 10 and 50.
  • ⁇ -transglucosylase denotes an enzyme capable of polymerizing glucosyl units along ⁇ bonds, by catalyzing the transfer of a glucosyl unit from a glucosyl donor sugar to an acceptor compound.
  • the expression “of the GH70 family”, relating to otransglucosylase, means that Ga-transglucosylase according to the invention belongs to the 70 family of glycoside hydrolases according to the CAZy classification (www.cazy.orq) .
  • CAZy standing for "Carbohydrate-Active enZYmes” (abbreviated "CAZy"), is a bioinformatic database for the classification of enzymes active on sugars, ie capable of catalyzing their dissociation or their synthesis according to sequence or sequence homologies. structure, in particular their catalytic and carbohydrate binding moduli.
  • the group of glycoside hydrolases (GH) has 167 families composed of enzymes active on sugars capable of catalyzing reactions of hydrolysis of glycosidic bonds or of transglucosylation.
  • the ⁇ -transglucosylases of the GH70 family are typically produced naturally by lactic acid bacteria of the genera Streptococcus, Leuconostoc (abbreviated “L”), Weisella or Lactobacillus (abbreviated “Lb.”).
  • the ⁇ -transglucosylases of the GH70 family according to the invention are active on sucrose, which means that the ⁇ -transglucosylases according to the invention specifically use sucrose or one of its analogues as a glucosyl donor.
  • the sucrose analogs can in particular be chosen from the group comprising ⁇ -D-glucopyranosyl fluoride (in English "aD-Glucopyranosyl fluoride”), 0-3-D-galactopyranosyl- (1 4) -3-D-fructofuranosyl -aD-glucopyranoside (in English "Lactulosucrose”), p-nitrophenyl aD-glucopyranoside, aD-glucopyranosyl aL-sorbofuranoside and mixtures thereof.
  • Ga-transglucosylase of the GH70 family is selected from one of the ⁇ -transglucosylases described below.
  • the enzyme DSR-M D1 of amino acid sequence SEQ ID NO: 1 is a fragment ranging from the amino acid at position 42 to the amino acid at position 1433 of the amino acid sequence of the form wild type of the DSR-M enzyme (derived from the strain L. citreum NRRL B-1299) having for reference GenBank CDX668951.1.
  • the enzyme DSR-M D5 of amino acid sequence SEQ ID NO: 2 is a fragment ranging from the amino acid at position 421 to the amino acid at position 1315 of the amino acid sequence of the form wild type of the DSR-M enzyme (derived from the strain L. citreum NRRL B-1299) having for reference GenBank CDX668951.1.
  • the DSR-M D5 W624A enzyme of amino acid sequence SEQ ID NO: 3 is a mutant at position 624 of the amino acid sequence fragment SEQ ID NO: 2.
  • the GS-A enzyme with the amino acid sequence SEQ ID NO: 4 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the Lb strain. kunkeei MP2 having for reference GenBank ALJ31412.
  • the GS-B enzyme of amino acid sequence SEQ ID NO: 5 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the Lb strain. animalis DSM 20602 having for reference GenBank KRM57462.1.
  • the GS-C enzyme with the amino acid sequence of SEQ ID NO: 6 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the Lb strain. Apodemi with reference GenBank WP_056957205.
  • the GS-D enzyme of amino acid sequence SEQ ID NO: 7 corresponds to the amino acid sequence of the wild form of the enzyme derived from the Lb strain. animalis DSM 20602 with GenBank reference KRM57463.
  • the GS-E enzyme of amino acid sequence SEQ ID NO: 8 corresponds to the amino acid sequence of the wild form of the enzyme derived from the Lb strain. capillatus DSM 19910 having for reference GenBank KRL03580.
  • the enzyme GS-F D1 of amino acid sequence SEQ ID NO: 9 is a fragment ranging from the amino acid at position 166 to the amino acid at position 1874 of the amino acid sequence of the form wild type of the enzyme derived from the strain L. fallax KCTC 3537 having for reference GenBank WP_010006777.1.
  • the GS-FS enzyme with the amino acid sequence SEQ ID NO: 10 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from Streptococcus salivarus HSISS4 having for reference GenBank ALR80278.
  • the DSR-G enzyme with amino acid sequence SEQ ID NO: 11 corresponds to the amino acid sequence of the wild form of the enzyme derived from Lb. kunkeei DSM 12361 with GenBank reference KRK22577.1.
  • the enzyme DSR-G CD1 of amino acid sequence SEQ ID NO: 12 is a fragment ranging from the amino acid at position 1 to the amino acid at position 1407 of the amino acid sequence of the form wild type of the enzyme derived from the strain Lb. kunkeei DSM 12361 having for reference GenBank KRK22577.
  • the enzyme ASR D1 of amino acid sequence SEQ ID NO: 13 is a fragment ranging from the amino acid at position 1 to the amino acid at position 1425 of the amino acid sequence of the wild form of the enzyme derived from the strain L. mesenteroides NRRL B-1355 having for reference GenBank CAB65910.2.
  • the GTF-SI enzyme with the amino acid sequence SEQ ID NO: 14 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the strain Streptococcus mutans with reference GenBank BAA26114.1.
  • the GTF-J enzyme of amino acid sequence SEQ ID NO: 15 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the strain Streptococcus mutans with reference GenBank AAA26896.1.
  • the enzyme GBDCD2 of amino acid sequence SEQ ID NO: 16 is a fragment ranging from the amino acid at position 1758 to the amino acid at position 2862 of the amino acid sequence of the wild form of l enzyme derived from the strain L. citreum NRRL B-1299 having for reference GenBank CDX66820.1.
  • the BRS-A enzyme of amino acid sequence SEQ ID NO: 17 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the strain L. citreum NRRL B-1299 having for reference GenBank CDX66896. 1.
  • the BRS-B D1 enzyme of amino acid sequence SEQ ID NO: 18 is a fragment ranging from the amino acid at position 39 to the amino acid at position 1313 of the amino acid sequence of the form wild type of the enzyme derived from the strain L. citreum NRRL B-742 having for reference GenBank CDX65123.1.
  • the BRS-C enzyme of amino acid sequence SEQ ID NO: 19 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the strain L. fallax KCTC 3537 having for reference GenBank ZP_08312597.1.
  • the BRS-D D1 enzyme of amino acid sequence SEQ ID NO: 20 is a fragment ranging from the amino acid at position 88 to the amino acid at position 1453 of the amino acid sequence of the form wild type of the enzyme derived from the strain Lb. kunkei EFB6 having for reference GenBank WP_051592287.
  • the BRS-E D1 enzyme of amino acid sequence SEQ ID NO: 21 is a fragment ranging from the amino acid at position 32 to the amino acid at position 1264 of the amino acid sequence of the form wild type of the enzyme derived from the strain L. mesenteroides KFRI-MG having for reference GenBank AHF19404.1.
  • the BRS-F enzyme of amino acid sequence SEQ ID NO: 22 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the strain Fructobacillus tropaeoli having for reference GenBank GAP05007.1.
  • the DSR-G CD2 enzyme of amino acid sequence SEQ ID NO: 23 is a fragment ranging from the amino acid at position 928 to the amino acid at position 2621 of the amino acid sequence of the form wild type of the enzyme derived from the strain Lb. kunkeei DSM 12361 with GenBank reference KRK22577.1.
  • the ⁇ -transglucosylases of the GH70 family according to the invention can be obtained according to methods known to those skilled in the art, in particular by the method consisting in cultivating a cell naturally expressing Ga-transglucosylase or a host cell comprising a transgene encoding Ga -transglucosylase and expressing said ⁇ -transglucosylase, and in extracting said ⁇ -transglucosylase from these cells or from the culture medium in which Ga-transglucosylase has been secreted.
  • Recombinant bacterial strains for example strains of bacillus subtillis or lactobacillus, secreting said ⁇ -transglucosylases of the GH70 family can be used.
  • the ⁇ -transglucosylases of the GH70 family can also be obtained via cell free protein expression systems.
  • Ga-transglucosylase of the GH70 family is preferably a glucansucrase of the GH70 family, a branching sucrase of the GH70 family, or a mixture thereof.
  • GS glucansucrase of the GH70 family
  • GS ⁇ -transglucosylase of the GH70 family capable of catalyzing the synthesis of ⁇ -glucans, ie of polysaccharides composed exclusively of. glucosyl units linked together by alpha bonds.
  • dextransucrases (sometimes abbreviated as DSR), which synthesize dextrans, ie glucans in which the residues of the main chain are linked mainly in alpha-1,6; reuteranesucrases, which synthesize reuterans, ie glucans whose main chain residues are linked in alpha-1, 4 and alpha-1, 6; mutans-sucrases, which synthesize mutans, i.e.
  • DSR dextransucrases
  • G ⁇ -transglucosylase of the GH70 family is not an alternansucrase.
  • branching sucrase of the GH70 family in English “branching sucrase”, sometimes abbreviated as BRS
  • ⁇ -transglucosylase of the GH70 family with branching activity or “branching enzyme of the family GH70 ”are used interchangeably and refer to an ⁇ -transglucosylase of the GH70 family capable of catalyzing the addition of glucosyl units in the main chain of a pre-existing dextran-type glucan forming branches (or in other words branches ).
  • the nature of the branching bond (alpha-1, 2; alpha-1, 3; alpha-1, 4 or alpha-1, 6 bonds) and the length of the chains of glucosyl units constituting the branches vary according to the specificity of the branching sucrase considered.
  • the branching sucrases of the GH70 family according to the invention catalyze branches preferably comprising 1 to 3 glucosyl units, preferably 1 or 2 glucosyl units according to alpha-1, 2 and / or alpha-bonds.
  • the carbohydrate part of the alkyl polyglucoside of formula (I) is in the form of a linear chain comprising branches comprising from 1 to 3 glucosyl units, preferably 1 or 2 glucosyl units, said branches being linked to the linear chain via alpha-1, 2 bonds and / or alpha-1, 3 bonds.
  • ⁇ -transglucosylases of the GH70 family are capable of transferring glucosyl units onto the carbohydrate part of an alkyl glucoside of formula (II) as described above via a mechanism glucosidic elongation.
  • Ga-transglucosylase of the GH70 family is a glucansucrase of the GH70 family.
  • Ga-transglucosylase of the GH70 family is a glucansucrase of the GH70 family
  • an alkyl polyglucoside is advantageously obtained, the carbohydrate part of which comprises at least 50%, preferably 60%, more preferably 80% of bonds chosen from the group comprising alpha-1, 6 bonds and alpha- bonds
  • the remainder of the bonds being advantageously alpha-1, 4 bonds.
  • the glucansucrases of the GH70 family according to the invention make it possible to obtain an alkyl polyglucoside having a long glucosidic chain, with n + m as defined above.
  • the glucansucrase of the GH70 family of the invention preferably has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,, SEQ ID NO : 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.
  • the glucansucrases of the GH70 family of the invention are capable of extending the alkyl glucosides of formula (II) by more than 3 glucosyl units from sucrose.
  • the glucansucrase of the GH70 family according to the invention has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO : 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 12, an alkyl polyglucoside of formula (I) is obtained in which the carbohydrate part comprises at least 80% of bonds alpha-1, 6, the remainder of the bonds being advantageously alpha-1, 3 bonds.
  • the glucansucrase of the GH70 family according to the invention has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO : 5, SEQ ID NO: 6, and SEQ ID NO: 12, an alkyl polyglucoside of formula (I) is obtained, the carbohydrate part of which comprises at least 90%, preferably at least 95% of alpha-1 bonds, 6, the rest of the bonds being advantageously alpha-1, 3 bonds.
  • the glucansucrase of the GH70 family according to the invention has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 15, a alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 90% of alpha-1, 3 bonds, the remainder of the bonds advantageously being alpha-1, 6 bonds.
  • the glucansucrase of the GH70 family according to the invention has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 8 and SEQ ID NO: 9, an alkyl polyglucoside of formula is obtained.
  • the carbohydrate part comprises at least 70% of alpha-1, 6 bonds, the remainder of the bonds advantageously being alpha-1, 3 bonds.
  • glucansucrase of the GH70 family according to the invention has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 4, an alkyl polyglucoside of formula (I) is obtained, the carbohydrate part of which comprises at least 50% of alpha-1, 6 bonds, the remainder of the bonds advantageously being alpha-1, 4 bonds.
  • Ga-transglucosylase of the GH70 family according to the invention is a GH70 branching sucrase.
  • an alkyl polyglucoside of formula (I) is advantageously obtained, the carbohydrate part of which comprises at least 50%, preferably 60%, more preferably 80% of bonds in the group comprising alpha-1, 2, alpha-1, 3 and mixtures thereof; the rest of the bonds being advantageously alpha-1,6 bonds.
  • the branching sucrase of the GH70 family according to the invention preferably has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23.
  • the branching sucrase of the GH70 family has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22, obtains an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 80% of alpha-1, 2 bonds, preferably 100% of alpha-1, 2 bonds.
  • the GH70 branching sucrase has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 23, an alkyl polyglucoside of formula ( I) in which the carbohydrate part comprises at least 80%, preferably 90%, more preferably 95%, more preferably 98%, of alpha-1, 3 bonds, more preferably 100% of alpha-1, 3 bonds.
  • the GH70 branching sucrase has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 21, an alkyl polyglucoside of formula (I) is obtained, the carbohydrate part of which comprises at least 60% of bonds alpha-1, 2, the remainder of the bonds being advantageously alpha-1, 6 bonds.
  • Step i) can be carried out by bringing the alkyl glucoside of formula (II) into contact simultaneously or successively with several ⁇ -transglucosylases of the GH70 family.
  • step i) comprises a sub-step io) of bringing the alkyl glucoside of formula (II) into contact with a mixture of one or more glucansucrase (s) GH70 and of one or more GH70 branching enzyme (s).
  • a ratio (glucansucrase): (branching sucrase), expressed in units of enzymatic activity, of between 0.01 and 10, is preferably used, the activities of each of the enzymes used varying advantageously between 0.2 and 2 U. ml 1 .
  • step i) comprises step h) of contacting the alkyl glucoside of formula (II) with one or more glucansucrase (s) of the GH70 family.
  • this embodiment firstly makes it possible to promote the elongation of the carbohydrate part in the form of a long linear chain.
  • step i) comprises bringing the alkyl glucoside of formula (II) into contact with one or more glucansucrase (s) of the GH70 family, then a step b) of bringing the alkyl glucoside into contact.
  • this embodiment firstly makes it possible to promote the elongation of the carbohydrate part in the form of a long linear chain, then secondly to promote the elongation of the carbohydrate part in the form of ramifications.
  • This embodiment also has the advantage of increasing the diversity of chemical structures obtained.
  • branching sucrases of the GH70 family in fact makes it possible to control the number of branches introduced, as a function of the reaction conditions, so as to obtain an alkyl polyglucoside of formula (I) of which the carbohydrate part comprises at least 50% of alpha-1, 6 bonds, the rest of the glucosidic bonds being alpha-1, 3 bonds and / or alpha-1, 2 bonds, the sum of the percentages of alpha-1, 6 bonds, of the percentages of alpha-1, 3 bonds and percentages of alpha-1 bonds, 2 being equal to 100%.
  • an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 60% of alpha-1, 6 bonds, at least 2% of alpha-1, 3 bonds and at least 2% of bonds alpha-1, 6, preferably between 2% and 35% of alpha-1, 3 bonds and at least 1% of alpha-1, 2 bonds, preferably between 2% and 35% of alpha-1, 2 bonds, the sum of the percentages of alpha-1, 6 bonds, of the percentages of alpha-1, 3 bonds and of percentages of alpha-1 bonds, 2 being equal to 100%.
  • step i) is carried out in solution at a controlled pH, in particular by the use of a buffer solution.
  • Step i) is advantageously carried out at a pH value of between 5 and 8.
  • Step i) is advantageously carried out with an initial concentration of sucrose or of a sucrose analogue of between 20 and 660 gL 1 .
  • Step i) is preferably carried out with an alkyl polyglucoside molar ratio of formula (II): sucrose or sucrose analog of between 0.001 and 10, preferably between 0.001 and 5, preferably 0.001 and 0.3, of preferably between 0.002 and 0.006.
  • the inventors have shown that the elongation of the chain can be modulated depending on the molar ratio of alkyl polyglucoside of formula (II): sucrose or sucrose analog. Without wishing to be bound by a particular theory, the inventors consider that the greater the molar ratio of alkyl polyglucosides of formula (II): sucrose or sucrose analog, the less sucrose is available to be a donor and therefore the less the acceptor is. extension.
  • step i) is carried out with an alkyl polyglucoside molar ratio of formula (II): sucrose or sucrose analog of between 0.05 and 5.
  • Step i) is preferably carried out at a temperature between 10 ° C and 80 ° C.
  • step i) is carried out with Ga-transglucosylase of the GH70 family in solid form, in solution, in suspension, or immobilized.
  • the method can advantageously further comprise a step ii) of purification of the alkyl polyglucoside of formula (I) obtained at the end of step i).
  • the alkyl polyglucoside of formula (I) obtained by carrying out the process as defined according to the first subject of the invention, in particular at the end of step i) constitutes a second subject of the invention .
  • a second subject of the invention is therefore an alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention, said alkyl polyglucoside being characterized in that it is of formula ( I):
  • R represents a linear or branched, saturated or unsaturated alkyl group comprising between 8 and 20 carbon atoms
  • [Glc] m - [Glc] n represents a linear or branched carbohydrate part comprising n + m glucosyl units, n + m being between 3 and 200.
  • the alkyl polyglucoside of formula (I) capable of being obtained by carrying out the process in accordance with the first subject of the invention may exhibit great structural diversity, in particular at the level of its carbohydrate part, in particular in terms of size, structure (branched or not) and nature of the osidic bonds.
  • the alkyl polyglucoside of formula (I) of the second subject of the invention is as defined in detail in the first subject of the invention, in particular, the number of carbon atoms of the alkyl group R, the number n + m of glucosyl units within the carbohydrate part [Glc] m - [Glc] n , as well as the nature of the bonds within the fractions of the carbohydrate part [Glc] m - [Glc] n and that of the bond between the alkyl group R and the carbohydrate moiety [Glc] n are as previously mentioned.
  • the alkyl polyglucoside of formula (I) obtainable by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which R represents an alkyl group comprising from 8 to 12 carbon atoms and in which the number n + m is between 7 and 200, preferably between 8 and 200, preferably between 9 and 200, more preferably between 10 and 200.
  • n + m can in particular be between 7 and 50, preferably between 8 and 50, preferably between 9 and 50, and more preferably between 10 and 50.
  • the alkyl polyglucoside of formula (I) obtainable by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which R represents an alkyl group comprising from 12 to 20 carbon atoms and in which n + m is between 3 and 200, preferably between 4 and 200, preferably between 5 and 200, and more preferably between 10 and 200.
  • n + m can in particular be between 3 and 50, preferably between 4 and 50, preferably between 5 and 50, and more preferably between 10 and 50.
  • the alkyl polyglucoside of formula (I) obtainable by the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which:
  • n + m is between 7 and 200, preferably between 8 and 200, preferably between 9 and 200, more preferably between 10 and 200 and n + m may in particular be between 7 and 50, preferably between 8 and 50, preferably between 9 and 50, and more preferably between 10 and 50; and
  • n + m is between 3 and 200, preferably between 4 and 200, preferably between 5 and 200, and more preferably between 10 and 200 and can in particular be between 3 and 50, preferably between 4 and 50, preferably between 5 and 50, and more preferably between 10 and 50.
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) consisting essentially of a mixture of molecules of alkyl polyglucoside of formula (I), said mixture exhibiting an average degree of polymerization between 3 and 25, preferably between 5 and 25, preferably between 6 and 25, preferably between 7 and 25, preferably still between 8 and 25.
  • This mixture consists essentially of alkyl polyglucoside molecules of formula (I), that is to say that it consists of at least 90% by weight of said alkyl polyglucosides, preferably at least 95% by weight of said alkyl polyglucosides, more preferably at least 97% by weight of said alkyl polyglucosides.
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 50 %, preferably 60%, more preferably 80% of alpha-1, 6 bonds or alpha-1, 3 bonds, the remainder of the bonds advantageously being alpha-1, 4 bonds and / or alpha-1 bond, 2.
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 50 %, preferably at least 70% of alpha-1, 6 bonds, the remainder of the bonds being advantageously alpha-1, 3 bonds.
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 60 % alpha-1, 6 bonds, at least 2% alpha-1, 3 bonds and at least 2% alpha-1, 6 bonds, preferably between 2% and 35% alpha-1, 3 bonds and at least minus 1% alpha- bonds
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 90 % alpha-1, 3 bonds, the remainder of the bonds being advantageously alpha-1, 6 bonds.
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 50 % alpha-1, 6 bonds, the remainder of the bonds being advantageously alpha-1, 4 bonds.
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 70 % of alpha-1, 6 bonds, the remainder of the bonds being advantageously alpha-1, 3 bonds.
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 80 % alpha-1, 2 bonds, the remainder of the bonds being advantageously alpha-1, 3 bonds.
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 80 %, preferably 90%, more preferably 95%, more preferably 98%, alpha- bonds
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 80 %, preferably 90%, more preferably 95%, more preferably 98%, alpha- bonds
  • the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 60 % alpha-1, 2 bonds, the remainder of the bonds being advantageously alpha-1, 6 bonds.
  • the third subject of the invention is the use of an alkyl polyglucoside obtainable according to the process according to the invention or as defined in the second subject of the invention, as a surfactant.
  • a fourth subject of the invention is the use of an ⁇ -transglucosylase of the GH70 family as defined in the first subject of the invention, in particular of a glucansucrase of the GH70 family or of a branching sucrase. of the GH70 family as defined in the first subject of the invention, for the glucosidic elongation of an alkyl glucoside, in particular of an alkyl glucoside of formula (II) as defined in the first subject of 'invention.
  • Example 1 Enzymes, organisms of origin and specificity
  • sequence SEQ ID NO: 3 corresponds to the mutant of the DSR-M D5 enzyme at position 624 where the wild-type tryptophan is replaced by an alanine.
  • Table 1 GH70 ⁇ -transglucosylases and specificities of bonds during the synthesis of the natural polymer.
  • P polymerase i.e. glucansucrase GH70;
  • B GH70 branching sucrase;
  • Bif Bifunctional (enzyme having two catalytic domains);
  • nd not determined.
  • the genes encoding the enzymes mentioned above are cloned into vectors allowing recombinant expression in E. coli, under the control of pBad or pET promoters.
  • Recombinant enzymes are produced from the cells of E. coli BL21 DE3 A1 or BL21 DE3 Star transformed with the plasmid containing the gene for the targeted enzymes (see Table 2).
  • Table 2 plasmids of the enzymes used and expression systems adapted to the different enzymes.
  • Gly Glycerol
  • Glu Glucose
  • a-Lac a-Lactose
  • L-Ara L- Arabinose
  • OD optical density
  • the culture media are centrifuged for 15 min at 6500 rpm and at a temperature of 4 ° C.
  • Cell pellets are concentrated to an OD of 80 in activity buffer (see Table 2).
  • the cells are then broken with ultrasound according to the following protocol: 5 cycles of 20 seconds at 30% of the maximum power of the probe, cold (ice bath), spaced 4 minutes apart in ice.
  • the Sonication supernatants containing the soluble enzymes of interest are then recovered after 30 minutes of centrifugation (10,000 rpm, 10 ° C) and stored at 4 ° C.
  • Example 3 Determination of enzymatic activity by determination of reducing sugars in DNS.
  • the enzymatic activity of glucansucrases and branching sucrases is determined by measuring the initial rate of production of reducing sugars using the dinitrosalicilic acid (DNS) method (Miller, 1959).
  • DNS dinitrosalicilic acid
  • One enzymatic unit represents the quantity of enzyme which releases one pmole of fructose per minute, at 30 ° C., for an initial sucrose concentration of 100 gL 1 under the conditions of adequate activity buffer.
  • Acceptor reactions are carried out in a volume of between 1 mL and 15 mL and for a final sucrose concentration of between 146 and 730 mM.
  • concentrations of alkylpoly-glucosides are dependent on their solubility and vary between: 20 and 50 mM for octyl-monoglucoside,
  • the reaction is initiated by the addition of a volume of cell lysate sufficient to obtain an enzymatic activity in reaction of 1 U.mL 1 .
  • the reactions are incubated at 37 ° C. and stirred at 800 rpm. After 24 h, the enzymes are denatured at 95 ° C. for 5 min.
  • the reactions are stored at -18 ° C. before analysis of the reaction products by high performance liquid chromatography (HPLC).
  • a step of prepurification of the glucosylated hydroxy-lipids obtained from 11-hydroxy-undecanoic acid is carried out by flash chromatography using a REVELERIS® X2 Flash Chromatography System (GRACE, USA) equipped with a C18 column of 80 g.
  • the glucosylation products are separated from the residual free sugars under the following conditions:
  • fractions containing the reaction products eluted between 4 and 10 VC are collected, partially evaporated on a rotary evaporator and then lyophilized. Samples are stored at room temperature and protected from moisture.
  • reaction media are diluted halfway in absolute ethanol. This dilution allows, among other things, the elimination of potential polymers of high molar mass by precipitation.
  • the separation of the lipid acceptors and their glucosylated forms is carried out by reverse phase chromatography with a Synergi TM Fusion-RP column (porosity of 80 ⁇ , particle size of 4 ⁇ m, C18 grafting with polar termination, Phenomenex, USA). This column is maintained at 30 ° C. on a Thermo U3000 HPLC system coupled to a Corona CAD Véo detector (Charged Aerosol Detector) (Thermo Scientific, USA). The nebulization temperature is set at 50 ° C and the filter set at 3.2 seconds.
  • the mobile phase is composed of a mixture of ultrapure water (solvent A) / acetonitrile of HPLC grade (solvent B) both containing 0.05% (v / v) of formic acid. Elution is carried out at a flow rate of 1 mL.min 1 according to the following gradient:
  • a first elution phase with 0% (v / v) of path B allows the elimination of residual simple sugars (fructose, glucose, leucrose, residual sucrose or possible short oligosaccharides);
  • a second phase of gradient ranging from 0% is carried out at 100% of path B for 25 minutes makes it possible to separate the various glucosylated lipid compounds; - A final phase of 5 minutes at 100% of channel B allows the regeneration of the column.
  • the characterization of the glucosylation products of the different APGs is carried out by NMR.
  • the products are diluted in D2O and in the presence of deuterated sodium trimethylsilylpropanoate (TSP-d4) used as internal reference.
  • TSP-d4 deuterated sodium trimethylsilylpropanoate
  • the 1 H spectra were recorded on Bruker Avance 500MHz equipment at 298K with a 5 mm z-gradient TBI probe. The data were acquired and processed with TopSpin 3 software.
  • 26 ⁇ -transglucosylases of the GH70 family were tested for their ability to lengthen alkyl-glycosides of different structures and sizes:
  • Triton CG110 (DuPont, USA), mixture mainly containing CsGi, C10G1 and mainly APGs of higher degree of polymerization.
  • the enzymes tested are listed in Table 3. They were all produced in recombinant form and expressed in Escherichia coli.
  • the enzymatic extracts obtained by fermentation are used crude after breaking the cells and centrifuging the cell debris.
  • the acceptor reactions involving GSs were carried out in a volume of 1 to 10 mL at variable concentrations of APG depending on their solubility in water (between 5 mM for C12G1 and 30 mM for CsGi) and concentrations of Sucrose varying between 146 mM (50 gL 1 ) and 1316 mM (450 gL ⁇ 1 ).
  • the separation of the various reaction products is carried out by reverse phase chromatography with a Synergi TM Fusion-RP 250 mm ⁇ 2 mm column (porosity: 80 ⁇ , particle size: 4 ⁇ m, Phenomenex, USA). This column is maintained at 30 ° C on a Thermo Ultimate 3000 HPLC system equipped with a Corona Véo detector.
  • the mobile phase is composed of an ultrapure water (solvent A) / acetonitrile mixture of LC-MS grade (solvent B) each containing 0.05% (v / v) formic acid.
  • solvent B a linear gradient, in solvent B, defined as follows: 0 min, 0% (v / v); 5 min, 0%; 35 min, 100%.
  • FIG. 2A Figures 2A and [Fig. 2B] 2B shows the elongation profiles of the C8G1 substrate obtained with the ⁇ -transglucosylases of the GH70 family which use sucrose as a substrate.
  • FIG. 2B Branching enzymes [Fig. 2B] (FIG. 2B) allow the addition of 1 to 7 glucosyl units to the CsGi depending on the enzymes considered, with conversion rates varying from 20% to 60% under the conditions tested.
  • glucansucrases FIG. 2A
  • FIG. 2A the sizes of the APGs obtained are much larger, the products carrying a large number of glucosyl units.
  • FIG. 3 shows the elongation profiles of CsGi obtained with i) the glucansucrase GS-C (SEQ ID NO: 6) in the presence of sucrose and ⁇ -cyclodextrins, and ii) the CGTase of Bacillus macerans (not in accordance with the invention) in the presence of ⁇ -cyclodextrins under conditions similar to the work of Svenson et al. (Svenson et al., 2009).
  • the APGs produced by the glucansucrases can themselves be used as substrates by the branching sucrases.
  • Figure 5 shows the elongation profiles obtained with the DSR-M D1 enzyme (SEQ ID NO: 1) with substrates of different geometries in terms of alkyl chain size (C8 to C16) and carbohydrate head ( mono, diglucoside or oligoglucosides).
  • acceptor alkyl glucoside molar ratio ie alkyl glucoside of formula (II): sucrose or sucrose analog on the elongation profile of the alkyl glucoside is studied.
  • acceptor CsGi or C12G2 by the enzyme DSR-M D1 (SEQ ID NO: 1).
  • the CsGi or C12G2 concentrations are set at 10 g / L (34.3 mM and 19.6 mM respectively) and the sucrose concentration is modulated between 233.4 g / L (684 mM ) and 2.3 g / L (6.8 mM) such that the molar ratio of acceptor alkyl glucosides: sucrose is 0.05; 0.1; 0.25; 0.5; from 1 ; of 2; or 5.
  • control reactions represent mixtures of C8G1 or C12G2 at a concentration of 10 g / L and 233.4g / L of sucrose without addition of enzyme
  • Figure 6 shows the elongation profiles obtained with the DSR-M D1 enzyme (SEQ ID NO: 1) with decreasing molar ratios between CsGi and Sucrose ( Figure 6A) and between C12G2 and sucrose ( Figure 6B).
  • the results show the effect of the alkyl acceptor glucoside: sucrose ratio on the elongation of the alkyl acceptor glucoside: the higher the molar ratio, the less the acceptor alkyl glucoside is extended.
  • the results therefore show that the average DP of the alkyl polyglucoside obtained is inversely proportional to the molar ratio of alkyl acceptor glucoside: sucrose.
  • Figure 6A shows that for CsGi, the elongation profile varies significantly for molar ratios between 0.05 and 2, while molar ratios greater than 2 have little influence on the elongation profile. /
  • Figure 6B shows that for C12G2, the elongation profile varies significantly for molar ratios between 0.05 and 5.
  • the glucansucrase is DSR-M D1 (SEQ ID NO: 1) and the branching sucrase BRS-A (SEQ ID NO: 18) or BRS-B D1 (SEQ ID NO: 19 ).
  • the concentration of CsGi is fixed at 10 g / L and the concentration of sucrose at 10Og / L.
  • the quantities of DSR-M D1 and BRS-A or BSR-B are modulated so that the ratio DSR-M D1: BRS-A and DSR-M D1: BRS-B D1 expressed in units of enzymatic activity is of 0.1; 0.25; 0.5; from 1 ; of 2; 5 or 10, while respecting a total activity (DSR-M D1 + BRS-A or BRS-B D1) of 1 U / mL.
  • control reactions represent the mixture of C8G1 at a concentration of 10 g / L and 100 g / L of sucrose without addition of enzyme
  • Figure 7 shows the elongation profiles obtained with the DSR-M D1 enzyme (SEQ ID NO: 1) in co-catalysis with the branching enzyme BRS-A (SEQ ID NO: 18) ( Figure 7A ) or with the BRS-B D1 branching enzyme (SEQ ID NO: 19) ( Figure 7B).
  • the results show the effect of the glucansucrase activity ratio: branching sucrase: the higher this activity ratio, the greater the acceptor alkyl glucoside. extension.
  • the results therefore show that the average DP of the alkyl polyglucoside obtained is proportional to the ratio of glucan-sucrase activity: branching sucrase.
  • thermostable a-transglucosidase from Talaromyces duponti-application to a-alkylglucoside synthesis. Enzyme and Microbial Technology 23, 83-90 (1998).
  • compositions including eg mixing lignocellulosic annual and perennial plant materials with water, contacting solution with strain or enzyme having xylanase activity to give composition of alkyl polypentosides. (2012).

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Abstract

The present invention relates to a process for preparing an alkyl polyglucoside by enzymatic catalysis, using sucrose or an analogue thereof as substrate and making it possible to obtain a large diversity of alkyl polyglucosides in terms of size and structure of the glucoside part thereof, making possible the obtaining of an alkyl polyglucoside with a number of glucosyl units that can be adjusted from 2 to 200 glucosyl units. The process also makes it possible to adjust the linear or branched structure of the carbohydrate part of the alkyl polyglucoside obtained, and also the nature of the glycosidic bonds linking the glucose residues within the carbohydrate part.

Description

DESCRIPTION DESCRIPTION
Titre : PROCÉDÉ DE PREPARATION DE POLYGLUCOSIDES D’ALKYLE ET POLYGLUCOSIDES D’ALKYLE OBTENUS SELON LE PROCÉDÉ Title: PROCESS FOR THE PREPARATION OF ALKYL POLYGLUCOSIDES AND ALKYL POLYGLUCOSIDES OBTAINED BY THE PROCESS
DOMAINE DE L'INVENTION FIELD OF THE INVENTION
La présente invention se rapporte au domaine de la préparation par voie enzymatique de polyglucosides d’alkyle. La présente invention se rapporte également aux polyglucosides d’alkyle susceptibles d’être obtenus par ledit procédé. The present invention relates to the field of the enzymatic preparation of alkyl polyglucosides. The present invention also relates to the alkyl polyglucosides which can be obtained by said process.
ARRIÈRE PLAN TECHNOLOGIQUE TECHNOLOGICAL BACKGROUND
Les surfactants sont utilisés depuis très longtemps dans un grand nombre de domaines tels que la pharmaceutique, cosmétique, les produits d’hygiène et d’entretien ou des formulations industrielles du fait de leurs propriétés variées : moussantes, émulsifiantes, dispersantes, détergentes, etc. Surfactants have been used for a very long time in a large number of fields such as pharmaceuticals, cosmetics, hygiene and cleaning products or industrial formulations due to their various properties: foaming, emulsifying, dispersing, detergents, etc.
Les surfactants sont structurellement constitués d’une partie hydrophobe et d’une partie hydrophile, cette dernière pouvant être chargée positivement ou négativement ou encore être non chargée. Surfactants are structurally made up of a hydrophobic part and a hydrophilic part, the latter of which may be positively or negatively charged or even be uncharged.
Les polyglucosides d’alkyle (en anglais « alkyl polyglucosides », abrégé APGs) sont des tensioactifs glucolipidiques dont la tête polaire est constituée de résidus glucosyle et d’une chaîne carbonée plus ou moins longue ayant typiquement 1 à 18 atomes de carbone.Alkyl polyglucosides (in English "alkyl polyglucosides", abbreviated APGs) are glucolipidic surfactants whose polar head consists of glucosyl residues and a more or less long carbon chain typically having 1 to 18 carbon atoms.
Les APGs présentent des propriétés de surface intéressantes, une biodégradabilité et une innocuité vis à vis de la peau et des muqueuses ce qui leur confère un intérêt certain pour de nombreuses applications industrielles. APGs exhibit interesting surface properties, biodegradability and harmlessness to the skin and to the mucous membranes, which gives them a certain interest for numerous industrial applications.
Les APGs sont synthétisés classiquement via des voies chimiques. Les procédures les plus communes de synthèse d’APGs incluent la méthode de Fischer, de Koenig-Knorr ou celles de Schmidt. APGs are conventionally synthesized via chemical routes. The most common procedures for synthesizing APGs include the Fischer, Koenig-Knorr, or Schmidt method.
La méthode de Fischer est la plus simple à mettre en oeuvre et est classiquement utilisée industriellement lorsque la sélectivité de synthèse n’est pas un critère recherché. Selon la nature du carbohydrate et de l’alcool gras, la réaction de Fischer peut être conduite en une ou deux étapes. Le carbohydrate est solubilisé dans un excès d’alcool en présence d’un catalyseur acide et à température élevée. The Fischer method is the simplest to implement and is conventionally used industrially when the selectivity of synthesis is not a desired criterion. Depending on the nature of the carbohydrate and the fatty alcohol, the Fischer reaction can be carried out in one or two stages. The carbohydrate is dissolved in excess alcohol in the presence of an acid catalyst and at elevated temperature.
L’obtention d’APGs dont la chaîne carbonée est de grande taille nécessite une étape dans laquelle un alkyle de petite taille, typiquement butyl-, est échangé avec un alcool dont la chaîne carbonée est de plus grande taille permettant ainsi de contourner les problèmes de solubilité des réactifs. La synthèse de Fischer conduit à l’obtention d’un mélange pouvant être extrêmement complexe de mono-, di-, tri- et oligoglucosides d’alkyle. Obtaining APGs whose carbon chain is large requires a step in which an alkyl of small size, typically butyl-, is exchanged with an alcohol whose carbon chain is larger, thus making it possible to bypass the problems of solubility of reagents. The Fischer synthesis leads to the production of a mixture which can be extremely complex of alkyl mono-, di-, tri- and oligoglucosides.
Les unités glucosyle constituant l’APG peuvent être présentes sous la forme d’anomères a ou b, et d’isomères pyranosides et furanosides. The glucosyl units constituting APG can be present as a or b anomers, and pyranoside and furanoside isomers.
Pour des raisons de cinétiques réactionnelles et d’incompatibilités des produits de réaction (faible solubilité des carbohydrates dans les alcools gras), le degré de polymérisation moyen de la partie glucidique des APGs commerciaux reste actuellement inférieur à 2 (Ulvenlund ét al., 2016), et est typiquement compris entre 1 ,3 et 1 ,6. For reasons of reaction kinetics and incompatibilities of the reaction products (low solubility of carbohydrates in fatty alcohols), the average degree of polymerization of the carbohydrate part of commercial APGs currently remains below 2 (Ulvenlund et al., 2016) , and is typically between 1, 3 and 1, 6.
Or, pour certaines applications, des APGs dont la partie glucidique est particulièrement longue sont requis et demandés par les acteurs industriels. However, for certain applications, APGs whose carbohydrate portion is particularly long are required and requested by industrial players.
Au vu de ce qui précède, le développement de voies de synthèse efficaces permettant d'accéder à des APGs dont la partie glucidique est particulièrement longue est un enjeu majeur. In view of the foregoing, the development of efficient synthetic routes allowing access to APGs in which the carbohydrate portion is particularly long is a major issue.
Ces dernières années, des efforts notables ont été consacrés au développement de voies enzymatiques de synthèse d’APGs. In recent years, significant efforts have been devoted to the development of enzyme pathways for the synthesis of APGs.
Les enzymes les plus étudiées dans cette optique sont des enzymes appartenant à la famille des glycoside hydrolases et plus particulièrement celles de la famille des b- glycosidases. Ces dernières sont des enzymes sans co-facteurs qui hydrolysent naturellement des liaisons osidiques de type b présentes dans les polysaccharides pour produire des mono- ou des oligosacharides. In vitro, ces enzymes sont capables d’utiliser un donneur de glycosylé et de catalyser le transfert du glycosylé sur l’hydroxyle libre d’une molécule acceptrice. The enzymes most studied in this perspective are enzymes belonging to the family of glycoside hydrolases and more particularly those of the family of b-glycosidases. The latter are enzymes without co-factors which naturally hydrolyze type b osidic bonds present in polysaccharides to produce mono- or oligosaccharides. In vitro, these enzymes are able to use a glycosyl donor and catalyze the transfer of the glycosyl to the free hydroxyl of an acceptor molecule.
La synthèse des alkyl-polyglucosides catalysée par les b-glycosidases conserve en général une liaison de configuration b entre les sucres et la partie alkyle (on parlera de b- alkyl-polyglucosides). Cette synthèse est possible soit par inversion d’hydrolyse (où le donneur de glucosyle peut être la cellulose ou des b-glucanes), soit par transglycosylation (où les donneurs de glycosylé sont des polysaccharides naturels ou des carbohydrates activés tels que le méthyl^-D-glucopyranoside, le rNR-b-D- glucopyranoside ou autres aryl-glucosides). The synthesis of alkyl-polyglucosides catalyzed by b-glycosidases generally retains a bond of configuration b between the sugars and the alkyl part (we will speak of b-alkyl-polyglucosides). This synthesis is possible either by hydrolysis reversal (where the glucosyl donor can be cellulose or b-glucans), or by transglycosylation (where the glycosyl donors are natural polysaccharides or activated carbohydrates such as methyl ^ - D-glucopyranoside, rNR-bD-glucopyranoside or other aryl-glucosides).
D’autres familles d’enzymes ont permis l’obtention d’alkyl-polyglucosides. Alkyl polyglucosides have been produced from other families of enzymes.
Bousquet et al. ont montré la possibilité d’obtenir des a-alkyl-polyglucosides en utilisant une a-transglucosylase à partir de maltodextrines sur du butanol (Bousquet et al., 1998 ; Bousquet ét al., 1999). Bousquet et al. have shown the possibility of obtaining α-alkyl-polyglucosides using an α-transglucosylase from maltodextrins on butanol (Bousquet et al., 1998; Bousquet et al., 1999).
Dahiya et collaborateurs ont réussi à produire des 1-O-hexyl-a-D-mono, di- et tri- a- glucopyranosides à partir d’hexanol ou d’octanol et de saccharose en utilisant une souche de Microbacterium paraoxydans présentant une activité de transglucosylation membranaire de type amylo-saccharase (Dahiya ét al., 2015). Dahiya et al. Successfully produced 1-O-hexyl-aD-mono, di- and tri- a-glucopyranosides from hexanol or octanol and sucrose using a strain of Microbacterium paraoxydans exhibiting amylosucrase-type membrane transglucosylation activity (Dahiya et al., 2015).
Ochs et collaborateurs ont produit des pentyl- et octyl-polyxylosides (alkyl-polyxylosides) par réaction de transglycosylation entre du pentanol, de l’octanol et des xylanes catalysée par différentes xylanases (Ochs et al., 2011 ; Rémond et al., 2012). Ochs et al. Produced pentyl- and octyl-polyxylosides (alkyl-polyxylosides) by transglycosylation reaction between pentanol, octanol and xylans catalyzed by different xylanases (Ochs et al., 2011; Rémond et al., 2012 ).
Dans chaque cas, la différence de nature chimique des réactants (carbohydrates et alcools gras) ainsi que le caractère très hydrophile des sucres et, en contraste, très hydrophobe des acides gras ne permettent pas l’obtention de concentrations suffisantes des deux substrats dans la même phase, ce qui limite grandement les rendements de production et les tailles des têtes glycosidiques à quelques unités. In each case, the difference in the chemical nature of the reactants (carbohydrates and fatty alcohols) as well as the very hydrophilic character of the sugars and, in contrast, very hydrophobic of the fatty acids do not allow sufficient concentrations of the two substrates to be obtained in the same. phase, which greatly limits the production yields and sizes of the glycosidic heads to a few units.
Une voie alternative est d’utiliser des APGs commerciaux présentant une partie glucidique de taille modérée (typiquement degré de polymérisation inférieur à 3) et de rallonger la partie glucidique par voie enzymatique. An alternative route is to use commercial APGs having a carbohydrate part of moderate size (typically degree of polymerization less than 3) and to lengthen the carbohydrate part enzymatically.
Dans cette optique, les cyclodextrine glucano-transferases (CGTases) ont largement été décrites pour leur capacité de production d’APGs de faible DP par réaction de transglucosylation. From this perspective, cyclodextrin glucano-transferases (CGTases) have been widely described for their capacity to produce low DP APGs by transglucosylation reaction.
Les CGTases appartiennent aux familles des glycoside hydrolases GH 13 et GH57 et sont spécifiques de la formation de liaisons a-1 ,4 glucosidiques. Ces enzymes catalysent quatre types de réaction à partir d’amidon ou d’amylose : la cyclisation, le couplage, la disproportionation et l’hydrolyse. Dans le cas où une molécule non glucidique porteuse d’un ou plusieurs résidus glucosyle est introduite dans le milieu, les CGTases sont susceptibles d’orienter leur activité vers une réaction de couplage et/ou disproportionation sur cet accepteur. CGTases belong to the glycoside hydrolase families GH 13 and GH57 and are specific for the formation of α-1, 4 glucosidic bonds. These enzymes catalyze four types of reactions from starch or amylose: cyclization, coupling, disproportionation and hydrolysis. In the case where a non-carbohydrate molecule carrying one or more glucosyl residues is introduced into the medium, the CGTases are capable of directing their activity towards a coupling and / or disproportionation reaction on this acceptor.
Okada et collaborateurs furent les premiers à utiliser les capacités de couplage de telles enzymes pour l’élongation de glycolipides (sucro-esters). Ils ont obtenu l’addition de 1 à 3 unités glucosyle sur du laurate de saccharose (Okada ét al., 2007). Okada et al. Were the first to use the coupling capacities of such enzymes for the elongation of glycolipids (sucro-esters). They obtained the addition of 1 to 3 glucosyl units on sucrose laurate (Okada et al., 2007).
La capacité des CGTases à rallonger des maltosides de butanol, d’octanol et de dodécanol a été montrée par Zhao et collaborateurs en 2008 (Zhao et al., 2008). The ability of CGTases to lengthen maltosides of butanol, octanol and dodecanol was demonstrated by Zhao et al. In 2008 (Zhao et al., 2008).
Les APGs utilisés n’ont pu être rallongés que de 2 à 3 unités glucosyle par la CGTase de Bacillus stearothermophilus à partir de dextrines de 10 à 15 unités glucosyle. The APGs used could only be lengthened by 2 to 3 glucosyl units by the Bacillus stearothermophilus CGTase from dextrins of 10 to 15 glucosyl units.
Svensson et collaborateurs ont comparé les activités de couplage et de disproportionation des CGTases de Bacillus macerans et de Thermoanaerobacter (Toruzyme 3.0L, Novozymes A S, Danemark) sur du dodécyl-maltoside (C12G2, c’est à dire un APG composé d’un alkyle en C12 et d’une partie glucidique comprenant deux unités glucosyle) à partir d’a-cyclodextrines (Svensson et al., 2009). Il a pu être mis en évidence que les différences de spécificités réactionnelles couplage/disproportionation des deux CGTases, permettaient la modulation des produits de réaction. Ainsi, des mélanges de produits porteurs de 1 à 20 unités glucosyle ont été obtenus avec des profils variant selon les CGTases (C12G8 et C12G14 pour la CGTase de B. macerans, enzyme peu disproportionante et une distribution +/- homogène de C12G1 à C12G20 pour la CGTase de Thermoanaerobacter, enzyme très disproportionante). Svensson et al. Compared the coupling and disproportionation activities of the CGTases of Bacillus macerans and Thermoanaerobacter (Toruzyme 3.0L, Novozymes AS, Denmark) on dodecyl-maltoside (C12G2, i.e. an APG composed of an alkyl in C12 and a carbohydrate part comprising two glucosyl units) from α-cyclodextrins (Svensson et al., 2009). It could be demonstrated that the differences in the coupling / disproportionation reaction specificities of the two CGTases, allowed modulation of the reaction products. Thus, mixtures of products carrying 1 to 20 glucosyl units were obtained with profiles varying according to the CGTases (C12G8 and C12G14 for the CGTase of B. macerans, enzyme little disproportionate and a distribution +/- homogeneous from C12G1 to C12G20 for Thermoanaerobacter CGTase, a very disproportionate enzyme).
L’obtention des différents produits est contrôlée par la cinétique de réaction (Svensson et al., 2011 ), les réactions de couplage étant plus rapides que celles de disproportionation. Ainsi, il a été possible de réguler le profil de produits en APGs rallongés en contrôlant le débit traversant un réacteur à lit fixe contenant la CGTase de B. macerans immobilisée sur Eupergit C : un débit rapide favorise le produit de couplage (C12G8) tandis qu’un débit lent défavorise le C12G8 et favorise les produits de couplage double ou triple et les produits de disproportionation (C12G14, C12G20). The production of the various products is controlled by the reaction kinetics (Svensson et al., 2011), the coupling reactions being faster than those of disproportionation. Thus, it was possible to regulate the profile of products in extended APGs by controlling the flow rate passing through a fixed bed reactor containing the CGTase from B. macerans immobilized on Eupergit C: a rapid flow rate favors the coupling product (C12G8) while 'slow flow disadvantages C12G8 and favors double or triple coupling products and disproportionation products (C12G14, C12G20).
Paul et collaborateurs ont utilisé une glucanotransférase de la famille GH57 pour allonger du dodécanol maltoside à partir d’amidon. Des APGs porteurs de 24 à 26 unités glucosyle (C12G24 à C12G26) ont été identifiés par analyse MS (Paul, ét al., 2015). Paul et al used a glucanotransferase of the GH57 family to extend dodecanol maltoside from starch. APGs carrying 24 to 26 glucosyl units (C12G24 to C12G26) were identified by MS analysis (Paul, et al., 2015).
Des glucane-saccharases (GS) des familles GH 13 et GH70 peuvent aussi être utilisées pour de telles réactions de glucosylation. Glucan sucrases (GS) of the GH 13 and GH70 families can also be used for such glucosylation reactions.
À partir de saccharose, les glucane-saccharases catalysent la synthèse de glucanes présentant généralement de très hautes masses molaires et des structures variées en raison de la présence de différents types de liaisons osidiques (a-1 ,2, a-1 ,3, a-1 ,4 et/ou a- 1 ,6) ainsi qu’à leur localisation dans le polymère. Des isomères du saccharose ainsi que du glucose sont aussi produits à partir de saccharose mais généralement en beaucoup plus faible quantité que le polymère. From sucrose, glucansucrases catalyze the synthesis of glucans generally having very high molar masses and varied structures due to the presence of different types of osidic bonds (a-1, 2, a-1, 3, a -1, 4 and / or a- 1, 6) as well as their location in the polymer. Sucrose isomers as well as glucose are also produced from sucrose but generally in much smaller quantities than the polymer.
Quelques glucane-saccharases des familles GH13 et GH70 ont été utilisées dans des réactions d’accepteurs dans le but d’allonger la partie glucidique d’alkyl monoglucosides à groupe alkyle courts (1 à 8 atomes de carbone) à partir de saccharose. Some glucansucrases of the GH13 and GH70 families have been used in acceptor reactions to extend the carbohydrate portion of short alkyl monoglucosides (1 to 8 carbon atoms) from sucrose.
Ainsi, les dextrane-saccharases extracellulaires de Leuconostoc mesenteroides NRRL- B512F ont permis dès 1966 le transfert de 1 à 4 unités glucosyle sur le 1-O-méthyl-a-D- glucoside. Thus, the extracellular dextransucrases of Leuconostoc mesenteroides NRRL-B512F allowed, as early as 1966, the transfer of 1 to 4 glucosyl units onto 1-O-methyl-a-D-glucoside.
L’alternane saccharase de L. mesenteroides NRRL B-1355 a catalysé le transfert d’une seule unité glucosyle sur les 1-O-méthyl-a- et b-D-glucosides (Côté et al., 1982). L. mesenteroides NRRL B-1355 alternansucrase catalyzed the transfer of a single glucosyl unit to 1-O-methyl-a- and b-D-glucosides (Côté et al., 1982).
Plus récemment, Richard et collaborateurs ont étudié l’effet de la longueur de la chaîne alkyle sur la capacité des dextrane-saccharases à glucosyler les alkyl-monoglucosides et ont montré que, si des glucane-saccharases de L. mesenteroides B-512F, B-1299, B-1355 et B-23192 sont capables de glucolyser le 1-O-butyl-a-glucoside (en ajoutant 1 à 3 unités glucosyle supplémentaires selon des liaisons a-1 ,6 glucosidiques), seules les alternane- saccharases de L. mesenteroides B-1355 et B-23192 ont allongé le 1-O-octyl-a-glucoside (Richard et al., 2003). More recently, Richard et al. Studied the effect of the length of the alkyl chain on the ability of dextransucrases to glucosylate alkyl monoglucosides and showed that, while glucansucrases from L. mesenteroides B-512F, B -1299, B-1355 and B-23192 are capable of glucolysing 1-O-butyl-α-glucoside (by adding 1 to 3 additional glucosyl units according to α-1, 6 glucosidic bonds), only the alternansucrases of L. mesenteroides B-1355 and B-23192 extended 1-O-octyl-α-glucoside (Richard et al., 2003).
Les procédés connus sont donc limités notamment en ce qu’ils ne permettent pas de moduler aisément à la fois le nombre et la nature des liaisons reliant les unités glucosyle au sein des polyglucosides d’alkyle obtenus. The known processes are therefore limited in particular in that they do not allow easy modulation of both the number and the nature of the bonds connecting the glucosyl units within the alkyl polyglucosides obtained.
Par ailleurs, la plupart des procédés connus ne peuvent avoir comme substrat des glucosides d’alkyle ayant de longues chaînes d’alkyle, typiquement supérieur à 8 atomes de carbone. Furthermore, most of the known processes cannot have as substrate alkyl glucosides having long alkyl chains, typically greater than 8 carbon atoms.
Il existe donc un besoin pour un procédé de préparation de glucosides d’alkyle permettant d'accéder à une grande diversité d'architectures moléculaires en utilisant un substrat renouvelable et bon marché tel que le saccharose. There is therefore a need for a process for the preparation of alkyl glucosides allowing access to a wide variety of molecular architectures using a renewable and inexpensive substrate such as sucrose.
En particulier, il existe également un besoin pour un procédé permettant de moduler à la fois le nombre d’unités glucosyle au sein de la partie glucidique, mais aussi la nature des liaisons osidiques reliant lesdites unités glucosyle, tout en rendant possible l’obtention de polyglucosides d’alkyle à longue chaîne glucosidique, et ce même si le glucoside d’alkyle utilisé comme substrat a un groupe alkyle de grande taille. In particular, there is also a need for a method making it possible to modulate both the number of glucosyl units within the carbohydrate part, but also the nature of the osidic bonds connecting said glucosyl units, while making it possible to obtain Long-chain alkyl glucoside polyglucosides, even if the alkyl glucoside used as a substrate has a large alkyl group.
BREVE DESCRIPTION DES FIGURES BRIEF DESCRIPTION OF THE FIGURES
[Fig. 1] La Figure 1 illustre le schéma de la réaction d’élongation enzymatique de la partie glucosidique d’un monoglucoside d’alkyle mettant en oeuvre des a-transglucosylases de la famille GH70 actives sur le saccharose. [Fig. 1] Figure 1 illustrates the diagram of the enzymatic elongation reaction of the glucosidic part of an alkyl monoglucoside using α-transglucosylases of the GH70 family active on sucrose.
Les Figures [Fig. 2A] 2A et [Fig. 2B] 2B montre les profils d’élongation pour le substrat C8G1 obtenus avec les a-transglucosylases de la famille GH70 qui utilisent le saccharose comme donneur de glucosyle. [Fig. 2A] La Figure 2A montre les profils obtenus avec les glucane saccharases à partir de [C8G1] = 30 mM et de [saccharose] = 585 mM pour les glucane-saccharases DSR-M D1 (SEQ ID NO : 1 ), DSR-M D5 (SEQ ID NO : 2), DSR-M D5 W624A (SEQ ID NO : 3), GS-B (SEQ ID NO : 5), GS-C (SEQ ID NO : 6), GS-D (SEQ ID NO : 9) et GS-FS D1 (SEQ ID NO : 10) et 1170 mM de saccharose pour les enzymes GS- A SEQ ID NO :4, GS-D (SEQ ID NO :7), DSR-G (SEQ ID NO :11 ), DSR-G CD1 (SEQ ID NO :12). Les glucane saccharases sont utilisées en réaction à 1 U.mL 1. [Fig. 2B] La Figure 2B montre les profils obtenus avec les enzymes de branchement BRS-A (SEQ ID NO : 17), BRS-B D1 (SEQ ID NO : 18), BRS-C (SEQ ID NO : 19), BRS-D D1 (SEQ ID NO : 20), BRS- E D1 (SEQ ID NO : 21 ), BRS-F (SEQ ID NO : 22), DSR-G CD2 (SEQ ID NO : 23), GBD- CD2 (SEQ ID NO : 16), à partir de [C8G1] = 20 mM et de [saccharose] = 585 mM. Les enzymes de branchement sont utilisées en réaction à 1 U.mL 1 The Figures [Fig. 2A] 2A and [Fig. 2B] 2B shows the elongation profiles for the C8G1 substrate obtained with the α-transglucosylases of the GH70 family which use sucrose as a glucosyl donor. [Fig. 2A] Figure 2A shows the profiles obtained with the glucan saccharases from [C8G1] = 30 mM and from [saccharose] = 585 mM for the glucan saccharases DSR-M D1 (SEQ ID NO: 1), DSR-M D5 (SEQ ID NO: 2), DSR-M D5 W624A (SEQ ID NO: 3), GS-B (SEQ ID NO: 5), GS-C (SEQ ID NO: 6), GS-D (SEQ ID NO: 9) and GS-FS D1 (SEQ ID NO: 10) and 1170 mM of sucrose for the enzymes GS- A SEQ ID NO: 4, GS-D (SEQ ID NO: 7), DSR-G (SEQ ID NO: 11), DSR-G CD1 (SEQ ID NO: 12). Glucan sucrases are used in reaction at 1 U.mL 1 . [Fig. 2B] Figure 2B shows the profiles obtained with the branching enzymes BRS-A (SEQ ID NO: 17), BRS-B D1 (SEQ ID NO: 18), BRS-C (SEQ ID NO: 19), BRS- D D1 (SEQ ID NO: 20), BRS- E D1 (SEQ ID NO: 21), BRS-F (SEQ ID NO: 22), DSR-G CD2 (SEQ ID NO: 23), GBD- CD2 (SEQ ID NO: 16), from [C8G1] = 20 mM and [sucrose] = 585 mM. Branching enzymes are used in reaction to 1 U.mL 1
[Fig. 3] La Figure 3 compare le profil de la réaction d’élongation du C8G1 catalysée par GS-C (SEQ ID NO : 6) en présence de 200 g.L 1 de saccharose et de 200 g.L 1 d’a- cyclodextrines et de la cyclodextrine glucanotransferases (CGTase) de Bacillus macerans (enzyme non conforme à l’invention) en présence de 200 g.L 1 d’a-cyclodextrines (substrat non conforme à l’invention). [Fig. 3] Figure 3 compares the profile of the C8G1 elongation reaction catalyzed by GS-C (SEQ ID NO: 6) in the presence of 200 gL 1 of sucrose and 200 gL 1 of α-cyclodextrins and cyclodextrin glucanotransferases (CGTase) from Bacillus macerans (enzyme not in accordance with the invention) in the presence of 200 gL 1 of α-cyclodextrins (substrate not in accordance with the invention).
[Fig. 4] La Figure 4 compare les profils de la réaction d’élongation du C8G1 catalysée par la glucane-saccharase DSR-M D1 seule (SEQ ID NO : 1 ) et catalysées par la glucane- saccharase DSR-M D1 et les enzymes de branchement BRS-A (SEQ ID NO :17) ou BRS- B D1 (SEQ ID NO :18) utilisées de manière séquentielle. [Fig. 4] Figure 4 compares the profiles of the C8G1 elongation reaction catalyzed by DSR-M D1 glucansucrase alone (SEQ ID NO: 1) and catalyzed by DSR-M D1 glucansucrase and branching enzymes. BRS-A (SEQ ID NO: 17) or BRS-B D1 (SEQ ID NO: 18) used sequentially.
[Fig. 5] La Figure 5 montre le profil d’élongation d’alkyle glucosides de tailles croissantes obtenu avec la glucane-saccharase DSR-M D1 (SEQ ID NO : 1 ) avec des concentrations initiales en saccharose de 200 g.L 1. Encart A : [C8G1]=30mM ; Encart B : [C10G1]=10mM ; Encart C : Triton CG110 (Dow Chemicals, USA) à 15 g.L 1 ; Encart D : [C12G1]=5 mM ; Encart E : [C12G2]=10mM, Encart F : [C16G2]=5mM. [Fig. 5] Figure 5 shows the lengthening profile of alkyl glucosides of increasing sizes obtained with the glucansucrase DSR-M D1 (SEQ ID NO: 1) with initial sucrose concentrations of 200 gL 1 . Inset A: [C8G1] = 30mM; Inset B: [C10G1] = 10mM; Box C: Triton CG110 (Dow Chemicals, USA) at 15 gL 1 ; Inset D: [C12G1] = 5mM; Insert E: [C12G2] = 10mM, Insert F: [C16G2] = 5mM.
DESCRIPTION DETAILLEE DE L’INVENTION DETAILED DESCRIPTION OF THE INVENTION
Le but de la présente invention est de pallier les inconvénients de l’art antérieur et de fournir un procédé de préparation d’un polyglucoside d’alkyle par catalyse enzymatique, ledit procédé étant économique car utilisant du saccharose ou l’un de ses analogues comme substrat et permettant d’obtenir une grande diversité de polyglucosides d’alkyle en termes de taille et de structure de leur partie glucosidique. The aim of the present invention is to overcome the drawbacks of the prior art and to provide a process for preparing an alkyl polyglucoside by enzymatic catalysis, said process being economical because using sucrose or one of its analogues as substrate and making it possible to obtain a great diversity of alkyl polyglucosides in terms of the size and structure of their glucosidic part.
Ainsi, le procédé de l’invention rend possible d’obtenir un polyglucoside d’alkyle avec un nombre d’unités glucosyle modulable de 2 à 200 unités glucosyle. Thus, the process of the invention makes it possible to obtain an alkyl polyglucoside with an adjustable number of glucosyl units of 2 to 200 glucosyl units.
Le procédé permet également de moduler la structure linéaire ou ramifiée de la partie glucidique du polyglucoside d’alkyle obtenu, ainsi que la nature des liaisons osidiques alpha-1 ,2 ; alpha-1 ,3 ; alpha-1 ,4 ou alpha-1 ,6 reliant les résidus glucosyle au sein de la partie glucidique. The process also makes it possible to modulate the linear or branched structure of the carbohydrate part of the alkyl polyglucoside obtained, as well as the nature of the alpha-1, 2 osidic bonds; alpha-1, 3; alpha-1, 4 or alpha-1, 6 linking the glucosyl residues within the carbohydrate part.
De manière importante, le procédé de l’invention permet de préparer des polyglucosides d’alkyle ayant un groupe alkyle de taille importante, par exemple supérieure à 8 atomes de carbone. Importantly, the process of the invention makes it possible to prepare polyalkylglucosides having an alkyl group of large size, for example greater than 8 carbon atoms.
Cette diversité est très avantageuse pour produire de nouveaux polyglucosides d’alkyle d'intérêt dans le secteur des détergents et des cosmétiques. Un autre but de l’invention est de fournir des alkyle polyglucosides d’une grande diversité en termes de taille et de structure de la partie glucosidique, et en particulier des polyglucosides d’alkyle dont la taille du groupe alkyle est importante, par exemple supérieure à 8 atomes de carbone. This diversity is very advantageous for producing new alkyl polyglucosides of interest in the detergents and cosmetics sector. Another aim of the invention is to provide alkyl polyglucosides of great diversity in terms of size and structure of the glucosidic part, and in particular alkyl polyglucosides in which the size of the alkyl group is large, for example greater. with 8 carbon atoms.
Ces buts sont atteints par l’invention qui va être décrite ci-après. These objects are achieved by the invention which will be described below.
La présente invention a pour premier objet un procédé de préparation d’un polyglucoside d’alkyle de formule (I) The first subject of the present invention is a process for the preparation of an alkyl polyglucoside of formula (I)
[Glc]m-[Glc]n(-0-R) (I) dans laquelle : [Glc] m - [Glc] n (-0-R) (I) in which:
R représente un groupe alkyle linéaire ou ramifié, saturé ou insaturé, comprenant entre 8 et 20 atomes de carbone, R represents a linear or branched, saturated or unsaturated alkyl group comprising between 8 and 20 carbon atoms,
[Glc]m-[Glc]n représente une partie glucidique linéaire ou ramifiée comprenant n+m unités glucosyle, n+m étant compris entre 3 et 200 ; ledit procédé comprenant au moins une étape i) d’élongation de la chaîne glucosidique d’un glucoside d’alkyle de formule (II) [Glc] m - [Glc] n represents a linear or branched carbohydrate part comprising n + m glucosyl units, n + m being between 3 and 200; said process comprising at least one step i) of elongation of the glucosidic chain of an alkyl glucoside of formula (II)
[Glc]n(-0-R) (II) dans laquelle : [Glc] n (-0-R) (II) in which:
R est tel que défini dans la formule (I), R is as defined in formula (I),
[Glcjn représente une partie glucidique comprenant n unités glucosyle n étant compris entre 1 et 15. ladite étape comprenant la mise en contact dudit glucoside d’alkyle de formule (II) avec au moins une a-transglucosylase de la famille GH70 en présence de saccharose ou d’un analogue de saccharose. [Glcj n represents a carbohydrate part comprising n glucosyl units n being between 1 and 15. said step comprising bringing said alkyl glucoside of formula (II) into contact with at least one α-transglucosylase of the GH70 family in the presence of sucrose or a sucrose analogue.
Grâce au procédé conforme à l’invention, il est maintenant possible de préparer une grande diversité de polyglucosides d’alkyle. Thanks to the process according to the invention, it is now possible to prepare a wide variety of alkyl polyglucosides.
De manière importante, le procédé permet de contrôler la taille, la structure ramifiée ou non et la nature des liaisons osidiques au sein de la partie glucidique du polyglucoside d’alkyle obtenu, et ce même lorsque le groupe alkyle du glucoside d’alkyle utilisé comme substrat est de grande taille. Importantly, the method makes it possible to control the size, the branched or unbranched structure and the nature of the osidic bonds within the carbohydrate part of the alkyl polyglucoside obtained, and this even when the alkyl group of the alkyl glucoside used as substrate is large.
Les a-transglucosylases de la famille GH70 sont des enzymes hydrosolubles catalysant naturellement des substances hydrophiles, et dont l’activité est a priori fortement limitée en présence de matière grasse. The α-transglucosylases of the GH70 family are water-soluble enzymes which naturally catalyze hydrophilic substances, and whose activity is a priori strongly limited in the presence of fat.
Les inventeurs ont découvert que, de manière surprenante, des a-transglucosylases de la famille GH70 sont capables d’accepter comme substrat un glucosyle d’alkyle en C8-C20, c’est-à-dire un composé peu soluble comprenant une partie hydrophobe défavorable à l’action de ce type d’enzyme, et de catalyser efficacement l’élongation de sa partie glucidique. The inventors have discovered that, surprisingly, α-transglucosylases of the GH70 family are capable of accepting as substrate a C8-C20 alkyl glucosyl, that is to say a poorly soluble compound comprising a hydrophobic part unfavorable to the action of this type of enzyme, and to effectively catalyze the elongation of its carbohydrate part.
Partie glucidique Carbohydrate part
Selon l’invention, l’expression « partie glucidique », désigne un polymère linéaire ou ramifié constitué d’unités glucosyle liées entre elles par des liaisons osidiques. According to the invention, the expression "carbohydrate portion" denotes a linear or branched polymer consisting of glucosyl units linked together by osidic bonds.
Selon l’invention, les termes « liaison glycosidique » ou « liaison glucosidique » ou « liaison osidique » peuvent être utilisés indifféremment et désignent le lien covalent qui lie un glucosyle à un autre glucosyle adjacent. According to the invention, the terms "glycosidic bond" or "glucosidic bond" or "osidic bond" can be used interchangeably and denote the covalent bond which links a glucosyl to another adjacent glucosyl.
La partie glucidique [Glc]m-[Glc]n du polyglucoside d’alkyle de formule (I) comprend deux fractions glucidiques [Glc]m et [Glc]n. The carbohydrate part [Glc] m - [Glc] n of the alkyl polyglucoside of formula (I) comprises two carbohydrate fractions [Glc] m and [Glc] n.
La fraction glucidique [Glc]n correspond aux n unités glucosyle de la partie glucidique du glucoside d’alkyle de formule (II) utilisé comme substrat dans le procédé de l’invention. The carbohydrate fraction [Glc] n corresponds to the n glucosyl units of the carbohydrate part of the alkyl glucoside of formula (II) used as substrate in the process of the invention.
La fraction glucidique [Glc]m correspond aux m unités glucosyle ajoutées sur la partie glucidique [Glc]n du glucoside d’alkyle de formule (II) lors de l’étape d’élongation glucosidique du procédé de l’invention. n+m, c’est-à-dire le nombre d'unités glucosyle dans la partie glucidique [Glc]m-[Glc]n du polyglucoside d’alkyle de formule (I) est avantageusement compris entre 3 et 150, de préférence entre 3 et 100, de préférence encore entre 3 et 30, de préférence encore entre 3 et 25. De manière préférée, n+m est compris entre 5 et 50, et peut notamment être compris entre 5 et 40, notamment encore entre 5 et 30, en particulier entre 5 et 25. Avantageusement, n+m est compris entre 7 et 200, de préférence entre 8 et 200, de préférence entre 9 et 200, de préférence entre 10 et 200. m, c’est-à-dire le nombre d'unités glucosyle dans la fraction glucidique [Glc]m de la partie glucidique [Glc]m-[Glc]n du polyglucoside d’alkyle de formule (I), ou autrement dit le nombre d’unités glucosyle ajoutés sur la partie glucidique [Glc]n du glucoside d’alkyle de formule (II) lors de la mise en oeuvre du procédé de l’invention, est avantageusement supérieur à 3. De manière préférée, m est compris entre 3 et 150, de préférence compris entre 3 et 100, de préférence encore compris entre 3 et 30. n, c’est-à-dire le nombre d'unités glucosyle dans la fraction glucidique [Glc]n de la partie glucidique [Glc]m-[Glc]n du polyglucoside d’alkyle de formule (I), ou autrement dit le nombre d'unités glucosyle de la partie glucidique [Glc]n du glucoside d’alkyle de formule (II), est avantageusement compris entre 1 et 8, de préférence entre 1 et 5, de préférence encore entre 1 et 3. De manière préférée, n est égal à 1 ou 2. Type de liaisons The carbohydrate fraction [Glc] m corresponds to the m glucosyl units added to the carbohydrate part [Glc] n of the alkyl glucoside of formula (II) during the glucosidic elongation step of the process of the invention. n + m, that is to say the number of glucosyl units in the carbohydrate part [Glc] m - [Glc] n of the alkyl polyglucoside of formula (I) is advantageously between 3 and 150, preferably between 3 and 100, more preferably between 3 and 30, more preferably between 3 and 25. Preferably, n + m is between 5 and 50, and can in particular be between 5 and 40, in particular still between 5 and 30, in particular between 5 and 25. Advantageously, n + m is between 7 and 200, preferably between 8 and 200, preferably between 9 and 200, preferably between 10 and 200. m, that is to say say the number of glucosyl units in the carbohydrate fraction [Glc] m of the carbohydrate part [Glc] m - [Glc] n of the alkyl polyglucoside of formula (I), or in other words the number of glucosyl units added to the carbohydrate part [Glc] n of the alkyl glucoside of formula (II) during the implementation of the process of the invention is advantageously greater than 3. Preferably, m is between 3 and 150, preferably between 3 and 100, more preferably between 3 and 30. n, that is to say the number of glucosyl units in the carbohydrate fraction [Glc] n of the carbohydrate part [Glc] m - [Glc] n of the alkyl polyglucoside of formula (I), or in other words the number of glucosyl units of the carbohydrate part [Glc] n of the alkyl glucoside of formula (II), is advantageously between 1 and 8, preferably between 1 and 5, more preferably between 1 and 3. Preferably, n is equal to 1 or 2. Type of connections
Selon l’invention, l’expression « liaison a » désigne un lien covalent qui lie l’atome de carbone 1 d’une unité glucosyle dans sa configuration a ; et l’expression « liaison b » désigne un lien covalent qui lie l’atome de carbone 1 d’une unité glucosyle dans sa configuration b. According to the invention, the expression "a bond" denotes a covalent bond which links the carbon atom 1 of a glucosyl unit in its a configuration; and the term "b bond" denotes a covalent bond which links carbon atom 1 of a glucosyl unit in its b configuration.
Au sein de la fraction glucidique [Glc]n du polyglucoside d’alkyle de formule (I) ou de la partie glucidique [Glc]n glucoside d’alkyle de formule (II), l’unité glucosyle adjacente au groupe alkyle est liée au groupe alkyl par une liaison a ou par une liaison b, de préférence par une liaison b. Within the carbohydrate moiety [Glc] n of the alkyl polyglucoside of formula (I) or the carbohydrate portion [Glc] n alkyl glucoside of formula (II), the glucosyl unit adjacent to the alkyl group is linked to the alkyl group through an a bond or through a b bond, preferably through a b bond.
En d’autres termes, dans le polyglucoside d’alkyle de formule (I) et le glucoside d’alkyle de formule (II), l’unité glucosyle adjacente au groupe alkyle est en configuration a ou b, de préférence en configuration b. In other words, in the alkyl polyglucoside of formula (I) and the alkyl glucoside of formula (II), the glucosyl unit adjacent to the alkyl group is in the a or b configuration, preferably in the b configuration.
Les autres unités glucosyle de la fraction glucidique [Glc]n du polyglucoside d’alkyle de formule (I) ou de la partie glucidique [Glc]n glucoside d’alkyle de formule (II) sont liés entres elles par des liaisons glucosidiques a et/ou b, de préférence par des liaisons glucosidiques a. The other glucosyl units of the carbohydrate fraction [Glc] n of the alkyl polyglucoside of formula (I) or of the carbohydrate part [Glc] n alkyl glucoside of formula (II) are linked to each other by a and / or b, preferably by glucosidic bonds a.
Les m unités glucosyles adjacentes au sein de [Glc]m sont liés entres elles par des liaisons glucosidiques a. The m adjacent glucosyl units within [Glc] m are linked together by α-glucosidic bonds.
Dans le présent document, le terme « liaison a-1,3 » désigne le lien covalent qui lie l’atome de carbone 1 d’une unité glucosyle dans sa configuration a et le carbone 3 d’une autre unité glucosyle adjacente. Le terme « liaison a-1 ,2 » désigne le lien covalent qui lie l’atome de carbone 1 d’une unité glucosyle dans sa configuration a et le carbone 2 d’une autre unité glucosyle adjacente. Le terme « liaison alpha-1,6 » désigne le lien covalent qui lie l’atome de carbone 1 d’une unité glucosyle dans sa configuration alpha et le carbone 6 d’une autre unité glucosyle adjacente. In this document, the term "α-1,3 bond" refers to the covalent bond that links the carbon 1 of one glucosyl unit in its α configuration and the 3 carbon of another adjacent glucosyl unit. The term "α-1, 2 bond" refers to the covalent bond that links the carbon 1 of one glucosyl unit in its α configuration and the 2 carbon of another adjacent glucosyl unit. The term "alpha-1,6 bond" refers to the covalent bond that links the carbon 1 of one glucosyl unit in its alpha configuration and the 6 carbon of another adjacent glucosyl unit.
Les unités alpha-glucosyle sont de préférence des unités alpha-D-glucosyle. The alpha-glucosyl units are preferably alpha-D-glucosyl units.
L’analyse des liaisons glycosidiques d’un polyglucoside d’alkyl peut être réalisée grâce à n’importe quelle méthode connue de l’homme du métier. The analysis of the glycosidic bonds of an alkyl polyglucoside can be carried out by any method known to those skilled in the art.
Par exemple, les liaisons glucosidiques peuvent être analysées par résonance magnétique nucléaire (RMN). For example, glucosidic bonds can be analyzed by nuclear magnetic resonance (NMR).
L’étape i) peut être conduite avec un glucoside d’alkyle de formule (II) essentiellement constitué de molécules de monoglucoside d’alkyle, de molécules de diglucloside d’alkyle, ou d’un mélange de celles-ci, ledit mélange présentant avantageusement un degré de polymérisation moyen compris entre 1 et 2, de préférence compris entre 1,3 et 1,6. Le mélange est essentiellement constitué de molécules de monoglucoside d’alkyle et de molécules de diglucloside d’alkyle, c’est-à-dire qu’il est constitué d’au moins 90% en poids de molécules de monoglucoside d’alkyle et de diglucloside d’alkyle, de préférence d’au moins 95% en poids de molécules de monoglucoside d’alkyle et de diglucloside d’alkyle, de préférence encore d’au moins 97% en poids de molécules de monoglucoside d’alkyle et de diglucloside d’alkyle. Step i) can be carried out with an alkyl glucoside of formula (II) essentially consisting of alkyl monoglucoside molecules, of alkyl diglucloside molecules, or of a mixture thereof, said mixture having advantageously an average degree of polymerization between 1 and 2, preferably between 1.3 and 1.6. The mixture consists essentially of molecules of alkyl monoglucoside and of molecules of alkyl diglucloside, i.e. it consists of at least 90% by weight of molecules of alkyl monoglucoside and of alkyl diglucloside, preferably at least 95% by weight of alkyl monoglucoside and alkyl diglucloside molecules, more preferably at least 97% by weight of alkyl monoglucoside and diglucloside molecules alkyl.
Le reste peut avantageusement être constitué de molécules de d’oligoglucosides d’alkyle, i.e. de glucosides d’alkyle dont le glucoside comprend de 3 à 8 unités glucosyle, de préférence 3 à 5 unités glucosyle. The remainder can advantageously consist of molecules of alkyl oligoglucosides, i.e. alkyl glucosides, the glucoside of which comprises 3 to 8 glucosyl units, preferably 3 to 5 glucosyl units.
Dans un mode de réalisation, le polyglucoside d’alkyle de formule (I) est constitué essentiellement d’un mélange de molécules de polyglucosides d’alkyle de formule (I), ledit mélange présentant avantageusement un degré de polymérisation moyen compris entre 3 et 25, de préférence compris entre 5 et 25, de préférence compris entre 6 et 25, de préférence entre 7 et 25, de préférence encore entre 8 et 25. In one embodiment, the alkyl polyglucoside of formula (I) consists essentially of a mixture of alkyl polyglucoside molecules of formula (I), said mixture advantageously exhibiting an average degree of polymerization of between 3 and 25 , preferably between 5 and 25, preferably between 6 and 25, preferably between 7 and 25, more preferably between 8 and 25.
Le mélange est essentiellement constitué de molécules de polyglucoside d’alkyle de formule (I), c’est-à-dire qu’il est de préférence constitué d’au moins 90% en poids desdites molécules de polyglucosides d’alkyle, de préférence d’au moins 95% en poids desdites molécules de polyglucosides d’alkyle, de préférence encore d’au moins 97% en poids desdites molécules de polyglucosides d’alkyle. The mixture consists essentially of alkyl polyglucoside molecules of formula (I), i.e. it preferably consists of at least 90% by weight of said alkyl polyglucoside molecules, preferably at least 95% by weight of said polyalkylglucoside molecules, more preferably at least 97% by weight of said polyalkylglucoside molecules.
Dans la présente invention, le « degré de polymérisation moyen » rend compte de la distribution moyenne du nombre d'unités glucosyle par molécule de polyglucoside d’alkyle de formule (I) ou de glucoside d’alkyle de formule (II) au sein d’un mélange de molécules de polyglucoside d’alkyle de formule (I) ou d’un mélange de molécules de glucoside d’alkyle de formule (II). In the present invention, the “average degree of polymerization” accounts for the average distribution of the number of glucosyl units per molecule of alkyl polyglucoside of formula (I) or of alkyl glucoside of formula (II) within a mixture of polyalkyl glucoside molecules of formula (I) or of a mixture of alkyl glucoside molecules of formula (II).
Dans l’invention, ce degré de polymérisation moyen est déterminé à partir de mesures réalisées par résonance magnétique nucléaire (RMN). In the invention, this average degree of polymerization is determined from measurements made by nuclear magnetic resonance (NMR).
Pour un mélange de molécules de polyglucoside d’alkyle de formule (I), le degré de polymérisation moyen est déterminé en calculant le ratio (masse molaire moyenne du mélange de molécules polyglucoside d’alkyle de formule (I) - masse molaire de la chaîne alkyle correspondante) : (masse molaire d’une unité glucosyle). For a mixture of alkyl polyglucoside molecules of formula (I), the average degree of polymerization is determined by calculating the ratio (average molar mass of the mixture of alkyl polyglucoside molecules of formula (I) - molar mass of the chain corresponding alkyl): (molar mass of a glucosyl unit).
Pour un mélange de molécules de glucoside d’alkyle de formule (II), le degré de polymérisation moyen est déterminé en calculant le ratio (masse molaire moyenne du mélange de molécules de glucoside d’alkyle de formule (II) - masse molaire de la chaîne alkyle correspondante) : (masse molaire d’une unité glucosyle). For a mixture of alkyl glucoside molecules of formula (II), the average degree of polymerization is determined by calculating the ratio (average molar mass of the mixture of alkyl glucoside molecules of formula (II) - molar mass of the corresponding alkyl chain): (molar mass of a glucosyl unit).
Groupe alkyle De manière surprenante, les inventeurs ont découvert que des a-transglucosylases de la famille GH70 permettent de glucosyler efficacement des glucosides d’alkyle de formule (II) ayant un groupe alkyle de taille importante. Alkyl group Surprisingly, the inventors have discovered that α-transglucosylases of the GH70 family make it possible to efficiently glucosylate alkyl glucosides of formula (II) having an alkyl group of large size.
Le groupe alkyl comprend de préférence au moins 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18 ou 19 atomes de carbone et au plus 20 atomes de carbone. The alkyl group preferably comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 carbon atoms and at most 20 carbon atoms.
Dans un mode de réalisation, le groupe alkyle R comprend avantageusement entre 8 et 16 atomes de carbone, de préférence encore comprend 8 et 12 atomes de carbone. In one embodiment, the alkyl group R advantageously comprises between 8 and 16 carbon atoms, more preferably comprises 8 and 12 carbon atoms.
Dans un mode de réalisation, le groupe alkyle R comprend entre 9 et 20 atomes de carbone, de préférence comprend entre 9 et 16 atomes de carbone, de préférence encore comprend entre 9 et 12 atomes de carbone. In one embodiment, the alkyl group R comprises between 9 and 20 carbon atoms, preferably comprises between 9 and 16 carbon atoms, more preferably comprises between 9 and 12 carbon atoms.
Dans un mode de réalisation, le groupe alkyle R comprend entre 10 et 20 atomes de carbone, de préférence comprend entre 10 et 16 atomes de carbone, de préférence encore comprend entre 10 et 12 atomes de carbone. In one embodiment, the alkyl group R comprises between 10 and 20 carbon atoms, preferably comprises between 10 and 16 carbon atoms, more preferably comprises between 10 and 12 carbon atoms.
Dans un mode de réalisation, le groupe alkyle R comprend entre 13 et 20 atomes de carbone, de préférence comprend entre 16 et 20 atomes de carbone. In one embodiment, the alkyl group R comprises between 13 and 20 carbon atoms, preferably comprises between 16 and 20 carbon atoms.
Plus particulièrement, les inventeurs ont montré que des a-transglucosylases de la famille GH70 sont capables de catalyser l’élongation de la partie glucosidique d’un glucoside d’alkyle de formule (II) présentant un groupe alkyle R comprenant entre 8 et 20 atomes de carbone, c’est-à-dire un substrat accepteur présentant un pôle alkyl beaucoup plus hydrophobe que des glucosides d’alkyl présentant un groupe alkyl tel qu’un groupe méthyle, éthyle, propyle ou butyle. More particularly, the inventors have shown that α-transglucosylases of the GH70 family are capable of catalyzing the elongation of the glucosidic part of an alkyl glucoside of formula (II) having an alkyl group R comprising between 8 and 20 atoms. carbon, that is to say an acceptor substrate having an alkyl pole much more hydrophobic than alkyl glucosides having an alkyl group such as a methyl, ethyl, propyl or butyl group.
Autrement dit, le procédé de l’invention est particulièrement avantageux en ce qu’il permet d’obtenir un polyglucoside d’alkyle de formule (I) ayant un groupe alkyle R compris entre 8 et 20 atomes de carbone et une partie glucidique [Glc]m-[Glc]n ayant un n+m tel que défini ci-dessus. In other words, the process of the invention is particularly advantageous in that it makes it possible to obtain an alkyl polyglucoside of formula (I) having an alkyl group R of between 8 and 20 carbon atoms and a carbohydrate part [Glc ] m - [Glc] n having an n + m as defined above.
Dans un mode de réalisation, R représente un groupe alkyle comprenant de 8 à 12 atomes de carbone, et n+m est compris entre 7 et 200, de préférence entre 8 et 200, de préférence entre 9 et 200, de préférence encore entre 10 et 200. Dans ce mode de réalisation, n+m peut avantageusement être compris entre 7 et 50, de préférence entre 8 et 50, de préférence entre 9 et 50, et de préférence encore entre 10 et 50. In one embodiment, R represents an alkyl group comprising from 8 to 12 carbon atoms, and n + m is between 7 and 200, preferably between 8 and 200, preferably between 9 and 200, more preferably between 10 and 200. In this embodiment, n + m can advantageously be between 7 and 50, preferably between 8 and 50, preferably between 9 and 50, and more preferably between 10 and 50.
Dans un mode de réalisation, R représente un groupe alkyle comprenant de 12 à 20 atomes de carbone, et n+m est compris entre 3 et 200, de préférence entre 4 et 200, de préférence entre 5 et 200, et de préférence encore entre 10 et 200. Dans ce mode de réalisation, n+m peut avantageusement être compris entre 3 et 50, de préférence entre 4 et 50, de préférence entre 5 et 50, et de préférence encore entre 10 et 50. Dans un mode de réalisation, lorsque R représente un groupe alkyle comprenant de 8 à 12 atomes de carbone, n+m est compris entre 7 et 200, de préférence entre 8 et 200, de préférence entre 9 et 200, de préférence encore entre 10 et 200 et peut notamment être compris entre 7 et 50, de préférence entre 8 et 50, de préférence entre 9 et 50, et de préférence encore entre 10 et 50 ; et lorsque R représente un groupe alkyle comprenant de 12 à 20 atomes de carbone, n+m est compris entre 3 et 200, de préférence entre 4 et 200, de préférence entre 5 et 200, et de préférence encore entre 10 et 200 et peut notamment être compris entre 3 et 50, de préférence entre 4 et 50, de préférence entre 5 et 50, et de préférence encore entre 10 et 50. a-transalucosylase In one embodiment, R represents an alkyl group comprising from 12 to 20 carbon atoms, and n + m is between 3 and 200, preferably between 4 and 200, preferably between 5 and 200, and more preferably between 10 and 200. In this embodiment, n + m can advantageously be between 3 and 50, preferably between 4 and 50, preferably between 5 and 50, and more preferably between 10 and 50. In one embodiment, when R represents an alkyl group comprising from 8 to 12 carbon atoms, n + m is between 7 and 200, preferably between 8 and 200, preferably between 9 and 200, more preferably between 10 and 200 and may in particular be between 7 and 50, preferably between 8 and 50, preferably between 9 and 50, and more preferably between 10 and 50; and when R represents an alkyl group comprising from 12 to 20 carbon atoms, n + m is between 3 and 200, preferably between 4 and 200, preferably between 5 and 200, and more preferably between 10 and 200 and may in particular be between 3 and 50, preferably between 4 and 50, preferably between 5 and 50, and more preferably between 10 and 50. a-transalucosylase
Selon l’invention, le terme « a-transglucosylase » désigne une enzyme capable de polymériser des unités glucosyle selon des liaisons a, en catalysant le transfert d'une unité glucosyle depuis un sucre donneur de glucosyle vers un composé accepteur. According to the invention, the term "α-transglucosylase" denotes an enzyme capable of polymerizing glucosyl units along α bonds, by catalyzing the transfer of a glucosyl unit from a glucosyl donor sugar to an acceptor compound.
Selon l’invention, l’expression « de la famille GH70 », relatif à l’otransglucosylase, signifie que Ga-transglucosylase selon l’invention appartient à la famille 70 des glycoside- hydrolases selon la classification CAZy (www.cazy.orq). CAZy, de l'anglais « Carbohydrate-Active enZYmes » (abrégé « CAZy »), est une base de données bio informatique de classification des enzymes actives sur les sucres i.e. capables de catalyser leur dissociation ou leur synthèse selon des homologies de séquences ou de structure, en particulier de leurs modules catalytiques et de liaison aux glucides. Dans la classification CAZy, le groupe des glycoside-hydrolases (GH) compte 167 familles composées d’enzymes actives sur les sucres capables de catalyser des réactions d’hydrolyse de liaisons glycosidique ou de transglucosylation. According to the invention, the expression “of the GH70 family”, relating to otransglucosylase, means that Ga-transglucosylase according to the invention belongs to the 70 family of glycoside hydrolases according to the CAZy classification (www.cazy.orq) . CAZy, standing for "Carbohydrate-Active enZYmes" (abbreviated "CAZy"), is a bioinformatic database for the classification of enzymes active on sugars, ie capable of catalyzing their dissociation or their synthesis according to sequence or sequence homologies. structure, in particular their catalytic and carbohydrate binding moduli. In the CAZy classification, the group of glycoside hydrolases (GH) has 167 families composed of enzymes active on sugars capable of catalyzing reactions of hydrolysis of glycosidic bonds or of transglucosylation.
Les a-transglucosylases de la famille GH70 sont typiquement produites de façon naturelles par des bactéries lactiques des genres Streptococcus, Leuconostoc (abrégé « L »), Weisella ou Lactobacillus (abrégé « Lb. »). The α-transglucosylases of the GH70 family are typically produced naturally by lactic acid bacteria of the genera Streptococcus, Leuconostoc (abbreviated "L"), Weisella or Lactobacillus (abbreviated "Lb.").
Les a-transglucosylases de la famille GH70 selon l’invention sont actives sur le saccharose, ce qui signifie que les a-transglucosylases selon l’invention utilisent spécifiquement le saccharose ou un de ses analogues comme donneur de glucosyle. The α-transglucosylases of the GH70 family according to the invention are active on sucrose, which means that the α-transglucosylases according to the invention specifically use sucrose or one of its analogues as a glucosyl donor.
Les analogues de saccharose peuvent notamment être choisis dans le groupe comprenant le fluorure d’a D-glucopyranosyle (en anglais « a-D-Glucopyranosyl fluoride »), le 0-3-D-galactopyranosyl-(1 4)-3-D-fructofuranosyl-a-D-glucopyranoside (en anglais « Lactulosucrose »), le p-nitrophenyl a-D-glucopyranoside, le a-D-glucopyranosyl a-L- sorbofuranoside et leurs mélanges. Selon un mode de réalisation, Ga-transglucosylase de la famille GH70 est sélectionnée parmi l’une des a-transglucosylases décrites ci-dessous. The sucrose analogs can in particular be chosen from the group comprising α-D-glucopyranosyl fluoride (in English "aD-Glucopyranosyl fluoride"), 0-3-D-galactopyranosyl- (1 4) -3-D-fructofuranosyl -aD-glucopyranoside (in English "Lactulosucrose"), p-nitrophenyl aD-glucopyranoside, aD-glucopyranosyl aL-sorbofuranoside and mixtures thereof. According to one embodiment, Ga-transglucosylase of the GH70 family is selected from one of the α-transglucosylases described below.
L’enzyme DSR-M D1 de séquence d’acides aminés SEQ ID NO : 1 est un fragment allant de l’acide aminé à la position 42 à l’acide aminé à la position 1433 de la séquence d’acides aminés de la forme sauvage de l'enzyme DSR-M (issue de la souche L. citreum NRRL B-1299) ayant pour référence GenBank CDX668951.1. The enzyme DSR-M D1 of amino acid sequence SEQ ID NO: 1 is a fragment ranging from the amino acid at position 42 to the amino acid at position 1433 of the amino acid sequence of the form wild type of the DSR-M enzyme (derived from the strain L. citreum NRRL B-1299) having for reference GenBank CDX668951.1.
L’enzyme DSR-M D5 de séquence d’acides aminés SEQ ID NO : 2 est un fragment allant de l’acide aminé à la position 421 à l’acide aminé à la position 1315 de la séquence d’acides aminés de la forme sauvage de l'enzyme DSR-M (issue de la souche L. citreum NRRL B-1299) ayant pour référence GenBank CDX668951.1. The enzyme DSR-M D5 of amino acid sequence SEQ ID NO: 2 is a fragment ranging from the amino acid at position 421 to the amino acid at position 1315 of the amino acid sequence of the form wild type of the DSR-M enzyme (derived from the strain L. citreum NRRL B-1299) having for reference GenBank CDX668951.1.
L’enzyme DSR-M D5 W624A de séquence d’acides aminés SEQ ID NO : 3 est un mutant en position 624 du fragment de séquence d’acides aminés SEQ ID NO :2. The DSR-M D5 W624A enzyme of amino acid sequence SEQ ID NO: 3 is a mutant at position 624 of the amino acid sequence fragment SEQ ID NO: 2.
L’enzyme GS-A de séquence d’acides aminés SEQ ID NO : 4 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Lb. kunkeei MP2 ayant pour référence GenBank ALJ31412. The GS-A enzyme with the amino acid sequence SEQ ID NO: 4 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the Lb strain. kunkeei MP2 having for reference GenBank ALJ31412.
L’enzyme GS-B de séquence d’acides aminés SEQ ID NO : 5 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Lb. animalis DSM 20602 ayant pour référence GenBank KRM57462.1. The GS-B enzyme of amino acid sequence SEQ ID NO: 5 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the Lb strain. animalis DSM 20602 having for reference GenBank KRM57462.1.
L’enzyme GS-C de séquence d’acides aminés SEQ ID NO : 6 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Lb. Apodemi ayant pour référence GenBank WP_056957205. The GS-C enzyme with the amino acid sequence of SEQ ID NO: 6 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the Lb strain. Apodemi with reference GenBank WP_056957205.
L’enzyme GS-D de séquence d’acides aminés SEQ ID NO : 7 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Lb. animalis DSM 20602 ayant pour référence GenBank KRM57463. The GS-D enzyme of amino acid sequence SEQ ID NO: 7 corresponds to the amino acid sequence of the wild form of the enzyme derived from the Lb strain. animalis DSM 20602 with GenBank reference KRM57463.
L’enzyme GS-E de séquence d’acides aminés SEQ ID NO : 8 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Lb. capillatus DSM 19910 ayant pour référence GenBank KRL03580. The GS-E enzyme of amino acid sequence SEQ ID NO: 8 corresponds to the amino acid sequence of the wild form of the enzyme derived from the Lb strain. capillatus DSM 19910 having for reference GenBank KRL03580.
L’enzyme GS-F D1 de séquence d’acides aminés SEQ ID NO : 9 est un fragment allant de l’acide aminé à la position 166 à l’acide aminé à la position 1874 de la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche L. fallax KCTC 3537 ayant pour référence GenBank WP_010006777.1. The enzyme GS-F D1 of amino acid sequence SEQ ID NO: 9 is a fragment ranging from the amino acid at position 166 to the amino acid at position 1874 of the amino acid sequence of the form wild type of the enzyme derived from the strain L. fallax KCTC 3537 having for reference GenBank WP_010006777.1.
L’enzyme GS-FS de séquence d’acides aminés SEQ ID NO : 10 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la Streptococcus salivarus HSISS4 ayant pour référence GenBank ALR80278. L’enzyme DSR-G de séquence d’acides aminés SEQ ID NO : 11 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la Lb. kunkeei DSM 12361 ayant pour référence GenBank KRK22577.1. The GS-FS enzyme with the amino acid sequence SEQ ID NO: 10 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from Streptococcus salivarus HSISS4 having for reference GenBank ALR80278. The DSR-G enzyme with amino acid sequence SEQ ID NO: 11 corresponds to the amino acid sequence of the wild form of the enzyme derived from Lb. kunkeei DSM 12361 with GenBank reference KRK22577.1.
L’enzyme DSR-G CD1 de séquence d’acides aminés SEQ ID NO : 12 est un fragment allant de l’acide aminé à la position 1 à l’acide aminé à la position 1407 de la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Lb. kunkeei DSM 12361 ayant pour référence GenBank KRK22577. The enzyme DSR-G CD1 of amino acid sequence SEQ ID NO: 12 is a fragment ranging from the amino acid at position 1 to the amino acid at position 1407 of the amino acid sequence of the form wild type of the enzyme derived from the strain Lb. kunkeei DSM 12361 having for reference GenBank KRK22577.
L’enzyme ASR D1 de séquence d’acides aminés SEQ ID NO : 13 est un fragment allant de l’acide aminé à la position 1 à l’acide aminé à la position 1425 de la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche L. mesenteroides NRRL B-1355 ayant pour référence GenBank CAB65910.2. The enzyme ASR D1 of amino acid sequence SEQ ID NO: 13 is a fragment ranging from the amino acid at position 1 to the amino acid at position 1425 of the amino acid sequence of the wild form of the enzyme derived from the strain L. mesenteroides NRRL B-1355 having for reference GenBank CAB65910.2.
L’enzyme GTF-SI de séquence d’acides aminés SEQ ID NO : 14 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Streptococcus mutans ayant pour référence GenBank BAA26114.1 . The GTF-SI enzyme with the amino acid sequence SEQ ID NO: 14 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the strain Streptococcus mutans with reference GenBank BAA26114.1.
L’enzyme GTF-J de séquence d’acides aminés SEQ ID NO : 15 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Streptococcus mutans ayant pour référence GenBank AAA26896.1 . The GTF-J enzyme of amino acid sequence SEQ ID NO: 15 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the strain Streptococcus mutans with reference GenBank AAA26896.1.
L’enzyme GBDCD2 de séquence d’acides aminés SEQ ID NO : 16 est un fragment allant de l’acide aminé à la position 1758 à l’acide aminé à la position 2862 de la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche L. citreum NRRL B- 1299 ayant pour référence GenBank CDX66820.1 . The enzyme GBDCD2 of amino acid sequence SEQ ID NO: 16 is a fragment ranging from the amino acid at position 1758 to the amino acid at position 2862 of the amino acid sequence of the wild form of l enzyme derived from the strain L. citreum NRRL B-1299 having for reference GenBank CDX66820.1.
L’enzyme BRS-A de séquence d’acides aminés SEQ ID NO : 17 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche L. citreum NRRL B-1299 ayant pour référence GenBank CDX66896.1. The BRS-A enzyme of amino acid sequence SEQ ID NO: 17 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the strain L. citreum NRRL B-1299 having for reference GenBank CDX66896. 1.
L’enzyme BRS-B D1 de séquence d’acides aminés SEQ ID NO : 18 est un fragment allant de l’acide aminé à la position 39 à l’acide aminé à la position 1313 de la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche L. citreum NRRL B- 742 ayant pour référence GenBank CDX65123.1. The BRS-B D1 enzyme of amino acid sequence SEQ ID NO: 18 is a fragment ranging from the amino acid at position 39 to the amino acid at position 1313 of the amino acid sequence of the form wild type of the enzyme derived from the strain L. citreum NRRL B-742 having for reference GenBank CDX65123.1.
L’enzyme BRS-C de séquence d’acides aminés SEQ ID NO : 19 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche L. fallax KCTC 3537 ayant pour référence GenBank ZP_08312597.1 . The BRS-C enzyme of amino acid sequence SEQ ID NO: 19 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the strain L. fallax KCTC 3537 having for reference GenBank ZP_08312597.1.
L’enzyme BRS-D D1 de séquence d’acides aminés SEQ ID NO : 20 est un fragment allant de l’acide aminé à la position 88 à l’acide aminé à la position 1453 de la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Lb. kunkei EFB6 ayant pour référence GenBank WP_051592287. L’enzyme BRS-E D1 de séquence d’acides aminés SEQ ID NO : 21 est un fragment allant de l’acide aminé à la position 32 à l’acide aminé à la position 1264 de la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche L. mesenteroides KFRI-MG ayant pour référence GenBank AHF19404.1 . The BRS-D D1 enzyme of amino acid sequence SEQ ID NO: 20 is a fragment ranging from the amino acid at position 88 to the amino acid at position 1453 of the amino acid sequence of the form wild type of the enzyme derived from the strain Lb. kunkei EFB6 having for reference GenBank WP_051592287. The BRS-E D1 enzyme of amino acid sequence SEQ ID NO: 21 is a fragment ranging from the amino acid at position 32 to the amino acid at position 1264 of the amino acid sequence of the form wild type of the enzyme derived from the strain L. mesenteroides KFRI-MG having for reference GenBank AHF19404.1.
L’enzyme BRS-F de séquence d’acides aminés SEQ ID NO : 22 correspond à la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Fructobacillus tropaeoli ayant pour référence GenBank GAP05007.1 . The BRS-F enzyme of amino acid sequence SEQ ID NO: 22 corresponds to the amino acid sequence of the wild-type form of the enzyme derived from the strain Fructobacillus tropaeoli having for reference GenBank GAP05007.1.
L’enzyme DSR-G CD2 de séquence d’acides aminés SEQ ID NO : 23 est un fragment allant de l’acide aminé à la position 928 à l’acide aminé à la position 2621 de la séquence d’acides aminés de la forme sauvage de l'enzyme issue de la souche Lb. kunkeei DSM 12361 ayant pour référence GenBank KRK22577.1. The DSR-G CD2 enzyme of amino acid sequence SEQ ID NO: 23 is a fragment ranging from the amino acid at position 928 to the amino acid at position 2621 of the amino acid sequence of the form wild type of the enzyme derived from the strain Lb. kunkeei DSM 12361 with GenBank reference KRK22577.1.
Les a-transglucosylases de la famille GH70 selon l'invention peuvent être obtenues selon des méthodes connues de l’homme du métier, notamment par la méthode consistant à cultiver une cellule exprimant naturellement Ga-transglucosylase ou une cellule hôte comprenant un transgène codant pour Ga-transglucosylase et exprimant ladite a- transglucosylase, et à extraire ladite a-transglucosylase à partir de ces cellules ou du milieu de culture dans lequel Ga-transglucosylase a été sécrétée. The α-transglucosylases of the GH70 family according to the invention can be obtained according to methods known to those skilled in the art, in particular by the method consisting in cultivating a cell naturally expressing Ga-transglucosylase or a host cell comprising a transgene encoding Ga -transglucosylase and expressing said α-transglucosylase, and in extracting said α-transglucosylase from these cells or from the culture medium in which Ga-transglucosylase has been secreted.
Des souches de bactéries recombinantes, par exemple des souches de bacillus subtillis ou de lactobacillus, sécrétant lesdites a-transglucosylases de la famille GH70 peuvent être utilisées. Recombinant bacterial strains, for example strains of bacillus subtillis or lactobacillus, secreting said α-transglucosylases of the GH70 family can be used.
On peut également obtenir les a-transglucosylases de la famille GH70 par l’intermédiaire de systèmes d’expression acellulaire (en anglais « cell free protein expression Systems). The α-transglucosylases of the GH70 family can also be obtained via cell free protein expression systems.
Dans le procédé de l’invention, Ga-transglucosylase de la famille GH70 est de préférence une glucane-saccharase de la famille GH70, une saccharase de branchement de la famille GH70, ou un mélange de celles-ci. In the method of the invention, Ga-transglucosylase of the GH70 family is preferably a glucansucrase of the GH70 family, a branching sucrase of the GH70 family, or a mixture thereof.
Dans le présent document, l’expression « glucane-saccharase de la famille GH70 » parfois abrégée « GS » se réfère à une a-transglucosylase de la famille GH70 capable de catalyser la synthèse d’a-glucanes i.e. de polysaccharides composés exclusivement d’unités glucosyle liées entre elles par des liaisons alpha. Parmi les glucane-saccharases, on peut citer les dextrane-saccharases (parfois abrégé DSR), qui synthétisent des dextranes, i.e. des glucanes dont les résidus de la chaîne principale sont liés majoritairement en alpha-1 ,6 ; les reuterane-saccharases, qui synthétisent des reuteranes, i.e. des glucanes dont les résidus de la chaîne principale sont liés en alpha-1 ,4 et alpha- 1 ,6 ; les mutanes-saccharases, qui synthétisent des mutanes, i.e. des glucanes dont les résidus de la chaîne principale sont liés majoritairement en alpha-1 ,3 ; des alternanes- saccharase, i.e. des glucanes dont les résidus de la chaîne principale sont liés alternativement en alpha-1 ,3 et alpha-1 ,6. De préférence, G a-transglucosylase de la famille GH70 n’est pas une alternane-saccharase. In the present document, the expression “glucansucrase of the GH70 family” sometimes abbreviated “GS” refers to an α-transglucosylase of the GH70 family capable of catalyzing the synthesis of α-glucans, ie of polysaccharides composed exclusively of. glucosyl units linked together by alpha bonds. Among the glucansucrases, mention may be made of dextransucrases (sometimes abbreviated as DSR), which synthesize dextrans, ie glucans in which the residues of the main chain are linked mainly in alpha-1,6; reuteranesucrases, which synthesize reuterans, ie glucans whose main chain residues are linked in alpha-1, 4 and alpha-1, 6; mutans-sucrases, which synthesize mutans, i.e. glucans whose Main chain residues are linked predominantly at alpha-1, 3; alternans-sucrase, ie glucans whose main chain residues are linked alternately in alpha-1, 3 and alpha-1, 6. Preferably, G α-transglucosylase of the GH70 family is not an alternansucrase.
Dans le présent document, les termes « saccharase de branchement de la famille GH70 » (en anglais « branching sucrase », parfois abrégé BRS) ou « a-transglucosylase de la famille GH70 à activité de branchement » ou « enzyme de branchement de la famille GH70 » sont utilisées indifféremment et se réfèrent à une a-transglucosylase de la famille GH70 capable de catalyser l’ajout d’unités glucosyle dans la chaîne principale d’un glucane de type dextrane pré-existant formant des branchements (ou autrement dit des ramifications). La nature de la liaison de branchement (liaisons alpha-1 ,2 ; alpha-1 ,3 ; alpha-1 ,4 ou alpha-1 ,6) et la longueur des chaînes d’unités glucosyle constituant les branchements varient selon la spécificité de la saccharase de branchement considérée. In the present document, the terms “branching sucrase of the GH70 family” (in English “branching sucrase”, sometimes abbreviated as BRS) or “α-transglucosylase of the GH70 family with branching activity” or “branching enzyme of the family GH70 ”are used interchangeably and refer to an α-transglucosylase of the GH70 family capable of catalyzing the addition of glucosyl units in the main chain of a pre-existing dextran-type glucan forming branches (or in other words branches ). The nature of the branching bond (alpha-1, 2; alpha-1, 3; alpha-1, 4 or alpha-1, 6 bonds) and the length of the chains of glucosyl units constituting the branches vary according to the specificity of the branching sucrase considered.
De manière préférée, les saccharases de branchement de la famille GH70 selon l’invention catalysent des ramifications comprenant de préférence 1 à 3 unités glucosyle, de préférence 1 ou 2 unités glucosyle selon des liaisons alpha-1 ,2 et/ou des liaisons alpha-Preferably, the branching sucrases of the GH70 family according to the invention catalyze branches preferably comprising 1 to 3 glucosyl units, preferably 1 or 2 glucosyl units according to alpha-1, 2 and / or alpha-bonds.
1.3. 1.3.
Ainsi, dans un mode de réalisation, la partie glucidique du polyglucoside d’alkyle de formule (I) se présente sous la forme d’une chaîne linéaire comprenant des ramifications comportant de 1 à 3 unités glucosyle, de préférence 1 ou 2 unités glucosyle, lesdites ramifications étant liées à la chaîne linéaire selon des liaisons alpha-1 ,2 et/ou des liaisons alpha-1 ,3. Thus, in one embodiment, the carbohydrate part of the alkyl polyglucoside of formula (I) is in the form of a linear chain comprising branches comprising from 1 to 3 glucosyl units, preferably 1 or 2 glucosyl units, said branches being linked to the linear chain via alpha-1, 2 bonds and / or alpha-1, 3 bonds.
Les inventeurs ont démontré que, de manière surprenante, certaines a- transglucosylases de la famille GH70 sont aptes à transférer des unités glucosyle sur la partie glucidique d’un glucoside d’alkyle de formule (II) tel que décrit ci-avant via un mécanisme d’élongation glucosidique. The inventors have demonstrated that, surprisingly, certain α-transglucosylases of the GH70 family are capable of transferring glucosyl units onto the carbohydrate part of an alkyl glucoside of formula (II) as described above via a mechanism glucosidic elongation.
Selon un mode de réalisation, Ga-transglucosylase de la famille GH70 est une glucane- saccharase de la famille GH70. According to one embodiment, Ga-transglucosylase of the GH70 family is a glucansucrase of the GH70 family.
Lorsque Ga-transglucosylase de la famille GH70 est une glucane-saccharase de la famille GH70, on obtient avantageusement un polyglucoside d’alkyle dont la partie glucidique comprend au moins 50%, de préférence 60%, de préférence encore 80% de liaisons choisies dans le groupe comprenant des liaisons alpha-1 ,6 et des liaisons alpha-When Ga-transglucosylase of the GH70 family is a glucansucrase of the GH70 family, an alkyl polyglucoside is advantageously obtained, the carbohydrate part of which comprises at least 50%, preferably 60%, more preferably 80% of bonds chosen from the group comprising alpha-1, 6 bonds and alpha- bonds
1 .3, le reste des liaisons étant avantageusement des liaisons alpha-1 ,4. En outre, les glucane-saccharases de la famille GH70 selon l’invention rendent possible l’obtention d’un polyglucoside d’alkyle ayant une longue chaîne glucosidique, avec n+m tel que défini ci-dessus. 1 .3, the remainder of the bonds being advantageously alpha-1, 4 bonds. In addition, the glucansucrases of the GH70 family according to the invention make it possible to obtain an alkyl polyglucoside having a long glucosidic chain, with n + m as defined above.
La glucane-saccharase de la famille GH70 de l’invention a de préférence pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 1 , SEQ ID NO : 2, SEQ ID NO : 3, , SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11 , SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14 et SEQ ID NO : 15. The glucansucrase of the GH70 family of the invention preferably has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,, SEQ ID NO : 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.
Les inventeurs ont pour la première fois montré que les glucane-saccharases de la famille GH70 de l’invention sont capables de rallonger des glucosides d’alkyle de formule (II) de plus de 3 unités glucosyle à partir de saccharose. The inventors have for the first time shown that the glucansucrases of the GH70 family of the invention are capable of extending the alkyl glucosides of formula (II) by more than 3 glucosyl units from sucrose.
Avantageusement, lorsque la glucane-saccharase de la famille GH70 selon l’invention a pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 1 , SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 8, SEQ ID NO : 9 et SEQ ID NO : 12, on obtient un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 80% de liaisons alpha-1 ,6, le reste des liaisons étant avantageusement des liaisons alpha-1 ,3. Advantageously, when the glucansucrase of the GH70 family according to the invention has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO : 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 12, an alkyl polyglucoside of formula (I) is obtained in which the carbohydrate part comprises at least 80% of bonds alpha-1, 6, the remainder of the bonds being advantageously alpha-1, 3 bonds.
Avantageusement, lorsque la glucane-saccharase de la famille GH70 selon l’invention a pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 1 , SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 5, SEQ ID NO : 6, et SEQ ID NO : 12, on obtient un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 90%, de préférence au moins 95% de liaisons alpha-1 ,6, le reste des liaisons étant avantageusement des liaisons alpha-1 ,3. Advantageously, when the glucansucrase of the GH70 family according to the invention has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO : 5, SEQ ID NO: 6, and SEQ ID NO: 12, an alkyl polyglucoside of formula (I) is obtained, the carbohydrate part of which comprises at least 90%, preferably at least 95% of alpha-1 bonds, 6, the rest of the bonds being advantageously alpha-1, 3 bonds.
Avantageusement, lorsque la glucane-saccharase de la famille GH70 selon l’invention a pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 10, SEQ ID NO : 14, et SEQ ID NO : 15 on obtient un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 90% de liaisons alpha-1 ,3, le reste des liaisons étant avantageusement des liaisons alpha-1 ,6. Advantageously, when the glucansucrase of the GH70 family according to the invention has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 15, a alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 90% of alpha-1, 3 bonds, the remainder of the bonds advantageously being alpha-1, 6 bonds.
Avantageusement, lorsque la glucane-saccharase de la famille GH70 selon l’invention a pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 8 et SEQ ID NO :9, on obtient un polyglucoside d’alkyle de formule (I) dont le la partie glucidique comprend au moins 70% de liaisons alpha-1 ,6, le reste des liaisons étant avantageusement des liaisons alpha-1 ,3. Advantageously, when the glucansucrase of the GH70 family according to the invention has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 8 and SEQ ID NO: 9, an alkyl polyglucoside of formula is obtained. (I) in which the carbohydrate part comprises at least 70% of alpha-1, 6 bonds, the remainder of the bonds advantageously being alpha-1, 3 bonds.
Avantageusement, lorsque la glucane-saccharase de la famille GH70 selon l’invention a pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 4 on obtient un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 50% de liaisons alpha-1 ,6, le reste des liaisons étant avantageusement des liaisons alpha-1 ,4. Advantageously, when the glucansucrase of the GH70 family according to the invention has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 4, an alkyl polyglucoside of formula (I) is obtained, the carbohydrate part of which comprises at least 50% of alpha-1, 6 bonds, the remainder of the bonds advantageously being alpha-1, 4 bonds.
Selon un autre mode de réalisation, Ga-transglucosylase de la famille GH70 selon l’invention est une saccharase de branchement GH70. According to another embodiment, Ga-transglucosylase of the GH70 family according to the invention is a GH70 branching sucrase.
Lorsque Ga-transglucosylase de la famille GH70 est une saccharase de branchement, on obtient avantageusement un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 50%, de préférence 60%, de préférence encore 80% de liaisons dans le groupe comprenant alpha-1 ,2, alpha-1 ,3 et leurs mélanges ; le reste des liaisons étant avantageusement des liaisons alpha-1 ,6. When Ga-transglucosylase of the GH70 family is a branching sucrase, an alkyl polyglucoside of formula (I) is advantageously obtained, the carbohydrate part of which comprises at least 50%, preferably 60%, more preferably 80% of bonds in the group comprising alpha-1, 2, alpha-1, 3 and mixtures thereof; the rest of the bonds being advantageously alpha-1,6 bonds.
La saccharase de branchement de la famille GH70 selon l’invention a de préférence pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21 , SEQ ID NO : 22, et SEQ ID NO : 23. The branching sucrase of the GH70 family according to the invention preferably has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23.
Avantageusement, lorsque la saccharase de branchement de la famille GH70 a pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 20, et SEQ ID NO : 22 on obtient un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 80% de liaisons alpha-1 ,2, de préférence 100% de liaisons alpha-1 ,2. Advantageously, when the branching sucrase of the GH70 family has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, and SEQ ID NO: 22, obtains an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 80% of alpha-1, 2 bonds, preferably 100% of alpha-1, 2 bonds.
Avantageusement, lorsque la saccharase de branchement GH70 a pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 18, SEQ ID NO : 19, et SEQ ID NO : 23 on obtient un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 80%, de préférence 90%, de préférence encore 95%, de préférence encore 98%, de liaisons alpha-1 ,3, de préférence encore 100% de liaisons alpha-1 ,3. Advantageously, when the GH70 branching sucrase has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 23, an alkyl polyglucoside of formula ( I) in which the carbohydrate part comprises at least 80%, preferably 90%, more preferably 95%, more preferably 98%, of alpha-1, 3 bonds, more preferably 100% of alpha-1, 3 bonds.
Avantageusement, lorsque la saccharase de branchement GH70 a pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 21 on obtient un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 60% de liaisons alpha-1 ,2, le reste des liaisons étant avantageusement des liaisons alpha- 1 ,6. Advantageously, when the GH70 branching sucrase has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 21, an alkyl polyglucoside of formula (I) is obtained, the carbohydrate part of which comprises at least 60% of bonds alpha-1, 2, the remainder of the bonds being advantageously alpha-1, 6 bonds.
Mise en œuyre du procédé Implementation of the process
L’étape i) peut être conduite en mettant en contact simultanément ou successivement le glucoside d’alkyle de formule (II) avec plusieurs a-transglucosylases de la famille GH70. Dans un mode de réalisation, l’étape i) comprend une sous-étape io) de mise en contact du glucoside d’alkyle de formule (II) avec un mélange d’une ou plusieurs glucane- saccharase(s) GH70 et d’une ou plusieurs enzyme(s) de branchement GH70. Step i) can be carried out by bringing the alkyl glucoside of formula (II) into contact simultaneously or successively with several α-transglucosylases of the GH70 family. In one embodiment, step i) comprises a sub-step io) of bringing the alkyl glucoside of formula (II) into contact with a mixture of one or more glucansucrase (s) GH70 and of one or more GH70 branching enzyme (s).
Dans cette sous-étape io), on utilise de préférence un ratio (glucane-saccharase) : (saccharase de branchement), exprimé en unité d’activité enzymatique, compris entre 0,01 et 10, les activités de chacune des enzymes utilisées variant avantageusement entre 0,2 et 2 U. ml 1. In this sub-step io), a ratio (glucansucrase): (branching sucrase), expressed in units of enzymatic activity, of between 0.01 and 10, is preferably used, the activities of each of the enzymes used varying advantageously between 0.2 and 2 U. ml 1 .
Sans vouloir être liés par une théorie particulière, les inventeurs pensent que la mise en contact d’un glucoside d’alkyle de formule (II) avec un mélange de glucane- saccharase^) et de saccharase(s) de branchement selon l’invention lors de l’étape io) favorise l’amorçage de la réaction d’élongation. Without wishing to be bound by a particular theory, the inventors believe that bringing an alkyl glucoside of formula (II) into contact with a mixture of glucansucrase ^) and of branching sucrase (s) according to the invention during step io) promotes the initiation of the elongation reaction.
Dans un mode de réalisation, l’étape i) comprend une étape h) de mise en contact du glucoside d’alkyle de formule (II) avec une ou plusieurs glucane-saccharase(s) de la famille GH70. In one embodiment, step i) comprises step h) of contacting the alkyl glucoside of formula (II) with one or more glucansucrase (s) of the GH70 family.
Sans vouloir être liés par une théorie, les inventeurs pensent que ce mode de réalisation permet dans un premier temps de favoriser l’élongation de la partie glucidique sous forme d’une longue chaîne linéaire. Without wishing to be bound by a theory, the inventors believe that this embodiment firstly makes it possible to promote the elongation of the carbohydrate part in the form of a long linear chain.
Dans un mode de réalisation, l’étape i) comprend la mise en contact du glucoside d’alkyle de formule (II) avec une ou plusieurs glucane-saccharase(s) de la famille GH70 puis une étape b) de mise en contact du glucoside d’alkyle obtenu à l’issue de l’étape h) avec une ou plusieurs saccharase(s) de branchement de la famille GH70. In one embodiment, step i) comprises bringing the alkyl glucoside of formula (II) into contact with one or more glucansucrase (s) of the GH70 family, then a step b) of bringing the alkyl glucoside into contact. alkyl glucoside obtained at the end of step h) with one or more branching sucrase (s) of the GH70 family.
Sans vouloir être liés par une théorie, les inventeurs pensent que ce mode de réalisation permet dans un premier temps de favoriser l’élongation de la partie glucidique sous forme d’une longue chaîne linéaire, puis dans un second temps de favoriser l’élongation de la partie glucidique sous forme de ramifications. Without wishing to be bound by a theory, the inventors believe that this embodiment firstly makes it possible to promote the elongation of the carbohydrate part in the form of a long linear chain, then secondly to promote the elongation of the carbohydrate part in the form of ramifications.
Ce mode de réalisation a également pour avantage d’augmenter la diversité de structures chimiques obtenues. This embodiment also has the advantage of increasing the diversity of chemical structures obtained.
L’utilisation de saccharases de branchement de la famille GH70 selon l’invention permet en effet de contrôler le nombre de branchements introduits, en fonction des conditions réactionnelles, de sorte à obtenir un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 50% de liaisons alpha-1 ,6, le reste des liaisons glucosidique étant des liaisons alpha-1 ,3 et/ou des liaisons alpha-1 ,2, la somme des pourcentages de liaisons alpha-1 ,6, des pourcentages de liaisons alpha-1 ,3 et de pourcentages de liaisons alpha-1 ,2 étant égale à 100%. On peut par exemple obtenir un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 60% de liaisons alpha-1 ,6, au moins 2% de liaisons alpha- 1 ,3 et au moins 2% de liaisons alpha-1 ,6, de préférence entre 2% et 35% de liaisons alpha- 1 ,3 et au moins 1 % de liaisons alpha-1 ,2, de préférence entre 2% et 35% de liaisons alpha- 1 ,2, la somme des pourcentages de liaisons alpha-1 ,6, des pourcentages de liaisons alpha- 1 ,3 et de pourcentages de liaisons alpha-1 ,2 étant égale à 100%. The use of branching sucrases of the GH70 family according to the invention in fact makes it possible to control the number of branches introduced, as a function of the reaction conditions, so as to obtain an alkyl polyglucoside of formula (I) of which the carbohydrate part comprises at least 50% of alpha-1, 6 bonds, the rest of the glucosidic bonds being alpha-1, 3 bonds and / or alpha-1, 2 bonds, the sum of the percentages of alpha-1, 6 bonds, of the percentages of alpha-1, 3 bonds and percentages of alpha-1 bonds, 2 being equal to 100%. It is possible, for example, to obtain an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 60% of alpha-1, 6 bonds, at least 2% of alpha-1, 3 bonds and at least 2% of bonds alpha-1, 6, preferably between 2% and 35% of alpha-1, 3 bonds and at least 1% of alpha-1, 2 bonds, preferably between 2% and 35% of alpha-1, 2 bonds, the sum of the percentages of alpha-1, 6 bonds, of the percentages of alpha-1, 3 bonds and of percentages of alpha-1 bonds, 2 being equal to 100%.
Dans un mode de réalisation particulier, l’étape i) est effectuée en solution à un pH contrôlé, en particulier par l’emploi d’une solution tampon. In a particular embodiment, step i) is carried out in solution at a controlled pH, in particular by the use of a buffer solution.
L’étape i) est avantageusement conduite à une valeur de pH comprise entre 5 et 8.Step i) is advantageously carried out at a pH value of between 5 and 8.
L’étape i) est avantageusement conduite avec une concentration initiale de saccharose ou d’un analogue de saccharose comprise entre 20 et 660 g.L 1. Step i) is advantageously carried out with an initial concentration of sucrose or of a sucrose analogue of between 20 and 660 gL 1 .
L’étape i) est de préférence réalisée avec un rapport molaire polyglucoside d’alkyle de formule (II) : saccharose ou analogue de saccharose compris entre 0,001 et 10, de préférence entre 0,001 et 5, de préférence 0,001 et 0,3, de préférence entre 0,002 et 0,006. Step i) is preferably carried out with an alkyl polyglucoside molar ratio of formula (II): sucrose or sucrose analog of between 0.001 and 10, preferably between 0.001 and 5, preferably 0.001 and 0.3, of preferably between 0.002 and 0.006.
De manière importante, les inventeurs ont montré que l’élongation de la chaîne peut être modulée en fonction du rapport molaire polyglucoside d’alkyle de formule (II) : saccharose ou analogue de saccharose. Sans vouloir être liés par une théorie particulière, les inventeurs considèrent que plus le rapport molaire polyglucosides d’alkyle de formule (II) : saccharose ou analogue de saccharose est grand, moins le saccharose est disponible pour être donneur et donc moins l’accepteur est rallongé. Importantly, the inventors have shown that the elongation of the chain can be modulated depending on the molar ratio of alkyl polyglucoside of formula (II): sucrose or sucrose analog. Without wishing to be bound by a particular theory, the inventors consider that the greater the molar ratio of alkyl polyglucosides of formula (II): sucrose or sucrose analog, the less sucrose is available to be a donor and therefore the less the acceptor is. extension.
De manière avantageuse, l’étape i) est réalisée avec un rapport molaire polyglucosides d’alkyle de formule (II) : saccharose ou analogue de saccharose compris entre 0,05 et 5. L’étape i) est de préférence mise en oeuvre à une température comprise entre 10°C et 80°C. Advantageously, step i) is carried out with an alkyl polyglucoside molar ratio of formula (II): sucrose or sucrose analog of between 0.05 and 5. Step i) is preferably carried out at a temperature between 10 ° C and 80 ° C.
De préférence, l’étape i) est conduite avec Ga-transglucosylase de la famille GH70 sous forme solide, en solution, en suspension, ou immobilisée. Preferably, step i) is carried out with Ga-transglucosylase of the GH70 family in solid form, in solution, in suspension, or immobilized.
Le procédé peut comprendre en outre avantageusement une étape ii) de purification du polyglucoside d’alkyle de formule (I) obtenu à l’issue de l’étape i). The method can advantageously further comprise a step ii) of purification of the alkyl polyglucoside of formula (I) obtained at the end of step i).
Polyglucosides d’alkyle susceptibles d’être obtenus selon le procédé de l’inventionPolyalkyl glucosides obtainable according to the process of the invention
Le polyglucoside d’alkyle de formule (I) obtenu par la mise en oeuvre du procédé tel que défini selon le premier objet de l’invention, en particulier à l’issue de l’étape i) constitue un deuxième objet de l’invention. L’invention a donc pour deuxième objet un polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention, ledit polyglucoside d’alkyle étant caractérisé en ce qu’il est de formule (I) : The alkyl polyglucoside of formula (I) obtained by carrying out the process as defined according to the first subject of the invention, in particular at the end of step i) constitutes a second subject of the invention . A second subject of the invention is therefore an alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention, said alkyl polyglucoside being characterized in that it is of formula ( I):
[Glc]m-[Glc]n(-0-R) (I) dans laquelle : [Glc] m - [Glc] n (-0-R) (I) in which:
R représente un groupe alkyle linéaire ou ramifié, saturé ou insaturé, comprenant entre 8 et 20 atomes de carbone, R represents a linear or branched, saturated or unsaturated alkyl group comprising between 8 and 20 carbon atoms,
[Glc]m-[Glc]n représente une partie glucidique linéaire ou ramifiée comprenant n+m unités glucosyle, n+m étant compris entre 3 et 200. [Glc] m - [Glc] n represents a linear or branched carbohydrate part comprising n + m glucosyl units, n + m being between 3 and 200.
Le polyglucoside d’alkyle de formule (I) susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention peut présenter une grande diversité structurale, notamment au niveau de sa partie glucidique, en particulier en termes de taille, de structure (ramifiée ou non) et de nature des liaisons osidiques. The alkyl polyglucoside of formula (I) capable of being obtained by carrying out the process in accordance with the first subject of the invention may exhibit great structural diversity, in particular at the level of its carbohydrate part, in particular in terms of size, structure (branched or not) and nature of the osidic bonds.
Ainsi, le polyglucoside d’alkyle de formule (I) du deuxième objet de l’invention est tel que défini de manière détaillée dans le premier objet de l’invention, en particulier, le nombre d’atomes de carbone du groupe alkyle R, le nombre n+m d’unités glucosyle au sein de la partie glucidique [Glc]m-[Glc]n , ainsi que la nature des liaison au sein des fractions de la partie glucidique [Glc]m-[Glc]n et celle de la liaison entre le groupe alkyle R et la fraction glucidique [Glc]n sont tels que précédemment mentionnés. Thus, the alkyl polyglucoside of formula (I) of the second subject of the invention is as defined in detail in the first subject of the invention, in particular, the number of carbon atoms of the alkyl group R, the number n + m of glucosyl units within the carbohydrate part [Glc] m - [Glc] n , as well as the nature of the bonds within the fractions of the carbohydrate part [Glc] m - [Glc] n and that of the bond between the alkyl group R and the carbohydrate moiety [Glc] n are as previously mentioned.
Dans un mode de réalisation, le polyglucoside d’alkyle de formule (I) susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention, est un polyglucoside d’alkyle de formule (I) dans lequel R représente un groupe alkyle comprenant de 8 à 12 atomes de carbone et dans lequel le nombre n+m est compris entre 7 et 200, de préférence entre 8 et 200, de préférence entre 9 et 200, de préférence encore entre 10 et 200. Dans ce mode de réalisation, n+m peut notamment être compris entre 7 et 50, de préférence entre 8 et 50, de préférence entre 9 et 50, et de préférence encore entre 10 et 50. In one embodiment, the alkyl polyglucoside of formula (I) obtainable by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which R represents an alkyl group comprising from 8 to 12 carbon atoms and in which the number n + m is between 7 and 200, preferably between 8 and 200, preferably between 9 and 200, more preferably between 10 and 200. In this embodiment, n + m can in particular be between 7 and 50, preferably between 8 and 50, preferably between 9 and 50, and more preferably between 10 and 50.
Dans un mode de réalisation, le polyglucoside d’alkyle de formule (I) susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention, est un polyglucoside d’alkyle de formule (I) dans lequel R représente un groupe alkyle comprenant de 12 à 20 atomes de carbone et dans lequel n+m est compris entre 3 et 200, de préférence entre 4 et 200, de préférence entre 5 et 200, et de préférence encore entre 10 et 200. Dans ce mode de réalisation, n+m peut notamment être compris entre 3 et 50, de préférence entre 4 et 50, de préférence entre 5 et 50, et de préférence encore entre 10 et 50. Dans un mode de réalisation, le polyglucoside d’alkyle de formule (I) susceptible d’être obtenu par du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dans lequel : In one embodiment, the alkyl polyglucoside of formula (I) obtainable by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which R represents an alkyl group comprising from 12 to 20 carbon atoms and in which n + m is between 3 and 200, preferably between 4 and 200, preferably between 5 and 200, and more preferably between 10 and 200. In this embodiment, n + m can in particular be between 3 and 50, preferably between 4 and 50, preferably between 5 and 50, and more preferably between 10 and 50. In one embodiment, the alkyl polyglucoside of formula (I) obtainable by the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which:
- lorsque R représente un groupe alkyle comprenant de 8 à 12 atomes de carbone, n+m est compris entre 7 et 200, de préférence entre 8 et 200, de préférence entre 9 et 200, de préférence encore entre 10 et 200 et n+m peut notamment être compris entre 7 et 50, de préférence entre 8 et 50, de préférence entre 9 et 50, et de préférence encore entre 10 et 50 ; et - when R represents an alkyl group comprising from 8 to 12 carbon atoms, n + m is between 7 and 200, preferably between 8 and 200, preferably between 9 and 200, more preferably between 10 and 200 and n + m may in particular be between 7 and 50, preferably between 8 and 50, preferably between 9 and 50, and more preferably between 10 and 50; and
- lorsque R représente un groupe alkyle comprenant de 12 à 20 atomes de carbone, n+m est compris entre 3 et 200, de préférence entre 4 et 200, de préférence entre 5 et 200, et de préférence encore entre 10 et 200 et peut notamment être compris entre 3 et 50, de préférence entre 4 et 50, de préférence entre 5 et 50, et de préférence encore entre 10 et 50. - When R represents an alkyl group comprising from 12 to 20 carbon atoms, n + m is between 3 and 200, preferably between 4 and 200, preferably between 5 and 200, and more preferably between 10 and 200 and can in particular be between 3 and 50, preferably between 4 and 50, preferably between 5 and 50, and more preferably between 10 and 50.
Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) constitué essentiellement d’un mélange de molécules de polyglucoside d’alkyle de formule (I), ledit mélange présentant un degré de polymérisation moyen compris entre 3 et 25, de préférence compris entre 5 et 25, de préférence compris entre 6 et 25, de préférence entre 7 et 25, de préférence encore entre 8 et 25. Ce mélange est essentiellement constitué de molécules de polyglucoside d’alkyle de formule (I), c’est-à-dire qu’il est constitué d’au moins 90% en poids desdits polyglucosides d’alkyle, de préférence d’au moins 95% en poids desdits polyglucosides d’alkyle, de préférence encore d’au moins 97% en poids desdits polyglucosides d’alkyle. In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) consisting essentially of a mixture of molecules of alkyl polyglucoside of formula (I), said mixture exhibiting an average degree of polymerization between 3 and 25, preferably between 5 and 25, preferably between 6 and 25, preferably between 7 and 25, preferably still between 8 and 25. This mixture consists essentially of alkyl polyglucoside molecules of formula (I), that is to say that it consists of at least 90% by weight of said alkyl polyglucosides, preferably at least 95% by weight of said alkyl polyglucosides, more preferably at least 97% by weight of said alkyl polyglucosides.
Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 50%, de préférence 60%, de préférence encore 80% de liaisons alpha-1 ,6 ou de liaisons alpha-1 ,3, le reste des liaisons étant avantageusement des liaisons alpha-1 ,4 et/ou de liaison alpha- 1 ,2. In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 50 %, preferably 60%, more preferably 80% of alpha-1, 6 bonds or alpha-1, 3 bonds, the remainder of the bonds advantageously being alpha-1, 4 bonds and / or alpha-1 bond, 2.
Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 50%, de préférence au moins 70% de liaisons alpha-1 ,6, le reste des liaisons étant avantageusement des liaisons alpha-1 ,3. Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 60% de liaisons alpha-1 ,6, au moins 2% de liaisons alpha-1 ,3 et au moins 2% de liaisons alpha- 1 ,6, de préférence entre 2% et 35% de liaisons alpha-1 ,3 et au moins 1 % de liaisons alpha-In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 50 %, preferably at least 70% of alpha-1, 6 bonds, the remainder of the bonds being advantageously alpha-1, 3 bonds. In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 60 % alpha-1, 6 bonds, at least 2% alpha-1, 3 bonds and at least 2% alpha-1, 6 bonds, preferably between 2% and 35% alpha-1, 3 bonds and at least minus 1% alpha- bonds
1.2, de préférence entre 2% et 35% de liaisons alpha-1 ,2, la somme des pourcentages de liaisons alpha-1 ,6, des pourcentages de liaisons alpha-1 ,3 et de pourcentages de liaisons alpha-1 ,2 étant égale à 100%. 1.2, preferably between 2% and 35% of alpha-1, 2 bonds, the sum of the percentages of alpha-1, 6 bonds, of the percentages of alpha-1, 3 bonds and of percentages of alpha-1, 2 bonds being equal to 100%.
Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 90% de liaisons alpha-1 ,3, le reste des liaisons étant avantageusement des liaisons alpha-1 ,6. In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 90 % alpha-1, 3 bonds, the remainder of the bonds being advantageously alpha-1, 6 bonds.
Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 50% de liaisons alpha-1 ,6, le reste des liaisons étant avantageusement des liaisons alpha-1 ,4. In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 50 % alpha-1, 6 bonds, the remainder of the bonds being advantageously alpha-1, 4 bonds.
Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 70% de liaisons alpha-1 ,6, le reste des liaisons étant avantageusement des liaisons alpha-1 ,3. In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 70 % of alpha-1, 6 bonds, the remainder of the bonds being advantageously alpha-1, 3 bonds.
Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 80% de liaisons alpha-1 ,2, le reste des liaisons étant avantageusement des liaisons alpha-1 ,3. In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 80 % alpha-1, 2 bonds, the remainder of the bonds being advantageously alpha-1, 3 bonds.
Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 80%, de préférence 90%, de préférence encore 95%, de préférence encore 98%, de liaisons alpha-In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 80 %, preferably 90%, more preferably 95%, more preferably 98%, alpha- bonds
1 .3, le reste des liaisons étant avantageusement des liaisons alpha-1 ,2. 1 .3, the remainder of the bonds being advantageously alpha-1, 2 bonds.
Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 80%, de préférence 90%, de préférence encore 95%, de préférence encore 98%, de liaisons alpha-In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 80 %, preferably 90%, more preferably 95%, more preferably 98%, alpha- bonds
1 ,3, le reste des liaisons étant avantageusement des liaisons alpha-1 ,2. Dans un mode de réalisation, le polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé conforme au premier objet de l’invention est un polyglucoside d’alkyle de formule (I) dont la partie glucidique comprend au moins 60% de liaisons alpha-1 ,2, le reste des liaisons étant avantageusement des liaisons alpha-1 ,6. 1, 3, the remainder of the bonds being advantageously alpha-1, 2 bonds. In one embodiment, the alkyl polyglucoside capable of being obtained by carrying out the process in accordance with the first subject of the invention is an alkyl polyglucoside of formula (I) in which the carbohydrate part comprises at least 60 % alpha-1, 2 bonds, the remainder of the bonds being advantageously alpha-1, 6 bonds.
Utilisation du polyglucoside d’alkyle susceptible d’être obtenu par le procédé de l’inventionUse of the alkyl polyglucoside obtainable by the process of the invention
L’invention a pour troisième objet l’utilisation d’un polyglucoside d’alkyle susceptible d’être obtenu selon le procédé conforme à l’invention ou tel que défini dans le deuxième objet de l’invention, à titre de surfactant. The third subject of the invention is the use of an alkyl polyglucoside obtainable according to the process according to the invention or as defined in the second subject of the invention, as a surfactant.
Utilisation des a-transglucosylases selon l’invention pour l’élongation glucosidigue d’un glucoside d’alkyle. Use of α-transglucosylases according to the invention for the glucosidic extension of an alkyl glucoside.
L’invention a pour quatrième objet l’utilisation d’une a-transglucosylase de la famille GH70 telle que définie au premier objet de l’invention, en particulier d’une glucane-saccharase de la famille GH70 ou d’une saccharase de branchement de la famille GH70 telle que définie au premier objet de l’invention, pour l’élongation glucosidique d’un glucoside d’alkyle, en particulier d’un glucoside d’alkyle de formule (II) tel que défini au premier objet de l’invention. A fourth subject of the invention is the use of an α-transglucosylase of the GH70 family as defined in the first subject of the invention, in particular of a glucansucrase of the GH70 family or of a branching sucrase. of the GH70 family as defined in the first subject of the invention, for the glucosidic elongation of an alkyl glucoside, in particular of an alkyl glucoside of formula (II) as defined in the first subject of 'invention.
La présente invention est illustrée par les exemples de réalisation suivants, auxquels elle n’est cependant pas limitée. The present invention is illustrated by the following exemplary embodiments, to which it is, however, not limited.
EXEMPLES EXAMPLES
Exemple 1 : Enzymes, organismes d’origine et spécificité Example 1: Enzymes, organisms of origin and specificity
Les enzymes utilisées sont listées dans le Tableau 1. The enzymes used are listed in Table 1.
La séquence SEQ ID NO :3 correspond au mutant de l’enzyme DSR-M D5 sur la position 624 où le tryptophane sauvage est remplacé par une alanine. The sequence SEQ ID NO: 3 corresponds to the mutant of the DSR-M D5 enzyme at position 624 where the wild-type tryptophan is replaced by an alanine.
Tableau 1: a-transglucosylases GH70 et spécificités de liaisons lors de la synthèse du polymère naturel. P :polymerase i.e. glucane-saccharase GH70 ; B : saccharase de branchement GH70 (« Branching sucrase » en anglais) ; Bif : Bifonctionnelle (enzyme ayant deux domaines catalytiques) ; nd= non déterminé. Table 1: GH70 α-transglucosylases and specificities of bonds during the synthesis of the natural polymer. P: polymerase i.e. glucansucrase GH70; B: GH70 branching sucrase; Bif: Bifunctional (enzyme having two catalytic domains); nd = not determined.
[Table 1] [Table 1]
Exemple 2 : Expression hétérologue des enzymes chez Escherichia coli Example 2: Heterologous expression of enzymes in Escherichia coli
Les gènes codant pour les enzymes citées ci-dessus sont clonés dans des vecteurs permettant l’expression recombinante chez E. coli, sous le contrôle de promoteurs pBad ou pET. The genes encoding the enzymes mentioned above are cloned into vectors allowing recombinant expression in E. coli, under the control of pBad or pET promoters.
Les enzymes recombinantes sont produites à partir des cellules d’E. coli BL21 DE3 Al ou BL21 DE3 Star transformées avec le plasmide contenant le gène des enzymes ciblées (voir Tableau 2). Recombinant enzymes are produced from the cells of E. coli BL21 DE3 A1 or BL21 DE3 Star transformed with the plasmid containing the gene for the targeted enzymes (see Table 2).
[Table 2] [Table 2]
Tableau 2 : plasmides des enzymes utilisées et systèmes d’expression adaptés aux différentes enzymes. (Gly : Glycérol ; Glu : Glucose ; a-Lac : a-Lactose ; L-Ara : L- Arabinose) Trois cents microlitres du mélange de transformation permettent d’ensemencer un volume de 30 ml_ de préculture en milieu LB (abrégé de l’anglais « Lysogeny Broth »), supplémenté avec 100 pg.mL 1 d’ampicilline. Chaque préculture est incubée à 37°C pendant 10 heures sous une agitation de 150 rpm. Des cultures d’1L en milieu ZYM5052 modifié dont les caractéristiques sont présentées dans le Tableau 2 sont ensemencées à une densité optique (DO) DOA=6oonm initiale de 0,05 à partir de la préculture de la veille, puis mises à incuber 26 heures à 21 °C et à 150 rpm. Table 2: plasmids of the enzymes used and expression systems adapted to the different enzymes. (Gly: Glycerol; Glu: Glucose; a-Lac: a-Lactose; L-Ara: L- Arabinose) Three hundred microliters of the transformation mixture make it possible to inoculate a volume of 30 ml of preculture in LB medium (abbreviated from l (English "Lysogeny Broth"), supplemented with 100 pg.mL 1 of ampicillin. Each preculture is incubated at 37 ° C. for 10 hours with stirring at 150 rpm. Cultures of 1L in modified ZYM5052 medium, the characteristics of which are presented in Table 2, are inoculated at an optical density (OD) OD A = 6,000 nm initial of 0.05 from the preculture of the day before, then set to incubate 26 hours at 21 ° C and 150 rpm.
En fin de fermentation, les milieux de culture sont centrifugés pendant 15 min à 6500 rpm et à une température de 4°C. Les culots cellulaires sont concentrés à une DO de 80 dans du tampon d’activité (voir Tableau 2). Les cellules sont ensuite cassées aux ultrasons selon le protocole suivant : 5 cycles de 20 secondes à 30% de la puissance maximale de la sonde, à froid (bain de glace), espacés de 4 minutes de repos dans la glace. Les surnageants de sonication contenant les enzymes d’intérêt solubles sont ensuite récupérés après 30 minutes de centrifugation (10000 rpm, 10°C) et conservés à 4°C. At the end of fermentation, the culture media are centrifuged for 15 min at 6500 rpm and at a temperature of 4 ° C. Cell pellets are concentrated to an OD of 80 in activity buffer (see Table 2). The cells are then broken with ultrasound according to the following protocol: 5 cycles of 20 seconds at 30% of the maximum power of the probe, cold (ice bath), spaced 4 minutes apart in ice. The Sonication supernatants containing the soluble enzymes of interest are then recovered after 30 minutes of centrifugation (10,000 rpm, 10 ° C) and stored at 4 ° C.
Exemple 3 : Détermination de l’activité enzymatique par dosage des sucres réducteurs au DNS. Example 3: Determination of enzymatic activity by determination of reducing sugars in DNS.
L’activité enzymatique des glucane-saccharases et des saccharases de branchement est déterminée en mesurant la vitesse initiale de production des sucres réducteurs à l’aide de la méthode à l’acide dinitrosalicilique (DNS) (Miller, 1959). The enzymatic activity of glucansucrases and branching sucrases is determined by measuring the initial rate of production of reducing sugars using the dinitrosalicilic acid (DNS) method (Miller, 1959).
Une unité enzymatique représente la quantité d’enzyme qui libère une pmole de fructose par minute, à 30 °C, pour une concentration initiale de saccharose de 100 g.L 1 dans les conditions de tampon d’activité adéquate. One enzymatic unit represents the quantity of enzyme which releases one pmole of fructose per minute, at 30 ° C., for an initial sucrose concentration of 100 gL 1 under the conditions of adequate activity buffer.
Au cours d’une cinétique d’1 mL de volume, 100 pL de milieu réactionnel sont prélevés et la réaction est stoppée par ajout d’un volume équivalent de DNS. Les échantillons sont ensuite chauffés 5 min à 95 °C, refroidis dans la glace, dilués au demi dans de l’eau, et l’absorbance est lue à 540 nm. Une gamme étalon de 0 à 2 g.L 1 de fructose permet d’établir le lien entre la valeur d’absorbance et la concentration en sucres réducteurs. During a kinetics of 1 ml of volume, 100 μL of reaction medium are taken and the reaction is stopped by adding an equivalent volume of DNS. The samples are then heated for 5 min at 95 ° C., cooled in ice, diluted to half in water, and the absorbance is read at 540 nm. A standard range of 0 to 2 gL 1 of fructose makes it possible to establish the link between the absorbance value and the concentration of reducing sugars.
Réactions d’accepteur Acceptor reactions
Les réactions d’accepteurs sont réalisées dans un volume compris entre 1 mL et 15 mL et pour une concentration finale en saccharose comprise entre 146 et 730 mM. Les concentrations en alkylpoly-glucosides sont dépendantes de leur solubilité et varient entre : 20 et 50 mM pour l’octyl-monoglucoside, Acceptor reactions are carried out in a volume of between 1 mL and 15 mL and for a final sucrose concentration of between 146 and 730 mM. The concentrations of alkylpoly-glucosides are dependent on their solubility and vary between: 20 and 50 mM for octyl-monoglucoside,
10 et 30 mM pour le décyl-monoglucoside, 10 and 30 mM for decyl-monoglucoside,
1 et 5 mM pour le dodécyl-monoglucoside, 1 and 5 mM for dodecyl-monoglucoside,
10 et 20 mM pour le dodecyl diglucoside. 10 and 20 mM for dodecyl diglucoside.
Dans le cas du triton CG110 (mélange commercial d’APGs en C8 et C10), la réaction a été réalisée en présence de 2 mg à 10 mg de Triton CG110 par mL de réaction. In the case of triton CG110 (commercial mixture of C8 and C10 APGs), the reaction was carried out in the presence of 2 mg to 10 mg of Triton CG110 per mL of reaction.
La réaction est initiée par l’addition d’un volume de lysat cellulaire suffisant pour obtenir une activité enzymatique en réaction de 1 U.mL 1. Les réactions sont incubées à 37°C et agitées à 800 rpm. Au bout de 24h, les enzymes sont dénaturées à 95°C pendant 5 min. Les réactions sont conservées à -18°C avant analyse des produits de la réaction par chromatographie liquide haute performance (CLHP). The reaction is initiated by the addition of a volume of cell lysate sufficient to obtain an enzymatic activity in reaction of 1 U.mL 1 . The reactions are incubated at 37 ° C. and stirred at 800 rpm. After 24 h, the enzymes are denatured at 95 ° C. for 5 min. The reactions are stored at -18 ° C. before analysis of the reaction products by high performance liquid chromatography (HPLC).
Purification des produits de réaction Une étape de prépurification des hydroxy-lipides glucosylés issus de l’acide 11-hydroxy- undécanoïque est réalisée par flash chromatographie grâce à un système REVELERIS® X2 Flash Chromatography System (GRACE, USA) équipé d’une colonne C18 de 80 g. Les produits de glucosylation sont séparés des sucres libres résiduels dans les conditions suivantes : Purification of reaction products A step of prepurification of the glucosylated hydroxy-lipids obtained from 11-hydroxy-undecanoic acid is carried out by flash chromatography using a REVELERIS® X2 Flash Chromatography System (GRACE, USA) equipped with a C18 column of 80 g. The glucosylation products are separated from the residual free sugars under the following conditions:
-débit de 60 ml. min 1 , détection ELSD -0,5 volumes de colonne (VC) à 100% H2O -flow of 60 ml. min 1 , ELSD detection -0.5 column volumes (CV) at 100% H2O
-gradient passant de 0% H2O ultra-pure à 50% acétonitrile en 8,5 volumes de colonne (VC)-gradient from 0% ultra-pure H2O to 50% acetonitrile in 8.5 column volumes (CV)
-1 VC à 50% acétonitrile -1 VC at 50% acetonitrile
-retour à 100% H2O ultra pure en 0,2 VC -return to 100% ultra pure H2O in 0.2 VC
-équilibration à 100% H2O ultra pure pendant 1 ,5 VC - 100% ultra pure H2O equilibration for 1.5 VC
Les fractions contenant les produits de réactions élués entre 4 et 10 VC sont collectées, partiellement évaporés à l’évaporateur rotatif puis lyophilisés. Les échantillons sont stockés à température ambiante et à l’abri de l’humidité. The fractions containing the reaction products eluted between 4 and 10 VC are collected, partially evaporated on a rotary evaporator and then lyophilized. Samples are stored at room temperature and protected from moisture.
Techniques analytiques Analytical techniques
Pour l’analyse en CLHP, les milieux réactionnels sont dilués au demi dans de l’éthanol absolu. Cette dilution permet entre autres l’élimination des potentiels polymères de haute masse molaire par précipitation. For HPLC analysis, the reaction media are diluted halfway in absolute ethanol. This dilution allows, among other things, the elimination of potential polymers of high molar mass by precipitation.
La séparation des accepteurs lipidiques et de leurs formes glucosylées est effectuée par chromatographie en phase inverse avec une colonne Synergi™ Fusion-RP (porosité de 80 Â, granulométrie de 4 pm, greffage C18 avec terminaison polaire, Phenomenex, USA). Cette colonne est maintenue à 30°C sur un système CLHP Thermo U3000 couplé à un détecteur Corona CAD Véo (Charged Aérosol Detector) (Thermo Scientific, USA). La température de nébulisation est fixée à 50°C et le filtre réglé à 3,2 secondes. The separation of the lipid acceptors and their glucosylated forms is carried out by reverse phase chromatography with a Synergi ™ Fusion-RP column (porosity of 80 Å, particle size of 4 μm, C18 grafting with polar termination, Phenomenex, USA). This column is maintained at 30 ° C. on a Thermo U3000 HPLC system coupled to a Corona CAD Véo detector (Charged Aerosol Detector) (Thermo Scientific, USA). The nebulization temperature is set at 50 ° C and the filter set at 3.2 seconds.
La phase mobile est composée d’un mélange eau ultrapure (solvant A) / acétonitrile de qualité CLHP (solvant B) tous deux contenant 0,05% (v/v) d’acide formique. L’élution est réalisée à un débit de 1 mL.min 1 selon le gradient suivant : The mobile phase is composed of a mixture of ultrapure water (solvent A) / acetonitrile of HPLC grade (solvent B) both containing 0.05% (v / v) of formic acid. Elution is carried out at a flow rate of 1 mL.min 1 according to the following gradient:
- Entre 0 min et 5 min, une première phase d’élution avec 0% (v/v) de la voie B permet l’élimination des sucres simples résiduels (fructose, glucose, leucrose, saccharose résiduels ou éventuels oligosaccharides courts) ; - Between 0 min and 5 min, a first elution phase with 0% (v / v) of path B allows the elimination of residual simple sugars (fructose, glucose, leucrose, residual sucrose or possible short oligosaccharides);
- Une seconde phase de gradient allant de 0% est réalisée à 100% de la voie B pendant 25 minutes permet de séparer les différents composés lipidiques glucosylés ; - Une dernière phase de 5 minutes à 100% de la voie B permet la régénération de la colonne. - A second phase of gradient ranging from 0% is carried out at 100% of path B for 25 minutes makes it possible to separate the various glucosylated lipid compounds; - A final phase of 5 minutes at 100% of channel B allows the regeneration of the column.
Analyse par RMN NMR analysis
La caractérisation des produits de glucosylation des différents APG est réalisée par RMN. Les produits sont dilués dans D2O et en présence de triméthylsilylpropanoate de sodium deutéré (TSP-d4) utilisé comme référence interne. Les spectres 1 H ont été enregistrés sur un équipement Bruker Avance 500MHz à 298K avec une sonde 5 mm z-gradient TBI. Les données ont été acquises et traitées avec le logiciel TopSpin 3. The characterization of the glucosylation products of the different APGs is carried out by NMR. The products are diluted in D2O and in the presence of deuterated sodium trimethylsilylpropanoate (TSP-d4) used as internal reference. The 1 H spectra were recorded on Bruker Avance 500MHz equipment at 298K with a 5 mm z-gradient TBI probe. The data were acquired and processed with TopSpin 3 software.
Exemple 4 : Synthèse d’alkyle glucosides Example 4: Synthesis of alkyl glucosides
4.1 . Protocole général 4.1. General protocol
26 a-transglucosylases de la famille GH70 ont été testées pour leur aptitude à rallonger des alkyl-glycosides de structures et de tailles différentes : 26 α-transglucosylases of the GH70 family were tested for their ability to lengthen alkyl-glycosides of different structures and sizes:
- Octyl-3-D-glucoside, CsGi - Octyl-3-D-glucoside, CsGi
- Decyl- b -D-Glucoside, C10G1, - Decyl- b -D-Glucoside, C10G1,
- Dodecyl- b -D-Glucoside, C12G1 - Dodecyl- b -D-Glucoside, C12G1
- Dodécyl- b -D-maltoside, C12G2, - Dodecyl- b -D-maltoside, C12G2,
- Hexadecyl maltoside, C16G2 - Hexadecyl maltoside, C16G2
- Triton CG110 (DuPont, USA), mélange contenant majoritairement du CsGi, du C10G1 et minoritairement des APGs de degré de polymérisation supérieur. - Triton CG110 (DuPont, USA), mixture mainly containing CsGi, C10G1 and mainly APGs of higher degree of polymerization.
Les enzymes testées sont répertoriées dans le Tableau 3. Elles ont toutes été produites sous forme recombinante et exprimées chez Escherichia coli. The enzymes tested are listed in Table 3. They were all produced in recombinant form and expressed in Escherichia coli.
Les extraits enzymatiques obtenus par fermentation sont utilisés bruts après cassage des cellules et centrifugation des débris cellulaires . The enzymatic extracts obtained by fermentation are used crude after breaking the cells and centrifuging the cell debris.
Les réactions d’accepteurs mettant en jeu des GSs ont été réalisées dans un volume de 1 à 10 mL à des concentrations variables en APG dépendantes de leur solubilité dans l’eau (entre 5mM pour C12G1 et 30 mM pour CsGi) et des concentrations de saccharose variant entre 146 mM (50 g.L 1) et 1316 mM (450 g.L·1). The acceptor reactions involving GSs were carried out in a volume of 1 to 10 mL at variable concentrations of APG depending on their solubility in water (between 5 mM for C12G1 and 30 mM for CsGi) and concentrations of Sucrose varying between 146 mM (50 gL 1 ) and 1316 mM (450 gL · 1 ).
Toutes les réactions ont été réalisées en présence d’1 U.mL 1 d’enzyme, d’un tampon Acétate Na 50mM pH=5,75, incubées à 37°C et sous une agitation de 800 rpm. Au bout de 24 heures les enzymes sont dénaturées par incubation à 95°C pendant 5 minutes. En vue de leurs analyses par CLHP-CAD, les milieux réactionnels sont dilués au 1/2 dans de l’éthanol et centrifugés 5 min à 11 000 g afin d’éliminer les glucanes co-produits de réaction et les protéines floculées. All the reactions were carried out in the presence of 1 U.mL 1 of enzyme, of a 50 mM Na acetate buffer, pH = 5.75, incubated at 37 ° C. and with agitation at 800 rpm. After 24 hours the enzymes are denatured by incubation at 95 ° C. for 5 minutes. With a view to their analyzes by HPLC-CAD, the reaction media are diluted 1/2 in ethanol and centrifuged for 5 min at 11,000 g in order to remove the glucans reaction co-products and the flocculated proteins.
La séparation des différents produits de réaction est effectuée par chromatographie en phase inverse avec une colonne Synergi™ Fusion-RP 250 mm x 2 mm (porosité : 80 Â, granulométrie : 4 pm, Phenomenex, USA). Cette colonne est maintenue à 30°C sur un système CLHP Thermo Ultimate 3000 équipé d’un détecteur Corona Véo. La phase mobile est composée d’un mélange eau ultrapure (solvant A) / acétonitrile de qualité LC-MS (solvant B) contenant chacun 0,05% (v/v) d’acide formique. La séparation est assurée en 35 min par un gradient linéaire, en solvant B, défini comme suit : 0 min, 0% (v/v) ; 5 min, 0% ; 35 min, 100%. The separation of the various reaction products is carried out by reverse phase chromatography with a Synergi ™ Fusion-RP 250 mm × 2 mm column (porosity: 80 Å, particle size: 4 μm, Phenomenex, USA). This column is maintained at 30 ° C on a Thermo Ultimate 3000 HPLC system equipped with a Corona Véo detector. The mobile phase is composed of an ultrapure water (solvent A) / acetonitrile mixture of LC-MS grade (solvent B) each containing 0.05% (v / v) formic acid. The separation is ensured in 35 min by a linear gradient, in solvent B, defined as follows: 0 min, 0% (v / v); 5 min, 0%; 35 min, 100%.
4.2. Résultats 4.2. Results
Les résultats obtenus lors de la glucosylation du CsGi par les a-transglucosylases de la famille GH70 utilisatrice de saccharose sont présentés dans les [Fig. 2A] Figures 2A et [Fig. 2B] 2B. The results obtained during the glucosylation of CsGi by the α-transglucosylases of the GH70 family using sucrose are presented in [Fig. 2A] Figures 2A and [Fig. 2B] 2B.
Les [Fig. 2A] Figures 2A et [Fig. 2B] 2B montre les profils d’élongation du substrat C8G1 obtenus avec les a-transglucosylases de la famille GH70 qui utilisent le saccharose comme substrat., The [Fig. 2A] Figures 2A and [Fig. 2B] 2B shows the elongation profiles of the C8G1 substrate obtained with the α-transglucosylases of the GH70 family which use sucrose as a substrate.,
[Fig. 2A] La Figure 2A montre les profils obtenus avec les glucane saccharases dans le Chromatogramme A : réactions avec [C8G1] = 30 mM, [saccharose] = 585 mM pour les glucane-saccharases DSR-M D1 (SEQ ID NO : 1), DSR-M D5 (SEQ ID NO : 2), DSR-M D5 W624A (SEQ ID NO : 3), GS-B (SEQ ID NO : 5), GS-C (SEQ ID NO : 6), GS-D (SEQ ID NO : 9) et GS-FS D1 (SEQ ID NO : 10) et 1170 mM de saccharose pour les enzymes GS- A SEQ ID NO :4, GS-D (SEQ ID NO :7), DSR-G (SEQ ID NO :11), DSR-G CD1 (SEQ ID NO :12). La [Fig. 2B] Figure 2B montre les profils obtenus avec les saccharases de branchement BRS-A (SEQ ID NO : 17), BRS-B D1 (SEQ ID NO : 18), BRS-C (SEQ ID NO : 19), BRS-D D1 (SEQ ID NO : 20), BRS-E D1 (SEQ ID NO : 21 ), BRS-F (SEQ ID NO : 22), DSR-G CD2 (SEQ ID NO : 23), GBD-CD2 (SEQ ID NO : 16) illustrés dans le chromatogramme B, à partir de [C8G1] = 20 mM et de [saccharose] = 585 mM. L’ensemble des enzymes testées a permis l’élongation de l’APG substrat. [Fig. 2A] Figure 2A shows the profiles obtained with the glucan saccharases in Chromatogram A: reactions with [C8G1] = 30 mM, [sucrose] = 585 mM for the glucan saccharases DSR-M D1 (SEQ ID NO: 1), DSR-M D5 (SEQ ID NO: 2), DSR-M D5 W624A (SEQ ID NO: 3), GS-B (SEQ ID NO: 5), GS-C (SEQ ID NO: 6), GS-D (SEQ ID NO: 9) and GS-FS D1 (SEQ ID NO: 10) and 1170 mM of sucrose for the enzymes GS- A SEQ ID NO: 4, GS-D (SEQ ID NO: 7), DSR-G (SEQ ID NO: 11), DSR-G CD1 (SEQ ID NO: 12). The [Fig. 2B] Figure 2B shows the profiles obtained with the branching sucrases BRS-A (SEQ ID NO: 17), BRS-B D1 (SEQ ID NO: 18), BRS-C (SEQ ID NO: 19), BRS-D D1 (SEQ ID NO: 20), BRS-E D1 (SEQ ID NO: 21), BRS-F (SEQ ID NO: 22), DSR-G CD2 (SEQ ID NO: 23), GBD-CD2 (SEQ ID NO: 16) shown in chromatogram B, from [C8G1] = 20 mM and [sucrose] = 585 mM. All the enzymes tested allowed the extension of the substrate APG.
On observe que les profils de produits obtenus sont différents selon la classe d’enzymes utilisées (glucane saccharases GH70 et/ou saccharases de branchement GH70). It is observed that the product profiles obtained are different depending on the class of enzymes used (glucan sucrases GH70 and / or branching sucrases GH70).
Les enzymes de branchement [Fig. 2B] (Figure 2B) permettent l’ajout de 1 à 7 unités glucosyle sur le CsGi selon les enzymes considérées, avec des taux de conversion variant de 20% à 60% dans les conditions testées. Avec les glucane-saccharases [Fig. 2A] (Figure 2A), les tailles des APG obtenus sont beaucoup plus étendues, les produits portant un nombre important d’unités glucosyle.Branching enzymes [Fig. 2B] (FIG. 2B) allow the addition of 1 to 7 glucosyl units to the CsGi depending on the enzymes considered, with conversion rates varying from 20% to 60% under the conditions tested. With glucansucrases [Fig. 2A] (FIG. 2A), the sizes of the APGs obtained are much larger, the products carrying a large number of glucosyl units.
À titre de comparaison, [Fig. 3] la Figure 3 présente les profils d’élongation de CsGi obtenu avec i) la glucane-saccharase GS-C (SEQ ID NO :6) en présence de saccharose et d’a- cyclodextrines, et ii) la CGTase de Bacillus macerans (non conforme à l’invention) en présence d’a-cyclodextrines dans des conditions similaires aux travaux de Svenson et collaborateurs (Svenson et al., 2009). Les profils montrent clairement le caractère polymérique de la tête glucosidique synthétisée par la glucane-saccharase DSR-M D1 (SEQ ID NO :1 ) (et l’ensemble des glucane saccharases testées), en contraste avec le profil oligomérique des APG obtenus avec la CGTase. For comparison, [Fig. 3] Figure 3 shows the elongation profiles of CsGi obtained with i) the glucansucrase GS-C (SEQ ID NO: 6) in the presence of sucrose and α-cyclodextrins, and ii) the CGTase of Bacillus macerans (not in accordance with the invention) in the presence of α-cyclodextrins under conditions similar to the work of Svenson et al. (Svenson et al., 2009). The profiles clearly show the polymeric character of the glucosidic head synthesized by the glucansucrase DSR-M D1 (SEQ ID NO: 1) (and all of the glucan saccharases tested), in contrast with the oligomeric profile of the APGs obtained with the CGTase.
Saccharases de branchement Branching sucrases
Par ailleurs, les APG produits par les glucane-saccharases peuvent eux-mêmes être utilisés en tant que substrats par les saccharases de branchement. Furthermore, the APGs produced by the glucansucrases can themselves be used as substrates by the branching sucrases.
Ces dernières permettent alors de « décorer » les têtes glucidiques par des unités glucosyle liées en a-1 ,2 ou a-1 ,3. The latter then make it possible to “decorate” the carbohydrate heads with glucosyl units linked at a-1, 2 or a-1, 3.
Ceci est illustré par [Fig. 4] la Figure 4 où l’on observe une modification du profil de produits obtenus avec la glucane-saccharase DSR-M D1 (SEQ ID NO :1 ) seule par rapport au profil de produits obtenus avec les enzymes de branchement BRS-A (SEQ ID NO :17) et BRS- B D1 (SEQ ID NO :18) utilisées dans une seconde étape après ajout de saccharose supplémentaire. This is illustrated by [Fig. 4] Figure 4 where we observe a modification of the profile of products obtained with the glucansucrase DSR-M D1 (SEQ ID NO: 1) alone compared to the profile of products obtained with the BRS-A branching enzymes ( SEQ ID NO: 17) and BRS-B D1 (SEQ ID NO: 18) used in a second step after addition of additional sucrose.
Les APG produits par l’ensemble des enzymes de type polymérase ont été purifiés et caractérisés par RMN 1H, enregistrés sur un équipement Bruker Avance 500MHz à 298K avec une sonde 5 mm z-gradient TBI. Les données ont été acquises et traitées grâce au logiciel TopSpin3. Le pourcentage de chaque liaison est calculé grâce aux intensités relatives des protons anomériques des unités glucosyle engagées dans une liaison avec l’APG par intégration de l’aire des pics. Le DP moyen est déterminé par la somme de l’intensité relative de ces mêmes protons anomériques en prenant le proton H1 du premier sucre lié à la chaîne alkyle en conformation beta (d = 4,5 ppm) comme référence. The APGs produced by all of the polymerase-type enzymes were purified and characterized by 1 H NMR, recorded on a Bruker Avance 500 MHz equipment at 298 K with a 5 mm z-gradient TBI probe. The data were acquired and processed using TopSpin3 software. The percentage of each bond is calculated by virtue of the relative intensities of the anomeric protons of the glucosyl units engaged in a bond with the APG by integration of the area of the peaks. The average DP is determined by the sum of the relative intensity of these same anomeric protons taking the H1 proton of the first sugar linked to the alkyl chain in beta conformation (d = 4.5 ppm) as a reference.
Les résultats obtenus présentés en Tableau 3 montrent la possibilité de moduler le DP moyen par le choix des concentrations initiales en substrats et le ratio molaire (saccharose/APG) et l’enzyme utilisée. Tableau 3 : caractéristiques structurales des APG à très longues têtes glucidiques. Conditions de synthèse [CsGi] = 30 mM, [saccharose] = 585 mM pour les enzymes SEQ ID 1 , SEQ ID NO :2, SEQ ID NO :3, SEQ ID NO :5, SEQ ID NO :6, SEQ ID NO :9, SEQ ID NO :10 et 1170 mM pour les enzymes SEQ ID NO :4, SEQ ID NO :7, SEQ ID NO :11 , SEQ ID NO :12. The results obtained presented in Table 3 show the possibility of modulating the mean DP by the choice of the initial concentrations of substrates and the molar ratio (sucrose / APG) and the enzyme used. Table 3: structural characteristics of APGs with very long carbohydrate heads. Synthesis conditions [CsGi] = 30 mM, [sucrose] = 585 mM for the enzymes SEQ ID 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 9, SEQ ID NO: 10 and 1170 mM for the enzymes SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12.
[Table 3] [Table 3]
[Fig. 5] La Figure 5 montre les profils d’élongation obtenus avec l’enzyme DSR-M D1 (SEQ ID NO :1 ) avec des substrats de géométries différentes en termes de taille de chaîne alkyle (C8 à C16) et de tête glucidique (mono, diglucoside ou oligoglucosides). [Fig. 5] Figure 5 shows the elongation profiles obtained with the DSR-M D1 enzyme (SEQ ID NO: 1) with substrates of different geometries in terms of alkyl chain size (C8 to C16) and carbohydrate head ( mono, diglucoside or oligoglucosides).
La glucane saccharase a permis l’élongation de l’ensemble de ces accepteurs. Glucan sucrase elongated all of these acceptors.
Les résultats montrent également l’influence de la taille des accepteurs. Plus la chaîne alkyle est longue (/.e. moins sa solubilité est grande) moins la glucosylation est efficace dans les conditions réactionnelles testées. Cet effet est contrebalancé par l’augmentation de la taille de la tête glucosidique (différence de glucosylation de C12G1 et de C12G2). Cela montre l’importance de la maîtrise des conditions de synthèse pour une élongation efficace des APG à chaîne alkyle longue. The results also show the influence of the size of the acceptors. The longer the alkyl chain (/.e. The lower its solubility), the less effective the glucosylation is under the reaction conditions tested. This effect is counterbalanced by the increase in the size of the glucosidic head (difference in glucosylation of C12G1 and C12G2). This shows the importance of controlling the synthetic conditions for efficient elongation of long alkyl chain APGs.
Exemple 5 : Contrôle du profil d’élongation Example 5: Control of the elongation profile
5.1 . Etude de l’influence du rapport molaire glucoside d’alkyle accepteur : saccharose sur le profil d’élongation 5.1. Study of the influence of the acceptor alkyl glucoside: sucrose molar ratio on the elongation profile
5.1.1 Matériel et méthodes 5.1.1 Materials and methods
Dans une première série d’expériences, on étudie l’influence du rapport molaire glucoside d’alkyle accepteur (i.e. glucoside d’alkyle de formule (II)) : saccharose ou analogue de saccharose sur le profil d’élongation du glucoside d’alkyle accepteur CsGi ou C12G2 par l’enzyme DSR-M D1 (SEQ ID NO : 1 ). In a first series of experiments, the influence of the acceptor alkyl glucoside molar ratio (ie alkyl glucoside of formula (II)): sucrose or sucrose analog on the elongation profile of the alkyl glucoside is studied. acceptor CsGi or C12G2 by the enzyme DSR-M D1 (SEQ ID NO: 1).
Dans cette première série d’expérience, les concentrations en CsGi ou C12G2 sont fixées à 10 g/L (respectivement 34,3 mM et 19,6 mM) et la concentration en saccharose est modulée entre 233,4 g/L (684 mM) et 2,3 g/L (6,8 mM) de telle manière que le ratio molaire glucosides d’alkyle accepteur : saccharose soit de 0,05 ; de 0,1 ; de 0,25 ; de 0,5 ; de 1 ; de 2 ; ou bien de 5. In this first series of experiments, the CsGi or C12G2 concentrations are set at 10 g / L (34.3 mM and 19.6 mM respectively) and the sucrose concentration is modulated between 233.4 g / L (684 mM ) and 2.3 g / L (6.8 mM) such that the molar ratio of acceptor alkyl glucosides: sucrose is 0.05; 0.1; 0.25; 0.5; from 1 ; of 2; or 5.
Témoin : les réactions témoin représentent les mélanges de C8G1 ou C12G2 à une concentration de 10 g/L et de 233.4g/L de saccharose sans ajout d’enzyme Control: the control reactions represent mixtures of C8G1 or C12G2 at a concentration of 10 g / L and 233.4g / L of sucrose without addition of enzyme
5.1.2. Résultats 5.1.2. Results
Les résultats de cette première série d’expériences sont présentés en Figure 6 et confirment la possibilité de moduler le profil d’élongation en fonction du rapport molaire glucoside d’alkyle accepteur : saccharose entre 0.05 et 5. The results of this first series of experiments are presented in Figure 6 and confirm the possibility of modulating the elongation profile as a function of the acceptor alkyl glucoside: sucrose molar ratio between 0.05 and 5.
[Fig. 6] La Figure 6 montre les profils d’élongation obtenus avec l’enzyme DSR-M D1 (SEQ ID NO : 1 ) avec des ratios molaires décroissants entre CsGi et Saccharose (Figure 6A) et entre C12G2 et saccharose (Figure 6B). Les résultats montrent l’effet du ratio glucoside d’alkyl accepteur : saccharose sur l’élongation du glucoside d’alkyl accepteur : plus le rapport molaire est élevé, moins le glucoside d’alkyle accepteur est rallongé. Les résultats montrent donc que le DP moyen du polyglucoside d’alkyle obtenu est inversement proportionnel au rapport molaire glucoside d’alkyl accepteur : saccharose. Par ailleurs, la Figure 6A montre que pour le CsGi, le profil d’élongation varie significativement pour des rapports molaires compris entre 0,05 et 2, tandis que les rapports molaires supérieurs à 2 ont peu d’influence sur le profil d’élongation/ [Fig. 6] Figure 6 shows the elongation profiles obtained with the DSR-M D1 enzyme (SEQ ID NO: 1) with decreasing molar ratios between CsGi and Sucrose (Figure 6A) and between C12G2 and sucrose (Figure 6B). The results show the effect of the alkyl acceptor glucoside: sucrose ratio on the elongation of the alkyl acceptor glucoside: the higher the molar ratio, the less the acceptor alkyl glucoside is extended. The results therefore show that the average DP of the alkyl polyglucoside obtained is inversely proportional to the molar ratio of alkyl acceptor glucoside: sucrose. Furthermore, Figure 6A shows that for CsGi, the elongation profile varies significantly for molar ratios between 0.05 and 2, while molar ratios greater than 2 have little influence on the elongation profile. /
La Figure 6B montre que pour le C12G2, le profil d’élongation varie significativement pour des rapports molaires compris entre 0,05 et 5. Figure 6B shows that for C12G2, the elongation profile varies significantly for molar ratios between 0.05 and 5.
5.2. Etude de l’influence du ratio d’activité glucane-saccharase: saccharase de branchement sur le profil d’élongation 5.2. Study of the influence of the glucan-sucrase activity ratio: branching sucrase on the elongation profile
5.2.1. Matériel et méthodes 5.2.1. Material and methods
Dans une deuxième série d’expériences, on étudie l’influence du ratio d’activité glucane- saccharase : saccharase de branchement sur le profil d’élongation du glucoside d’alkyle accepteur CsGi. In a second series of experiments, the influence of the glucansucrase: branching sucrase activity ratio on the elongation profile of the acceptor alkyl glucoside CsGi is studied .
Dans cette seconde série d’expérience, la glucane-saccharase est DSR-M D1 (SEQ ID NO : 1 ) et la saccharase de branchement BRS-A (SEQ ID NO : 18) ou BRS-B D1 (SEQ ID NO : 19). La concentration de CsGi est fixée à 10 g/L et la concentration de saccharose à 10Og/L. Les quantités de DSR-M D1 et de BRS-A ou de BSR-B sont modulés de sorte que les ratio DSR-M D1 : BRS-A et DSR-M D1 : BRS-B D1 exprimé en unité d’activité enzymatique soit de de 0,1 ; de 0,25 ; de 0,5 ; de 1 ; de 2 ; de 5 ou bien de 10, en respectant une activité totale (DSR-M D1 + BRS-A ou BRS-B D1 ) de 1 U/mL. In this second series of experiments, the glucansucrase is DSR-M D1 (SEQ ID NO: 1) and the branching sucrase BRS-A (SEQ ID NO: 18) or BRS-B D1 (SEQ ID NO: 19 ). The concentration of CsGi is fixed at 10 g / L and the concentration of sucrose at 10Og / L. The quantities of DSR-M D1 and BRS-A or BSR-B are modulated so that the ratio DSR-M D1: BRS-A and DSR-M D1: BRS-B D1 expressed in units of enzymatic activity is of 0.1; 0.25; 0.5; from 1 ; of 2; 5 or 10, while respecting a total activity (DSR-M D1 + BRS-A or BRS-B D1) of 1 U / mL.
Témoin : les réactions témoin représentent le mélange de C8G1 à une concentration de 10 g/L et de 100 g/L de saccharose sans ajout d’enzyme Control: the control reactions represent the mixture of C8G1 at a concentration of 10 g / L and 100 g / L of sucrose without addition of enzyme
5.2.2. Résultats 5.2.2. Results
Les résultats de cette seconde série d’expériences sont présntés en Figure 7 et confirment la possibilité de moduler le profil d’élongation en fonction du ratio d’activité enzyme d’élongation : enzyme de branchement. The results of this second series of experiments are presented in Figure 7 and confirm the possibility of modulating the elongation profile according to the ratio of elongation enzyme activity: branching enzyme.
[Fig. 7] La Figure 7 montre les profils d’élongation obtenus avec l’enzyme DSR-M D1 (SEQ ID NO : 1 ) en co-catalyse avec l’enzyme de branchement BRS-A (SEQ ID NO : 18) (Figure 7A) ou avec l’enzyme de branchement BRS-B D1 (SEQ ID NO : 19) (Figure 7B). Les résultats montrent l’effet du ratio d’activité glucane-saccharase : saccharase de branchement : plus ce ratio d’activité est élevé, plus le glucoside d’alkyle accepteur est rallongé. Les résultats montrent donc que le DP moyen du polyglucoside d’alkyle obtenu est proportionnel au ratio d’activité glucane-saccharase : saccharase de branchement. [Fig. 7] Figure 7 shows the elongation profiles obtained with the DSR-M D1 enzyme (SEQ ID NO: 1) in co-catalysis with the branching enzyme BRS-A (SEQ ID NO: 18) (Figure 7A ) or with the BRS-B D1 branching enzyme (SEQ ID NO: 19) (Figure 7B). The results show the effect of the glucansucrase activity ratio: branching sucrase: the higher this activity ratio, the greater the acceptor alkyl glucoside. extension. The results therefore show that the average DP of the alkyl polyglucoside obtained is proportional to the ratio of glucan-sucrase activity: branching sucrase.
REFERENCES REFERENCES
- Bousquet, M.-P., Willemot, R.-M., Monsan, P. F., Paul, F. & Boures, E. Enzymatic Synthesis of a-Butylglucoside in a Biphasic Butanol-Water System Using the o Transglucosidase from Aspergillus niger. in Carbohydrate Biotechnology Protocols (ed. Bucke, C.) 10, 291-296 (Humana Press, 1999). - Bousquet, M.-P., Willemot, R.-M., Monsan, PF, Paul, F. & Boures, E. Enzymatic Synthesis of a-Butylglucoside in a Biphasic Butanol-Water System Using the o Transglucosidase from Aspergillus niger . in Carbohydrate Biotechnology Protocols (ed. Bucke, C.) 10, 291-296 (Humana Press, 1999).
- Bousquet, M.-P., Willemot, R.-M., Monsan, P. & Boures, E. Production, purification, and characterization of thermostable a-transglucosidase from Talaromyces duponti-application to a-alkylglucoside synthesis. Enzyme and Microbial Technology 23, 83-90 (1998). - Bousquet, M.-P., Willemot, R.-M., Monsan, P. & Boures, E. Production, purification, and characterization of thermostable a-transglucosidase from Talaromyces duponti-application to a-alkylglucoside synthesis. Enzyme and Microbial Technology 23, 83-90 (1998).
- Dahiya, S., Ojha, S. & Mishra, S. Biotransformation of sucrose into hexyl-a-d- glucopyranoside and -polyglucosides by whole cells of Microbacterium paraoxydans. Biotechnol Lett 37, 1431-1437 (2015). - Dahiya, S., Ojha, S. & Mishra, S. Biotransformation of sucrose into hexyl-a-d- glucopyranoside and -polyglucosides by whole cells of Microbacterium paraoxydans. Biotechnol Lett 37, 1431-1437 (2015).
- Ochs, M., Muzard, M., Plantier-Royon, R., Estrine, B. & Rémond, C. Enzymatic synthesis of alkyle b-D-xylosides and oligoxylosides from xylans and from hydrothermally pretreated wheat bran. Green Chem. 13, 2380-2388 (2011 ). - Ochs, M., Muzard, M., Plantier-Royon, R., Estrine, B. & Rémond, C. Enzymatic synthesis of alkyl b-D-xylosides and oligoxylosides from xylans and from hydrothermally pretreated wheat bran. Green Chem. 13, 2380-2388 (2011).
- Rémond, Z. C., Ochs, M., Muzard, M., Plantier, R. R. & Estrine, B. Preparing surfactant compositions, comprises e.g. mixing lignocellulosic annual and perennial plant materials with water, contacting solution with strain or enzyme having xylanase activity to give composition of alkyle polypentosides. (2012). - Rémond, ZC, Ochs, M., Muzard, M., Plantier, RR & Estrine, B. Preparing surfactant compositions, including eg mixing lignocellulosic annual and perennial plant materials with water, contacting solution with strain or enzyme having xylanase activity to give composition of alkyl polypentosides. (2012).
- Svensson, D., Ulvenlund, S. & Adlercreutz, P. Efficient synthesis of a long carbohydrate chain alkyle glycoside catalyzed by cyclodextrin glycosyltransferase (CGTase). Biotechnol. Bioeng. 104, 854-861 (2009). - Svensson, D., Ulvenlund, S. & Adlercreutz, P. Efficient synthesis of a long carbohydrate chain alkyl glycoside catalyzed by cyclodextrin glycosyltransferase (CGTase). Biotechnol. Bioeng. 104, 854-861 (2009).
- Svensson, D. & Adlercreutz, P. Immobilisation of CGTase for continuous production of long-carbohydrate-chain alkyle glycosides: Control of product distribution by flow rate adjustment. Journal of Molecular Catalysis B: Enzymatic 69, 147-153 (2011 ). - Svensson, D. & Adlercreutz, P. Immobilisation of CGTase for continuous production of long-carbohydrate-chain alkyl glycosides: Control of product distribution by flow rate adjustment. Journal of Molecular Catalysis B: Enzymatic 69, 147-153 (2011).
- Paul, C. J. et al. A GH574-a-glucanotransferase of hyperthermophilic origin with potential for alkyle glycoside production. Appl Microbiol Biotechnol 99, 7101-7113 (2015). - Paul, C. J. et al. A GH574-a-glucanotransferase of hyperthermophilic origin with potential for alkyl glycoside production. Appl Microbiol Biotechnol 99, 7101-7113 (2015).
- Vuillemin, M. et al. Characterization of the First o(1 3) Branching Sucrases of the GH70 Family. Journal of Biological Chemistry 291 , 7687-7702 (2016). - Vuillemin, M. et al. Characterization of the First o (1 3) Branching Sucrases of the GH70 Family. Journal of Biological Chemistry 291, 7687-7702 (2016).
- Côté, G. L. & Fobyt, J. F. Acceptor reactions of alternansucrase from Leuconostoc mesenteroides NRRL B-1355. Carbohydrate Research 111 , 127-142 (1982). - Zhao, H. et al. Cyclomaltodextrin Glucanotransferase-Catalyzed Transglycosylation from Dextrin toAlkanol Maltosides. Bioscience, Biotechnology, and Biochemistry 72, 3006-3010 (2008). - Côté, GL & Fobyt, JF Acceptor reactions of alternansucrase from Leuconostoc mesenteroides NRRL B-1355. Carbohydrate Research 111, 127-142 (1982). - Zhao, H. et al. Cyclomaltodextrin Glucanotransferase-Catalyzed Transglycosylation from Dextrin toAlkanol Maltosides. Bioscience, Biotechnology, and Biochemistry 72, 3006-3010 (2008).

Claims

REVENDICATIONS
1. Procédé de préparation d’un polyglucoside d’alkyle de formule (I)1. Process for preparing an alkyl polyglucoside of formula (I)
[Glc]m-[Glc]n(-0-R) (I) dans laquelle : [Glc] m - [Glc] n (-0-R) (I) in which:
R représente un groupe alkyle linéaire ou ramifié, saturé ou insaturé, comprenant entre 8 et 20 atomes de carbone, R represents a linear or branched, saturated or unsaturated alkyl group comprising between 8 and 20 carbon atoms,
[Glc]m-[Glc]n représente une partie glucidique linéaire ou ramifiée comprenant n+m unités glucosyle, n+m étant compris entre 3 et 200 ; ledit procédé comprenant au moins une étape i) d’élongation de la chaîne glucosidique d’un glucoside d’alkyle de formule (II) [Glc] m - [Glc] n represents a linear or branched carbohydrate part comprising n + m glucosyl units, n + m being between 3 and 200; said process comprising at least one step i) of elongation of the glucosidic chain of an alkyl glucoside of formula (II)
[Glc]n(-0-R) (II) dans laquelle : [Glc] n (-0-R) (II) in which:
R est tel que défini dans la formule (I), R is as defined in formula (I),
[Glc]n représente une partie glucidique comprenant n unités glucosyle n étant compris entre 1 et 15. ladite étape comprenant la mise en contact dudit glucoside d’alkyle de formule (II) avec au moins une a-transglucosylase de la famille GH70 en présence de saccharose ou d’un analogue de saccharose. [Glc] n represents a carbohydrate part comprising n glucosyl units n being between 1 and 15. said step comprising bringing said alkyl glucoside of formula (II) into contact with at least one α-transglucosylase of the GH70 family in the presence of sucrose or a sucrose analogue.
2. Procédé selon la revendication 1 , dans lequel : 2. The method of claim 1, wherein:
- lorsque R représente un groupe alkyle comprenant entre 12 et 20 atomes de carbone, n+m est compris entre 3 et 200, et - when R represents an alkyl group comprising between 12 and 20 carbon atoms, n + m is between 3 and 200, and
- lorsque R représente un groupe alkyle comprenant entre 8 et 12 atomes de carbone, n+m est compris entre 7 et 200. - When R represents an alkyl group comprising between 8 and 12 carbon atoms, n + m is between 7 and 200.
3. Procédé selon la revendication 1 ou 2, dans lequel l’étape i) est conduite avec un glucoside d’alkyle de formule (II) essentiellement constitué de molécules de monoglucoside d’alkyle, de molécules de diglucloside d’alkyle, ou d’un mélange de celles- ci. 3. The method of claim 1 or 2, wherein step i) is carried out with an alkyl glucoside of formula (II) consisting essentially of alkyl monoglucoside molecules, of alkyl diglucloside molecules, or d a mixture of these.
4. Procédé selon l’une quelconque des revendications précédentes, dans lequel l’a- transglucosylase de la famille GH70 est une glucane-saccharase de la famille GH70, une saccharase de branchement de la famille GH70, ou un mélange de celles-ci. 4. A method according to any preceding claim, wherein the α-transglucosylase of the GH70 family is a glucansucrase of the GH70 family, a branching sucrase of the GH70 family, or a mixture thereof.
5. Procédé selon la revendication 4, dans lequel la glucane-saccharase de la famille GH70 a pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 1 , SEQ ID NO : 2, SEQ ID NO : 3, , SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO :5. The method of claim 4, wherein the glucansucrase of the GH70 family has for amino acid sequence a sequence chosen from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11 , SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14 et SEQ ID NO : 15. 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.
6. Procédé selon la revendication 4, dans lequel la saccharase de branchement de la famille GH70 pour séquence d’acides aminés une séquence choisie dans le groupe comprenant SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21 , SEQ ID NO : 22, et SEQ ID NO : 23. 6. The method of claim 4, wherein the branching sucrase of the GH70 family for amino acid sequence a sequence selected from the group comprising SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23.
7. Procédé selon l’une quelconque des revendications précédentes, dans lequel l’étape i) d’élongation glucosidique est conduite en mettant en contact simultanément ou successivement le glucoside d’alkyle de formule (II) avec plusieurs a-transglucosylases de la famille GH70. 7. Method according to any one of the preceding claims, wherein step i) of glucosidic elongation is carried out by simultaneously or successively bringing the alkyl glucoside of formula (II) into contact with several α-transglucosylases of the family. GH70.
8. Procédé selon l’une quelconque des revendications précédentes, dans lequel l’étape i) comprend une étape io) de mise en contact du glucoside d’alkyle de formule (II) avec un mélange d’une ou plusieurs glucane-saccharase(s) de la famille GH70 et d’enzyme(s) de branchement de la famille GH70. 8. Method according to any one of the preceding claims, wherein step i) comprises a step io) of contacting the alkyl glucoside of formula (II) with a mixture of one or more glucansucrase ( s) of the GH70 family and branching enzyme (s) of the GH70 family.
9. Procédé selon l’une quelconque des revendications précédentes, dans lequel l’étape i) comprend une étape h) de mise en contact du glucoside d’alkyle de formule (II) avec une ou plusieurs glucane-saccharase(s) de la famille GH70 puis une étape b) de mise en contact du polyglucoside d’alkyle obtenu à l’issue de l’étape h) avec une ou plusieurs saccharase(s) de branchement de la famille GH70. 9. Method according to any one of the preceding claims, wherein step i) comprises a step h) of contacting the alkyl glucoside of formula (II) with one or more glucansucrase (s) of the. GH70 family then a step b) of bringing the alkyl polyglucoside obtained at the end of step h) into contact with one or more branching sucrase (s) of the GH70 family.
10. Procédé selon l’une quelconque des revendications précédentes, dans lequel l’étape i) est effectuée en solution à un pH contrôlé, en particulier par l’emploi d’une solution tampon. 10. A method according to any preceding claim, wherein step i) is carried out in solution at a controlled pH, in particular by the use of a buffer solution.
11 . Procédé selon la revendication 10, dans lequel l’étape i) est conduite à une valeur de pH comprise entre 5 et 8. 11. Process according to Claim 10, in which step i) is carried out at a pH value of between 5 and 8.
12. Procédé selon l’une quelconque des revendications précédentes, dans lequel l’étape i) est conduite avec une concentration initiale de saccharose ou d’un analogue de saccharose comprise entre 20 et 660 g.L 1. 12. Process according to any one of the preceding claims, in which step i) is carried out with an initial concentration of sucrose or of a sucrose analogue of between 20 and 660 gL 1 .
13. Procédé selon l’une quelconque des revendications précédentes, dans lequel l’étape i) est de préférence réalisée avec un rapport molaire polyglucosides d’alkyle de formule (II) : saccharose ou analogue de saccharose compris entre 0,001 et 0,3, de préférence entre 0,002 et 0,006. 13. Process according to any one of the preceding claims, in which step i) is preferably carried out with an alkyl polyglucoside molar ratio of formula (II): sucrose or sucrose analog of between 0.001 and 0.3, preferably between 0.002 and 0.006.
14. Procédé selon l’une quelconque des revendications précédentes, dans lequel l’étape i) est de préférence mise en oeuvre à une température comprise entre 10°C et 80°C. 14. A method according to any preceding claim, wherein step i) is preferably carried out at a temperature between 10 ° C and 80 ° C.
15. Procédé selon l’une quelconque des revendications précédentes, dans lequel l’étape i) est conduite avec Ga-transglucosylase de la famille GH70 sous forme solide, en solution, en suspension, ou immobilisée. 15. A method according to any preceding claim, wherein step i) is carried out with Ga-transglucosylase of the GH70 family in solid, in solution, in suspension, or immobilized form.
16. Procédé selon l’une quelconque des revendications précédentes comprenant en outre une étape ii) de purification du polyglucoside d’alkyle de formule (I) obtenu à l’issue de l’étape i). 16. A method according to any one of the preceding claims further comprising a step ii) of purifying the alkyl polyglucoside of formula (I) obtained at the end of step i).
17. Polyglucoside d’alkyle susceptible d’être obtenu par la mise en oeuvre du procédé selon l’une quelconque des revendications 1 à 16, caractérisé en ce qu’il est de formule (I) : [Glc]m-[Glc]n(-0-R) (I) dans laquelle : 17. Alkyl polyglucoside obtainable by carrying out the process according to any one of claims 1 to 16, characterized in that it is of formula (I): [Glc] m - [Glc] n (-0-R) (I) in which:
R représente un groupe alkyle linéaire ou ramifié, saturé ou insaturé, comprenant entre 8 et 20 atomes de carbone, R represents a linear or branched, saturated or unsaturated alkyl group comprising between 8 and 20 carbon atoms,
[Glc]m-[Glc]n représente une partie glucidique linéaire ou ramifiée comprenant n+m unités glucosyle, n+m étant compris entre 3 et 200, de préférence dans lequel lorsque R représente un groupe alkyle comprenant de 8 à 12 atomes de carbone, n+m compris entre 7 et 200, et lorsque R représente un groupe alkyle comprenant de 12 à 20 atomes de carbone, n+m est compris entre 3 et 200. [Glc] m - [Glc] n represents a linear or branched carbohydrate part comprising n + m glucosyl units, n + m being between 3 and 200, preferably in which when R represents an alkyl group comprising from 8 to 12 atoms of carbon, n + m between 7 and 200, and when R represents an alkyl group comprising from 12 to 20 carbon atoms, n + m is between 3 and 200.
18. Polyglucoside d’alkyle selon la revendication 17, dans lequel le polyglucoside d’alkyle est constitué essentiellement d’un mélange de molécules de polyglucoside d’alkyle de formule (I), ledit mélange présentant un degré de polymérisation moyen compris entre 3 et 25. 18. The polyalkylglucoside of claim 17, wherein the polyalkylglucoside consists essentially of a mixture of polyalkylglucoside molecules of. formula (I), said mixture exhibiting an average degree of polymerization of between 3 and 25.
19. Utilisation d’un polyglucoside susceptible d’être obtenu selon un procédé tel que défini à l’une quelconque des revendications 1 à 16 ou tel que défini à l’une quelconque des revendications 17 ou 18, à titre de surfactant. 19. Use of a polyglucoside obtainable according to a process as defined in any one of claims 1 to 16 or as defined in any one of claims 17 or 18, as a surfactant.
20. Utilisation d’une a-transglucosylase de la famille GH70, de préférence d’une o transglucosylase de la famille GH70 telle que définie à l’une quelconque des revendications 4 ou 5, pour l’élongation glucosidique d’un glucoside d’alkyle de formule (II) tel que défini à la revendication 1. 20. Use of an α-transglucosylase of the GH70 family, preferably of a transglucosylase of the GH70 family as defined in any one of claims 4 or 5, for the glucosidic elongation of a glucoside of. alkyl of formula (II) as defined in claim 1.
EP21748921.0A 2020-07-02 2021-07-02 Process for preparing alkyl polyglucosides, and alkyl polyglucosides obtained according to the process Pending EP4176072A1 (en)

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