ESTOLIDES COPOLYMERS OF SATURATED AND UNSATURATED HYDROXYLATED FATTY ACIDS

Abstract

The invention relates to an estolide copolymer of a saturated hydroxylated fatty acid and an unsaturated hydroxylated fatty acid functionalized by a monoalcohol on the terminal acid function with an estolide number between 1 and 6 and its preparation process in the presence of a lipase.

Description

Title of the invention: SATURATED AND UNSATURATED HYDROXYLATED FATTY ACID COPOLYMER ESTOLIDES FIELD OF THE INVENTION

[0001] The present invention relates to estolides in the form of copolymers of a saturated hydroxylated fatty acid and an unsaturated hydroxylated fatty acid and their process for preparation by biocatalytic route in the presence of a lipase.

[0002] DESCRIPTION OF THE PRIOR ART

[0003] Estolides are oligomeric esters derived from fatty acids. They are biodegradable compounds that have industrial importance as lubricants, plasticizers, emulsifiers or moisturizers. They find their application in the automotive, cosmetic and food industries. This wide range of applications is linked to their thermo-oxidative stability, viscosity and low melting point. Estolides also have biological and texturizing properties and an adjustable hydrophilic / lipophilic balance. Estolides provide moisturizing power to products, they act as a thermal barrier, they improve the elasticity of fibers, and they give a shine effect. Estolides can represent up to 20% of the composition of a cosmetic product.

[0004] The preparation of estolides is mainly carried out by chemical processes from unsaturated fatty acids in the presence of a catalyst as described in patents and patent applications US5,380,894, WO2013 / 009471, WO2008 / 040864, ​​WO2011 / 037778 or US2,785,978. The estolides formed by these processes give rise to regioisomers and / or generally have a low purity and require long and expensive purification steps.

[0005] The preparation of estolides by biocatalytic means from hydroxylated fatty acids has also been described with various enzymes, in particular enzymes from Candida sp., Thermomyces sp., Pseudomonas sp. or even Rhizopus sp. Patent FR3073231 describes the synthesis of estolides in the presence of a lipase from Streptomyces avermitilis, in particular LpsA2.

[0006] A new lipase from Streptomyces sp. was identified by Wang et al. in 2016 and named MAS1 (Yuan D, Lan D, Xin R, Yang B & Wang Y (2016) Screening and characterization of a thermostable lipase from marine Streptomyces sp. strain W007. Biotechnol Appl Biochem 63, 41-50). Its hydrolytic activity and its ability to produce triacylglycerols have been demonstrated. However, it has never been demonstrated that it can catalyze the polymerization of fatty acids or their derivatives.

[0007] Unsaturated fatty acid based estolides have been shown to exhibit interesting properties in the field of cosmetics as described in patent application WO 2022 / 122719. In the interests of sustainable development, the cosmetics field is paying more and more attention to the biodegradability of its ingredients. Ricinoleic acid-based estolides have biodegradability that could be improved. There is a need to develop new types of estolides with improved biodegradability while maintaining acceptable cosmetic properties. Summary of the invention

[0008] The invention relates to copolymers based on a saturated hydroxylated fatty acid and an unsaturated hydroxylated fatty acid, the copolymer comprising in the terminal position an ester function and having a weight-average molar mass less than or equal to 5000 g / mol. The copolymers can be prepared in the presence of a lipase of Streptomyces sp., in particular a lipase comprising the amino acid sequence SEQ ID NO. 1 or an amino acid sequence having at least 75% identity with the sequence SEQ ID NO. 1.

[0009] Other aspects of the invention are as described below. DETAILED DESCRIPTION OF THE INVENTION

[0010] The inventors have developed copolymers having compatible criteria for use in a cosmetic formula and exhibiting improved biodegradability.

[0011] Cooolymer based on a saturated hydroxylated fatty acid and an unsaturated hydroxylated fatty acid

[0012] The copolymer according to the invention is a copolymer based on a saturated hydroxylated fatty acid and an unsaturated hydroxylated fatty acid, the copolymer comprising in the terminal position an ester function and having a weight-average molar mass (Mw) less than or equal to 5000 g / mol, in particular ranging from 1000 to 5000 g / mol.

[0013] The expression "based on" designates the product of the polymerization reaction between a saturated hydroxylated fatty acid and an unsaturated hydroxylated fatty acid.

[0014] The weight-average molar mass (Mw) of the copolymer can range from 1500 to 4950 g / mol, preferably from 2000 to 4900 g / mol, more preferably from 2200 to 4850 g / mol.

[0015] Typically, the copolymer has an estolide number EN ranging from 1.0 to 6.0, in particular from 1.5 to 5.8, preferably from 1.7 to 5.7, in particular from 2.0 to 5.5, more particularly from 2.5 to 5.0, for example between 2.7 and 4.5. The EN index can be determined by NMR, from the acid number (AI), or by GPC from the Mn (number-average molar mass).

[0016] Preferably, the copolymer has a number-average molar mass Mn ranging from 700 to 2700 g / mol, in particular from 1000 to 2600 g / mol, more preferably from 1200 to 2550 g / mol.

[0017] For example, the copolymer has a polydispersity index less than or equal to 3.0, preferably less than or equal to 2.5, more preferably less than or equal to 2.0.

[0018] The copolymer may have at least one or both of the following characteristics:

[0019] - An acid number of less than 20, preferably less than 15, in particular less than 10; - A pale yellow to amber color.

[0020] An estolide obtained by polymerization of one or more hydroxylated fatty acid(s) usually has at one end (or terminal position) an alcohol function and at another end (or terminal position) a carboxylic acid function.

[0021] The carboxylic acid function can be functionalized in the presence of an alcohol by esterification to access an estolide comprising an ester function in the terminal position.

[0022] The copolymer according to the invention comprises an ester function in the terminal position, precisely in the terminal acid position. In other words, it is functionalized by a hydrocarbon group on its terminal carboxylic acid function. The ester function may in particular correspond to the formula C(=O)-O-R1 with RI being a hydrocarbon chain comprising from 1 to 22 carbon atoms, or from 1 to 20 carbon atoms.

[0023] In particular, RI is a hydrocarbon chain, in particular aliphatic, comprising from 2 to 22 carbon atoms, in particular from 2 to 20 carbon atoms.

[0024] Typically, RI is a linear or branched alkyl group comprising from one to twenty-two carbon atoms (C1 to C22), for example from 2 to 20 carbon atoms (C2-C20) or a linear or branched alkenyl group comprising from 3 to twenty-two carbon atoms (C3 to C22), for example from 3 to 20 carbon atoms (C3 to C20).

[0025] Preferably, RI is unsubstituted, in particular unsubstituted by a hydroxy -OH group.

[0026] More preferably, RI is a linear or branched C1-C22 alkyl chain, in particular C2-C20, for example C2-C18. Without limitation, RI is ethyl, propyl, isopropyl, butyl, 2-butyl, pentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-methyl-pentyl, 3-methyl-pentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, tert-butyl, heptyl, octyl, 2-ethylhexyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, 2-butyloctyl, 2-octyldodecyl, or 2-hexyldecyl. Preferably, RI is ethyl, propyl, isopropyl, n-hexyl, octyl, 2-ethylhexyl, decyl or dodecyl group.

[0027] The copolymer according to the invention may also comprise an ester function in terminal alcohol position. In other words, it is functionalized by an acyl group on its terminal alcohol function. The ester function can notably correspond to the formula 0-C(0)-R6 with R6 being a hydrocarbon chain comprising from 1 to 22 carbon atoms.

[0028] Typically, the copolymer has the following chemical formula (I):

[0029] R8-(O-CHR7-Z-CO)n-OR1(I)

[0030] Where

[0031] R8 is a hydrogen atom or a -COR6 group,

[0032] Ri and R6 are as described above,

[0033] R7 is a hydrogen atom or a saturated, linear or branched, non-cyclic aliphatic group comprising from one to twelve carbon atoms (Cl to C12) optionally substituted by a hydroxyl group,

[0034] each Z is, independently of one another, a saturated divalent non-cyclic aliphatic group comprising from three to twelve carbon atoms (C3 to C12), optionally substituted by a hydroxyl group, or an unsaturated divalent non-cyclic aliphatic group comprising from three to twelve carbon atoms (C3 to C12), optionally substituted by a hydroxyl group,

[0035] at least one Z is an unsaturated aliphatic group and at least one Z is a saturated aliphatic group,

[0036] n is a value greater than or equal to 2.1, preferably greater than or equal to 2.5.

[0037] In particular, the copolymer has the following chemical formula (II):

[0038] R8-(O-CHR9-W-CO)n-OR1(n)

[0039] Where

[0040] R8 is a hydrogen atom or a -COR6 group,

[0041] Ri and R6 are as described above,

[0042] W is a saturated divalent non-cyclic aliphatic group comprising from three to twelve carbon atoms (C3 to C12), optionally substituted by a hydroxyl group, each R9 is independently of one another a saturated, linear or branched non-cyclic aliphatic group comprising from one to twelve carbon atoms (C1 to C12) optionally substituted by a hydroxyl group, or an unsaturated, linear or branched non-cyclic aliphatic group comprising from one to twelve carbon atoms (C1 to C12) optionally substituted by a hydroxyl group,

[0043] at least one R9 is an unsaturated aliphatic group and at least one R9 is a saturated aliphatic group,

[0044] n is a value greater than or equal to 2.1, preferably greater than or equal to 2.5.

[0045] The copolymer of the invention is typically obtained by biocatalysis. Saturated hydroxy fatty acid

[0046] The saturated hydroxy fatty acid may comprise from 12 to 24 carbon atoms, in particularly of 14 to 22 carbon atoms, more particularly of 16 to 20 carbon atoms, notably 18 carbon atoms.

[0047] Typically, the saturated hydroxy fatty acid is mono-hydroxylated.

[0048] The hydroxyl function of the saturated hydroxyl fatty acid is in particular in position 10, 12, 13 or 14 of the fatty acid when they exist, preferably in position 12. The hydroxyl function of the fatty acid can also be in the terminal position of the fatty acid, that is to say on the carbon furthest from the carboxylic acid function.

[0049] Typically, the saturated hydroxy fatty acid has the following chemical formula (III):

[0050] HO-CHR2-Q-COOH (III)

[0051] With R2 being a hydrogen atom or a saturated, linear or branched, non-cyclic aliphatic group comprising from one to twelve carbon atoms (Cl to C12), optionally substituted by a hydroxyl group,

[0052] Q being a saturated divalent non-cyclic aliphatic group of three to twelve carbon atoms (C3 to C12), optionally substituted by a hydroxyl group.

[0053] Typically R2 is an unsubstituted, linear, saturated, non-cyclic aliphatic group comprising from one to twelve carbon atoms (Cl to C12).

[0054] In particular, Q is a divalent, saturated, unsubstituted, non-cyclic aliphatic group of three to twelve carbon atoms (C3 to C12).

[0055] For example, the saturated hydroxy fatty acid is selected from the group consisting of 8-, 9-, 10, 11-, or 12-hydroxy-lauric acid, 8-, 9-, 10, 11-, or 12-hydroxy-tridecyl acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-myristic acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-pentadecyl acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-palmitic acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-margaric acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-stearic acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-nonadecyl acid, 5-, 6-, 7-, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-arachidic acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-heneicosanoic acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-behenic acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-tricosanoic acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-lignoceric acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-pentacosanoic acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-cerotic acid.

[0056] Preferably the saturated hydroxy fatty acid is selected from the group consisting of 10-hydroxystearic acid, 12-hydroxystearic acid, 13-hydroxystearic acid, and mixtures thereof. Unsaturated hydroxy fatty acid

[0057] The unsaturated hydroxylated fatty acid may comprise from 12 to 24 carbon atoms, in particular from 14 to 22 carbon atoms, more particularly from 16 to 20 carbon atoms, in particular 18 carbon atoms.

[0058] Typically, the unsaturated hydroxy fatty acid is mono-hydroxylated.

[0059] The hydroxyl function of the unsaturated hydroxylated fatty acid is in particular in position 10, 12, 13 or 14 of the fatty acid when they exist, preferably in position 12. The hydroxyl function of the fatty acid can also be in the terminal position of the fatty acid, that is to say on the carbon furthest from the carboxylic acid function.

[0060] Typically, the unsaturated hydroxy fatty acid has the following chemical formula (IV):

[0061] HO-CHR3-Y-COOH (IV)

[0062] With R3 being a hydrogen atom or a saturated or unsaturated, linear or branched, non-cyclic aliphatic group comprising from one to twelve carbon atoms (Cl to C12), optionally substituted by a hydroxyl group,

[0063] Y being a saturated or unsaturated divalent non-cyclic aliphatic group of three to twelve carbon atoms (C3 to C12), optionally substituted by a hydroxyl group, and

[0064] at least one of R3 or Y comprising at least one unsaturation.

[0065] Typically R3 is an unsubstituted linear, saturated or unsaturated, non-cyclic aliphatic group comprising from one to twelve carbon atoms (Cl to C12). In particular R3 is an unsaturated group, alternatively R3 is a saturated group.

[0066] In particular, Y is a divalent, saturated or unsaturated, unsubstituted, non-cyclic aliphatic group of three to twelve carbon atoms (C3 to C12). In particular Y is an unsaturated group, alternatively Y is a saturated group.

[0067] Preferably, one of R3 or Y comprises at least one unsaturation, more preferably one unsaturation.

[0068] Alternatively, R3 and Y each comprise at least one unsaturation, in particular one unsaturation each.

[0069] For example, the unsaturated hydroxy fatty acid is selected from the group consisting of 8-, 11-, 12-, or 13-hydroxy-myristoleic acid, 8-, 11-, 12-, or 13-hydroxy-palmitoleic acid, 8-, 9-, 10-, 11-, 12-, or 13-hydroxy-sapienic acid, 8-, 11-, 12-, or 13-hydroxy-oleic acid, ricinoleic acid, 10-hydroxy-12-octadecenoic acid, 13-hydroxy-9-octadecenoic acid, 8-, 11-, 12-, or 13-hydroxy-elaidic acid, 8- or 11-hydroxy-linoleic acid, 11-hydroxy-linolelaidic acid, 8- or 11-hydroxy-alpha-linolenic acid, 8- or 11-hydroxy-gammalinolenic acid, 8-, 9-, 10, 11-, or 12-hydroxy-erucic acid, 8-, 9-, 10, 11-, 12-, or 13-hydroxy-nervonic acid.

[0070] The unsaturated hydroxylated fatty acid is in particular selected from the group consisting of ricinoleic acid, 10-hydroxy-12-octadecenoic acid, 13-hydroxy-9-octadecenoic acid and a mixture of them.

[0071] Typically, the copolymer is based on a saturated hydroxy fatty acid and an unsaturated hydroxy fatty acid having from 12 to 24 carbon atoms each, in particular from 14 to 22 carbon atoms each, for example from 16 to 20 carbon atoms each.

[0072] In particular, the saturated hydroxy fatty acid and the unsaturated hydroxy fatty acid have the same number of carbon atoms each, in particular 18 carbon atoms each.

[0073] Preferably, the saturated hydroxy fatty acid is 12-hydroxy stearic acid and / or the unsaturated hydroxy fatty acid is ricinoleic acid. Process

[0074] The invention also relates to a process for the formation of copolymers as described above, characterized in that it is carried out in the presence of a saturated hydroxylated fatty acid and an unsaturated hydroxylated fatty acid, or their esters, and a lipase from Streptomyces sp.

[0075] Lipase useful in the context of the present invention

[0076] The lipase useful in the context of the present invention is a lipase from Streptomyces sp.

[0077] Typically, the lipase useful in the context of the present invention comprises the following amino acid sequence SEQ ID NO. 1, or a sequence having at least 75% identity with the sequence SEQ ID NO. 1:

[0078] ATATAATPAAEATSRGWNDYSCKPSAAHPRPVVLVHGTFGNSIDNWLVLAP YLVNRGYCVFSLDYGQLPGVPFFHGLGPIDKSAEQLDVFVDKVLDATGAPKA DLVGHSQGGMMPNYYLKFLGGADKVNALVGIAPDNHGTTLLGLTKLLPFFPG VEKFISDNTPGLADQVAGSPFITKLTAGGDTVPGVRYTVIATKYDQVVTPYRT QYLDGPNVRNVLLQDLCPVDLSEHVAIGTIDRIAFHEVANALDPARATPTTCA SVIG.

[0079] Preferably, the lipase useful in the context of the present invention is lipase MAS 1 isolated from the strain Streptomyces sp. Strain W007 having in particular the accession number WP_007448656.1 comprising the amino acid sequence SEQ ID NO. 1.

[0080] Alternatively, the lipase used in the method according to the invention may also be a lipase with a sequence having at least 80%, at least 82%, at least 83%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.3%, or at least 99.6% identity with the sequence SEQ ID NO. 1.

[0081] The identity percentages referred to in the context of the disclosure of the present invention are determined on the basis of a global alignment of the sequences to be compared, that is to say on an alignment of the sequences taken in their entirety over the entire length using any algorithm well known to those skilled in the art such as the algorithm of Needleman and Wunsch-1970. This comparison of sequences can be carried out using any software well known to those skilled in the art: for example, the needle software using the “Gap open” parameter equal to 10.0, the “Gap extend” parameter equal to 0.5 and a “Blosum 62” matrix. The needle software is for example available on the website ebi.ac.uk world wide under the name “Align”.

[0082] Preferably, the percentage of identity defined within the framework of the present invention is determined by means of a global alignment of the sequences to be compared over their entire length.

[0083] When the lipase useful in the context of the invention has an amino acid sequence which is not 100% identical to the reference sequence SEQ ID NO: 1 but which has at least 83%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.3%, or at least 99.6% identity with such a reference sequence, it may have insertions, deletions or substitutions with respect to the reference sequence. When substitutions are involved, the substitution is preferably made with an "equivalent" amino acid, i.e., any amino acid whose structure is close to that of the original amino acid and is therefore unlikely to alter the biological activities of lipase. Examples of such substitutions are shown in the following table:

[0084] [Tables 1] Original Amino Acid Substitution(s) Ala (A) Val, Gly, Pro Arg (R) Lys, His Asn (N) Gin Asp (D) Glu Cys (C) Ser Gin (Q) Asn Glu (G) Asp Gly (G) Ala His (H) Arg Ile (D Leu Leu (L) Ile, Val, Met Lys (K) Arg Met (M) Leu Phe(F) Tyr Pro (P) Ala Ser(S) Thr, Cys Thr (T) Ser Trp (W) Tyr Tyr(Y) Phe, Trp Val (V) Leu, Ala

[0085] The lipase comprising the amino acid sequence SEQ ID NO: 1 or a percentage of identity as described above typically has at least one of the following characteristics, in particular at least two, in particular at least three and preferably all of the following characteristics: • the amino acid sequence comprises the pentapeptide motif GHSQG (SEQ ID NO: 2); • the amino acid sequence also includes two cysteines positioned in the N-terminal region and two cysteines positioned in the C-terminal region. The N-terminal region corresponds to the 100 amino acids, preferably the 75 amino acids, of the amino acid sequence starting from the N-terminal end, i.e. the end ending in an amino acid with a free amine function (-NH2). The C-terminal region corresponds to the 100 amino acids, preferably the 75 amino acids, of the amino acid sequence from the C-terminal end, i.e. the end ending in an amino acid with a free carboxylic acid function (-COOH); • the amino acid sequence comprises the pentapeptide motif LLGLT (SEQ ID NO: 3); • the amino acid sequence comprises the hexapeptide motif LAPYLV (SEQ ID NO: 4); • the amino acid sequence comprises the hexapeptide motif FHGLGP (SEQ ID NO: 5); • the amino acid sequence comprises the oligopeptide motif YCVFSLDYGQL (SEQ ID NO: 6); • the amino acid sequence comprises the oligopeptide motif YLKFLGGA (SEQ ID NO: 7), • the amino acid sequence comprises the tripeptide motif PAR (SEQ ID NO: 8).

[0086] In particular, the lipase comprising the amino acid sequence SEQ ID NO: 1 or a percentage of identity as described above has at least one of the following two characteristics, in particular both: - the amino acid sequence comprises the pentapeptide motif LLGLT (SEQ ID NO: 3); - the amino acid sequence comprises the hexapeptide motif LAPYLV (SEQ ID NO: 4).

[0087] In general, lipase can be produced in recombinant form in expression systems such as bacteria, yeast or fungi. Recombinant protein expression systems are known to those skilled in the art and include, for example, strains of Bacillus type (eg, B. subtilis), Escherichia (eg, E. coli), Pseudomonas (eg, P. aeruginosa), Saccharomyces (eg, S. cerevisiae), Yarrowia (eg, Y. lipolytica), Pichia (eg, P. pastoris), Trichoderma (eg, T. reeseï), As-pergillus. The lipase is preferably produced in recombinant form in a host cell, such as Escherichia coli, by methods well known to those skilled in the art, and preferably it is produced as described in Example 1. The lipase may also advantageously be produced in recombinant form in a Yarrowia or Pichia host cell for applications in the food industry, such as Yarrowia lipolytica or Pichia pastoris.Lipase can also be immobilized on a support so that it can be recycled. For example, lipase can be deposited on a . resin or a mineral support. For example, the resins can be selected from polystyrene such as Lewatit® VP OC 1064 MD-PH from Lanxess, a copolymer of styrene and divinylbenzene such as Diaion® HP20 from Resindion Sri (Binasco, Italy), polymethyl methacrylate such as Diaion® HP2MG from Resindion Sri (Binasco, Italy), phenol-formaldehyde such as AMBERLITE® XAD761 from Rhom and Haas, polyvinyl chloride, polyethylene polyamides, silicone poly(hydroxyethyl) methacrylate, lignin, or polysaccharides such as starch, cellulose, or ethyl cellulose. Among the mineral supports, calcium carbonate, activated carbon, among others, can be used as immobilization supports. Enzyme immobilization supports and techniques are well known to those skilled in the art, such as those described in publication WO2016 / 151115.

[0088] Advantageously, the lipase used in the process of the present invention is thermostable. It can be used at a high temperature (between 40°C and 95°C) with or without immobilization on a solid support.

[0089] The lipase used in the process may also be a lipase from Streptomyces Avermitilis, in particular the lipase LpsA2 used in the process described in patent FR3073231.

[0090] The lipase useful in the context of the present invention comprises the following amino acid sequence SEQ ID NO. 9, or a sequence having at least 75% identity with the sequence SEQ ID NO. 9:

[0091] DS APSSGWNDYSCKPS AAHPRPVVLVHGTLGNSVDNWLGLAPYLEHRGYC VFSLDYGQLSGVPFFHGLGPIDKSAEQLQVFVDKVLTATGATKADLVGHSQG GMMPRYYLKFLGGAGKVNALVGIAPNNHGTTLSGLTNLLPYFPGAEDLLSTA TPGLADQVVGSAFMAKLNAGGDTVAGVHYTVIATQYDEVVTPYRTAFLSGSD VHNVLLQDLCPLDLSEHVAIGLIDRIAFHEVTNALDPAHATRTTCASVFS

[0092] Alternatively, the lipase used in the method according to the invention may also be a lipase with a sequence having at least 80%, at least 82%, at least 83%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.3%, or at least 99.6% identity with the sequence SEQ ID NO. 9.

[0093] The lipase comprising the amino acid sequence SEQ ID NO: 9 or a percentage identity as described above typically has at least one of the following characteristics, in particular at least two, in particular at least three and preferably all of the following characteristics: • the amino acid sequence comprises the pentapeptide motif GHSQG (SEQ ID NO: 2); • the amino acid sequence also includes two cysteines positioned in the N-terminal region and two cysteines positioned in the C-terminal region. The N-terminal region corresponds to the 100 amino acids, preferably to the 75 amino acids, of the amino acid sequence from the N-terminal end, i.e. from the end ending in an amino acid with a free amine function (-NH2). The C-terminal region corresponds to the 100 amino acids, preferably to the 75 amino acids, of the amino acid sequence from the C-terminal end, i.e. from the end ending in an amino acid with a free carboxylic acid function (-COOH); • the amino acid sequence comprises the hexapeptide motif FHGLGP (SEQ ID NO: 5); • the amino acid sequence comprises the oligopeptide motif YCVFSLDYGQL (SEQ ID NO: 6); • the amino acid sequence comprises the oligopeptide motif YLKFLGGA (SEQ ID NO: 7). Reaction conditions

[0094] The process for the formation of copolymer according to the invention is carried out in the presence

[0095] of a saturated hydroxy fatty acid and an unsaturated hydroxy fatty acid, or esters thereof, and a lipase of Streptomyces sp. as described above.

[0096] The saturated and unsaturated hydroxylated fatty acids are as described above. In the process, they can be introduced in acid form or in ester form.

[0097] The process of the present invention can advantageously be carried out from saturated and unsaturated hydroxylated fatty acid esters as described above. Examples of hydroxylated fatty acid esters include, but are not limited to, esters, such as (Cl-C12)alkyl esters or triglycerides of the hydroxylated fatty acids described above. In particular, the hydroxylated fatty acid esters are such that the ester function corresponds to the formula C(=O)-O-R5 with R5 being a hydrocarbon chain comprising from 1 to 22 carbon atoms.

[0098] Typically, R5 is a linear or branched alkyl group comprising from one to twenty-two carbon atoms (C1 to C22), for example from 1 to 20 carbon atoms (C1-C20) or a linear or branched alkenyl group comprising from 3 to twenty-two carbon atoms (C3 to C22), for example from 3 to 20 carbon atoms (C3 to C20).

[0099] Preferably, R5 is unsubstituted, in particular unsubstituted by a hydroxy -OH group.

[0100] More preferably, R5 is a linear or branched C1-C22 alkyl chain, in particular C1-C12, for example C1-C6. In a non-limiting manner, RI is an ethyl, propyl, isopropyl, butyl, 2-butyl, pentyl, 2-pentyl, 3-pentyl, n- hexyl, 2-methyl-pentyl, 3-methyl-pentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, tert-butyl, heptyl, octyl, 2-ethylhexyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, 2-butyloctyl, 2-octyldodecyl or 2-hexyldecyl. Preferably, R5 is ethyl, propyl, isopropyl, n-hexyl, octyl, 2-ethylhexyl, decyl or dodecyl.

[0101] The method can advantageously be carried out without solvent. However, in certain embodiments, the method can be carried out in the presence of an apolar solvent of the aliphatic or aromatic hydrocarbon type or of the ether type. For example, the solvent can be toluene, methyl tert-butyl ether (MBTE), hexane, isooctane, or di-isopropyl ether. In particular, the use of a solvent is advantageous when the hydroxylated fatty acid or its ester has a high melting point, such as above 95°C.

[0102] The reaction is typically initiated at a temperature above the melting point of the hydroxy fatty acid or ester. The temperature may then be reduced.

[0103] The process can also take place in the presence of water, in particular less than 10% m / m of water in the reaction medium.

[0104] At temperatures ranging from 20°C to 95°C, the process is typically carried out without solvent and without the addition of a drying agent. The temperature at which the process is carried out is chosen depending on the starting material. The temperature is selected so that the starting material is in the liquid phase. Thus, the reaction is advantageously initiated at a temperature above the melting point of the starting material.

[0105] An alcohol may be added to the reaction medium to esterify the terminal acid function of the estolide. The alcohol may be added at the beginning of the reaction or once the acid number of the reaction medium is less than 60. The amount of alcohol to be added depends on the degree of polymerization of the estolide desired or obtained. It may be evaluated according to the estolide number desired to add the alcohol at the beginning of polymerization or be evaluated according to the residual acid number of the reaction medium following polymerization. The lower the acid number, the higher the desired estolide number, the smaller the amount of alcohol to be added. In general, the alcohol is added at a rate of 10 to 35% equivalent of the starting material. Preferably, the alcohol is added at a rate of 15 to 25%, or 20 to 25%, or 25 to 30%, or 30 to 35%, or at about 25% equivalent of the starting material.

[0106] Typically, the alcohol is of formula R4-OH, R4 being a hydrocarbon chain comprising from 1 to 22 carbon atoms.

[0107] In particular, R4 is a hydrocarbon chain, in particular aliphatic, comprising from 2 to 22 carbon atoms, in particular from 2 to 20 carbon atoms.

[0108] Typically, R4 is a linear or branched alkyl group comprising from one to twenty-two carbon atoms (C1 to C22), for example from 2 to 20 carbon atoms (C2-C20) or a linear or branched alkenyl group comprising from 3 to twenty-two carbon atoms (C3 to C22), for example from 3 to 20 carbon atoms (C3 to C20).

[0109] Preferably, R4 is unsubstituted, in particular unsubstituted by a hydroxy -OH group.

[0110] More preferably, R4 is a linear or branched C1-C22 alkyl chain, in particular C2-C20, for example C2-C18. Without limitation, R4 is ethyl, propyl, isopropyl, butyl, 2-butyl, pentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-methyl-pentyl, 3-methyl-pentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, tert-butyl, heptyl, octyl, 2-ethylhexyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, 2-butyloctyl, 2-octyldodecyl or 2-hexyldecyl. Preferably, R4 is ethyl, propyl, isopropyl, n-hexyl, octyl, 2-ethylhexyl, decyl or dodecyl group.

[0111] Examples of alcohol include, but are not limited to, methanol, ethanol, propanol, isopropanol, butanol, 2-butanol, pentanol, 2-pentanol, 3-pentanol, n-hexanol, 2-methyl-pentanol, 3-methyl-pentanol, 2,2-dimethylbutanol, 2,3-dimethylbutanol, tert-butanol, heptanol, octanol, 2-ethylhexanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, no-nadecanol, eicosanol, heneicosanol, docosanol (behenyl alcohol), iso-octadecanol, 2-butyloctanol, 2-octyldodecanol, 2-hexyldecanol, isostearyl alcohol such as 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctan-l-ol (Fineoxocol®180) or 2-(3-methylhexyl)-7-decan-l-ol (Fineoxocol®180N), isopalmitic alcohol (Fineoxocol ®1600), 2-octyldecanol, glycerol, polyglycerol, glycol, polyethylene glycol (adjuvant E1521, PEG8, PEG400, PEG600, PEG1000, PEG2000, PEG4000, PEG8000), 1,2-propylene glycol, polypropylene glycol, 1,3-propylene glycol, 1,4-butanediol, threitol, tetritol,erythritol, xylitol, ribitol, arabinitol, arabitol, pentitol, pentaerythritol, mannitol, allitol, altritol, hexitol, glucitol, sorbitol, dulcitol, volemitol, polyerythritol, polypentaerythritol, polymannitol, or a mixture thereof. Preferably, the alcohol is methanol, ethanol, octanol, decanol, dodecanol, or 2-ethylhexanol, preferably 2-ethylhexanol.

[0112] Optionally also, an organic acid is added to the reaction medium to protect the free hydroxyl function of the estolide once the acid number of the estolide whose terminal acid function is esterified is between 10 and 40 inclusive, or once the acid number of the estolide whose terminal acid function is not esterified is below 60, or at the start of the reaction. The amount of acid to be added depends on the degree of polymerization of the estolide desired or obtained. It can be evaluated according to the desired estolide number to add the acid at the beginning of polymerization or be evaluated according to the residual acid number of the reaction medium. The lower the acid number, the higher the estolide number, the smaller the amount of acid to be added. Generally, the acid is added at a rate of 10 to 35% equivalent of the initial hydroxy fatty acid. Preferably, the acid is added at a rate of 15 to 20%, or 20 to 25%, or 25 to 30%, or 30 to 35%, or at about 25% equivalent of the initial hydroxy fatty acid.

[0113] Examples of organic acids include, but are not limited to, acetic, propionic, butyric, valeric, caproic, enanthic, caprylic, pelargonic, capric, undecyl, lauric, tridecyl, myristic, pentadecyl, palmitic, margaric, stearic, nonadecyl, arachidic, heneicosanoic, or behenic acid or a mixture thereof. Preferably, the organic acid is palmitic, stearic, or arachidic acid.

[0114] The process of the present invention is typically a one-pot process.

[0115] The amount of lipase to be introduced into the reaction can be readily determined by those skilled in the art. For example, the lipase may be used at a level of 5 to 10% by weight, such as 5, or 6, or 7, or 8, or 9, or 10% by weight, expressed in terms of the ratio of the mass of dry cells containing the lipase used to the mass of the starting hydroxy acid. EXAMPLES General Conditions

[0116] Ricinoleic acid (purity of more than 80%) is obtained from TCI Europe (Zwijndrecht, Belgium). 12-Hydroxystearic acid (purity of 85%) is obtained from Alfa Aesar (Karlsruhe, Germany). Methyl ricinoleate and ethyl ricinoleate (purity of more than 75% and 80% respectively) are obtained from TCI Europe (Zwijndrecht, Belgium R0029)

[0117] MAS1 and mutated MAS1 lipases were produced by Proteus by Seqens. Methods

[0118] The acid number (AI) and the estolide number (EN) are calculated as described below.

[0119] The acid number (AI) is calculated as the mass of KOH (mg) required to neutralize 1g of acid or reaction sample.

[0120] The EN value represents the number of hydroxy fatty acids added to a starting hydroxy fatty acid such that a dimer has an EN of 1, a trimer has an EN of 2, and an equimolar mixture of dimer and trimer has an EN of 1.5. The estolide number (EN) is therefore the average number of hydroxy fatty acids added to the starting hydroxy fatty acid (HFA). When the terminal carboxylic acid of the estolide is not not functionalized, EN is calculated according to the formula:

[0121] pu - (MWKOHxlOOO \ _! f IA x MW AG HJ 1

[0122] Where IA represents the acid number, MW KOH represents the molar mass of potash, and MW AGH represents the molar mass of the hydroxylated fatty acid.

[0123] When the terminal carboxylic acid of the estolide is functionalized, the acid number cannot be used to calculate the EN. The ENs are evaluated from GPC / SEC (gel permeation chromatography / size exclusion chromatography) analyses to determine the Mn and Mw. In particular, the GPC / SEC analysis was carried out by calibration with a polystyrene standard. It was previously verified that the EN determined by AI of analogous estolides whose carboxylic acid function has not been modified respond linearly to the Mn (number-average molar mass) determined by GPC (EN = Mn xa + b). The ENs announced by this method do not take into account the ester bond formed between the acid function of the estolide and the alcohol.

[0124] Example 1: Preparation of Lipases

[0125] The lipases were expressed in Escherichia coli. The genes were obtained synthetically and cloned into the pET26b+ expression vector. The cultures were prepared in 2L Erlenmeyer flasks as follows: 500mL of Autoinducing Medium (AIMTB0210 Formedium™) is inoculated with 5mL of a preculture (prepared the day before in LB medium with kanamycin), to which 1.2mL of a 25g / L kanamycin solution is added. The culture is then incubated in an Eppendorf Innova S44i incubator for 4 hours at 37°C, then 18 to 20 hours at 20°C at 1800rpm. The cultures are recovered, centrifuged for 10 minutes at 4°C and 5000rpm. Cell pellets are lyophilized before use in protocols.

[0126] The protein sequence SEQ ID NO. 1 of MAS1 lipase is as follows:

[0127] ATATAATPAAEATSRGWNDYSCKPSAAHPRPVVLVHGTFGNSIDNWLVLAP YLVNRGYCVFSLDYGQLPGVPFFHGLGPIDKSAEQLDVFVDKVLDATGAPKA DLVGHSQGGMMPNYYLKFLGGADKVNALVGIAPDNHGTTLLGLTKLLPFFPG VEKFISDNTPGLADQVAGSPFITKLTAGGDTVPGVRYTVIATKYDQVVTPYRT QYLDGPNVRNVLLQDLCPVDLSEHVAIGTIDRIAFHEVANALDPARATPTTCA SVIG

[0128] The protein sequence SEQ ID NO. 10 of the mutated MAS1 lipase is as follows (the mutation is F153A):

[0129] ATATAATPAAEATSRGWNDYSCKPSAAHPRPVVLVHGTFGNSIDNWLVLAP YLVNRGYCVFSLDYGQLPGVPFFHGLGPIDKSAEQLDVFVDKVLDATGAPKA DLVGHSQGGMMPNYYLKFLGGADKVNALVGIAPDNHGTTLLGLTKLLPFAPG VEKFISDNTPGLADQVAGSPFITKLTAGGDTVPGVRYTVIATKYDQVVTPYRT QYLDGPNVRNVLLQDLCPVDLSEHVAIGTIDRIAFHEVANALDPARATPTTCA SVIG

[0130] These two enzymes are expressed with the signal peptide MLPWIRAARVPRTRSL-LAALLLALTALVAPA (SEQ ID NO. 11).

[0131] Example 2: Preparation of estolide copolymer based on ricinoleic acid and 12-hydroxystearic acid 50 / 50 functionalized in the terminal acid position with 2-ethyl-1-hexanol

[0132] In a three-necked flask equipped with a bubbler, 25 of 12-hydroxystearic acid, 25g of ricinoleic acid and 5.4g of 2-ethyl-1-hexanol are introduced. The reaction medium is heated to 90°C until the mixture is completely dissolved, then the temperature is reduced to 60°C. At 60°C, 0.5g of water and 2g of mutated MAS1 lipase are added. The reaction medium is stirred under a nitrogen stream at 60°C for 46 hours until the two hydroxylated fatty acids are completely consumed. The reaction medium is diluted at room temperature with 150 mL of EtOAc then 5g of Clarcel ceca DIC B are added and stirred for 5 minutes. The reaction medium is filtered through a frit, washed with water and then the organic phase is evaporated and dried under vacuum. 45 g of a pale yellow viscous oil was recovered. Titration of one sample indicated an AI of 3.5, Mw of 2924 and EN of 3.3 by GPC analysis.

[0133] Example 3: Preparation of estolide copolymer based on ricinoleic acid and 12-hydroxystearic acid 90 / 10 functionalized in the terminal acid position by ethanol

[0134] In a three-necked flask equipped with a bubbler, 3g of ethyl 12-hydroxystearate and 27g of ethyl ri-cinoleate are introduced. The reaction medium is heated to 70°C until the mixture is completely dissolved. Then 0.3g of water and 0.75g of mutated MAS1 lipase are added. The reaction medium is stirred under a nitrogen stream at 70°C for 24 hours. The reaction medium is diluted at room temperature with 100 mL of EtOAc then 2g Clarcel ceca DIC B is added and stirred for 5 minutes. The reaction medium is filtered on a frit, evaporated and dried under vacuum. 25 g of a viscous amber-colored oil was recovered. Titration of one sample indicated an AI of 8.7, Mw of 2636, and EN of 3.3 by GPC analysis.

[0135] Example 4 (comparative): preparation of ricinoleic acid estolide functionalized in the terminal acid position by ethanol

[0136] In a three-necked flask equipped with a bubbler, 45.7g of ethyl ricinoleate are introduced followed by 0.75g of water and 2.28g of mutated MAS1 lipase. The reaction medium is stirred under a nitrogen stream at 60°C for 29 hours. The reaction medium is diluted at room temperature with 150 mL of EtOAc then 5g of Clarcel ceca DIC B is added and stirred for 5 minutes. The reaction medium is filtered through a frit, washed with water and then the organic phase is evaporated and dried under vacuum. 38g of a pale yellow viscous oil were recovered. Titration of a sample indicated an AI of 1.6, a Mw of 3678 and an EN of 4.1 by GPC analysis.

[0137] Example 6: study of the biodegradability of estolides according to examples 3, 4 and 5

[0138] The biodegradability of estolides is determined using the biodegradability test OECD 301F standardized reliability (manometric respirometry test).

[0139] The inoculum used in this experiment was composed of sludge collected from La Courly, Pierre-Bénite wastewater treatment plant (France). The inoculum was sampled and aerated under agitation overnight to decrease the organic carbon content. Then three successive centrifugation steps (9250 rpm, 15 min at room temperature) were performed to remove the liquid content and resuspend the inoculum at 5 g / L of MLSS (mixed liquor hanging solids) in the mineral medium of the biodegradation test. The composition of the biodegradation test medium was 85 mg / L KH2PO4, 217.5 mg / L K2HPO4, 334 mg / L Na2HPO4, 27.5 mg / L CaC12, 11.5 mg / L MgSO4 and 0.100 mg / L FeC13. The pH of the medium was adjusted to 7.2 by adding 1 M aqueous HCl solution.

[0140] The copolymers of Examples 2, 3 and 4 were prepared according to the following emulsion protocol. A 2 g / L stock solution in a non-biodegradable surfactant solution (Symperonic PE105 at 1 g / L) was prepared, then diluted by 2 with silicone oil and an emulsion was formed using an ultra-turax. The emulsion, kept under constant stirring, was introduced into the bottle containing the mineral medium.

[0141] A biodegradation test was performed by incubating the washed inoculum at a final concentration of 28.38 mg / L MLSS in the presence of the sample at approximately 140 mg / L 02 consumed to mineralize the entire sample expressed as ThOD. Biodegradation was assessed in BOD bottles incubated at 20 °C using TS608 / 4i incubation chambers (Xylem Analytics; France) by recording the variation of atmospheric pressure daily using OxiTop®-IDS sensor. Biodegradability was then expressed as a percentage based on the amount of oxygen required to mineralize the sample. Additional test conditions were performed using a reference substance used in the test to conclude both on the functionality of the inoculum and on irrelevant or low toxicity of the sample.

[0142] The biodegradability of the sample was monitored for more than 28 days and less than 60 days.

[0143] [Tables2] estolide Day 28 Day 60 1 Example 2 53.1% ±3.6% 73.1% ±3.6% 2 Example 3 51.4% ±2.1% 75.7% ± 3.4% 3 Example 4 44.7% ± 0.3% 67.7% ± 1.3%

Claims

Claims

1. Copolymer based on a saturated hydroxylated fatty acid and an unsaturated hydroxylated fatty acid, the copolymer comprising in the terminal position an ester function and having a weight-average molar mass less than or equal to 5000 g / mol, in particular ranging from 1000 to 5000 g / mol.

2. Copolymer according to claim 1 characterized in that its estolide number ranges from 1 to 6.

3. Copolymer according to any one of the preceding claims, characterized in that its polydispersity index is less than or equal to 3.0, preferably less than or equal to 2.5, more preferably less than or equal to 2.

0.

4. Copolymer according to any one of the preceding claims, characterized in that its estolide number ranges from 1.5 to 5.8, in particular from 2.0 to 5.5, for example between 2.7 and 4.

5.

5. Copolymer according to any one of the preceding claims, characterized in that the ester function corresponds to the formula C(=O)-O-R1 with RI being a hydrocarbon chain comprising from 1 to 20 carbon atoms.

6. Copolymer according to any one of the preceding claims, characterized in that the saturated hydroxylated fatty acid and the unsaturated hydroxylated fatty acid have the same number of carbons, in particular 18 carbon atoms.

7. Copolymer according to any one of the preceding claims, characterized in that the saturated hydroxylated fatty acid is 12-hydroxystearic acid and / or the unsaturated hydroxylated fatty acid is ricinoleic acid.

8. Copolymer according to any one of the preceding claims obtained by biocatalysis.

9. Process for the formation of a copolymer according to any one of the preceding claims, characterized in that it is carried out in the presence of a saturated hydroxylated fatty acid and an unsaturated hydroxylated fatty acid, or their esters, and a lipase of Streptomyces sp.

10. Method according to the preceding claim characterized in that the lipase comprises the amino acid sequence SEQ ID NO. 1 ATATAATPAAEATSRGWNDYSCKPSAAHPRPVVLVHGTFGNSI DNWLVLAPYLVNRGYCVFSLDYGQLPGVPFFHGLGPIDKSAEQL DVFVDKVLDATGAPKADLVGHSQGGMMPNYYLKFLGGADKV NALVGIAPDNHGTTLLGLTKLLPFFPPGVEKFISDNTPGLADQVAG SPFITKLTAGGDTVPGVRYTVIATKYDQVVTPYRTQYLDGPNVR NVLLQDLCPVDLSEHVAIGTIDRIAFHEVANALDPARATPTTCAS VIG or a sequence comprising at least 75% identity with the sequence SEQ ID NO.

1.

11. A method according to any one of claims 9 or 10, characterized in that the lipase has the amino acid sequence SEQ ID NO. 1 or a sequence having at least 80%, in particular at least 85%, especially at least 90% identity with the sequence SEQ ID NO.

1.

12. A method according to any one of claims 9 to 11, characterized in that the amino acid sequence comprises the pentapeptide motif GHSQG, in particular the amino acid sequence also comprises two cysteines positioned in the N-terminal region and two cysteines positioned in the C-terminal region.

13. Method according to any one of claims 1 to 3, characterized in that the amino acid sequence comprises one or more motifs chosen from the following group: the pentapeptide LLGLT (SEQ ID NO: 3), the hexapeptide LAPYLV (SEQ ID NO: 4), the hexapeptide FHGLGP (SEQ ID NO: 5), the oligopeptide YCVFSLDYGQL (SEQ ID NO: 6) and the oligopeptide YLKFLGGA (SEQ ID NO: 7), in particular one or more motifs chosen from the following group: the pentapeptide LLGLT (SEQ ID NO: 3) and the hexapeptide LAPYLV (SEQ ID NO: 4).

14. Method according to any one of claims 1 to 7, characterized in that it is carried out without solvent.

15. Process according to any one of claims 1 to 10, characterized in that the monoalcohol is added to the reaction medium when the fatty acids are in acid form.