Biocatalytic process for the formation of estolides

Abstract

The invention relates to a process for the formation of estolides from hydroxylated fatty acids or their esters, characterized in that it is carried out in the presence of a lipase, advantageously without solvent, quickly, efficiently and with a high number of estolides EN, or even advantageously greater than 1.1. Optionally, the terminal acid function of the estolide can be protected by the addition of an alcohol to the reaction medium, optionally followed by the addition of an organic acid to the reaction medium to protect the terminal hydroxyl function of the estolide, by a one-pot process.

Description

Title of the invention: Biocatalytic process for the formation of estolides FIELD OF THE INVENTION

[0001] The present invention relates to a process for preparing estolides from hydroxylated fatty acids or their esters and more particularly to a process 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 are in the form of regioisomers and / or generally have a low purity and require long and expensive purification steps.

[0005] The esterification of hydroxylated fatty acids can also be carried out by biocatalysis. For example, US7,125,694 describes the enzymatic esterification of 9,10-dihydroxystearic acid in the presence of Lypozyme™ lipase and an alcohol.

[0006] 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. This lipase has the advantage of making it possible to obtain estolides having a degree of oligomerization (also called "estolide number" or EN) greater than 1.1 with improved kinetics compared to lipases from other microorganisms. This lipase can be stable at high temperature for 5 hours.

[0007] 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.

[0008] If the prior art proposes processes for preparing estolides by biocatalytic means, a need remains to develop new processes for preparing estolides which are rapid and efficient, in particular under mild conditions, i.e. without solvent or drying agent and which can be implemented over wide temperature ranges, in particular at high temperature. Advantageously, the processes will make it possible to produce estolides in high yield, with rapid kinetics using low lipase concentrations. The estolides produced will advantageously have a high degree of purity, will be slightly colored, making the process economically attractive (in particular by limiting the purification steps). Summary of the invention

[0009] The invention relates to a process for preparing estolides from at least one hydroxylated fatty acid or hydroxylated fatty acid ester in the presence of a lipase comprising the amino acid sequence SEQ ID NO. 1 or an amino acid sequence having at least 83% identity with the sequence SEQ ID NO. 1.

[0010] Other aspects of the invention are as described below. DETAILED DESCRIPTION OF THE INVENTION

[0011] The inventors have developed a process meeting the expressed needs. Indeed, the inventors have demonstrated that estolides could be rapidly and efficiently produced from hydroxylated fatty acids or their esters in the presence of a lipase comprising the amino acid sequence SEQ ID NO. 1 or a sequence having at least 83% identity with the sequence SEQ ID NO. 1.

[0012] Lipase useful in the context of the present invention

[0013] 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 83% identity with the sequence SEQ ID NO. 1:

[0014] ATATAATPAAEATSRGWNDYSCKPSAAHPRPVVLVHGTFGNSIDNWLVLAP YLVNRGYCVFSLDYGQLPGVPFFHGLGPIDKSAEQLDVFVDKVLDATGAPKA DLVGHSQGGMMPNYYLKFLGGADKVNALVGIAPDNHGTTLLGLTKLLPFFPG VEKFISDNTPGLADQVAGSPFITKLTAGGDTVPGVRYTVIATKYDQVVTPYRT QYLDGPNVRNVLLQDLCPVDLSEHVAIGTIDRIAFHEVANALDPARATPTTCA SVIG.

[0015] Preferably, the lipase useful in the context of the present invention is the MAS 1 lipase 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.

[0016] Alternatively, the lipase used in the method according to the invention may also be a lipase with a sequence having 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 the sequence SEQ ID NO. 1.

[0017] 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”.

[0018] 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.

[0019] 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:

[0020] [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

[0021] The lipase comprising the amino acid sequence SEQ ID NO: 1 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 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).

[0022] 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).

[0023] 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.

[0024] 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.

[0025] The lipase used in the method of the present invention has numerous advantages over the LpsA2 lipase used in the method described in patent FR3073231.

[0026] Indeed, the lipase useful in the context of the present invention makes it possible in particular to obtain estolides having a high estolide number (EN), in particular greater than 4, in particular greater than 4.5.

[0027] The lipase useful in the context of the present invention also has faster estolide preparation kinetics than LpsA2 lipase. To obtain an analogous estolide, it is therefore possible to have a shorter reaction time and / or to add less lipase. The lipase useful in the context of the present invention is therefore more efficient and more productive.

[0028] The lipase useful in the context of the present invention makes it possible to prepare estolides from a hydroxylated fatty acid ester. The alcohol released during the reaction does not inhibit the activity of the lipase.

[0029] The lipase useful in the context of the present invention also allows the reaction to be carried out at high temperature for a long period without loss of lipase activity.

[0030] Finally, the lipase useful in the context of the present invention is compatible with the production of estolides on an industrial scale. Hydroxy fatty acids or their esters

[0031] The hydroxylated fatty acids useful in the context of the invention typically have the following chemical formula (I):

[0032] HO-CHRrQ-COOH (I)

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

[0034] Q being a divalent, saturated or unsaturated, non-cyclic aliphatic group comprising from three to twelve carbon atoms (C3 to C12), optionally substituted by a hydroxyl group.

[0035] Typically, the hydroxylated fatty acid is mono-hydroxylated.

[0036] Typically, the hydroxyl function of the fatty acid is 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.

[0037] In particular, the hydroxylated fatty acid comprises from 16 to 22 carbon atoms, more particularly from 16 to 20 carbon atoms, in particular 18 carbon atoms.

[0038] The hydroxy fatty acid may be saturated.

[0039] Alternatively, the hydroxy fatty acid may be monounsaturated or polyunsaturated, preferably monounsaturated.

[0040] Typically Ri is a non-cyclic, saturated or unsaturated, linear, unsubstituted aliphatic group comprising from one to twelve carbon atoms (Cl to C12).

[0041] Typically Ri is saturated. Alternatively, Ri is unsaturated, especially monounsaturated.

[0042] In particular, Q is a divalent, saturated or unsaturated, unsubstituted, non-cyclic aliphatic group of three to twelve carbon atoms (C3 to C12).

[0043] Q may be saturated. Alternatively, Q may be unsaturated, especially monounsaturated.

[0044] The hydroxylated fatty acids useful in the context of the invention may be natural fatty acids such as ricinoleic acid (also called 12-hydroxy-cis-9-octadecenoic acid), lesquerolic acid (also called 14-hydroxy-cis-11-eicosenoic acid), densipolic acid (also called 12-hydroxy-cis-9-cis-15-octadecadienoic acid), auricolic acid (also called 14-hydroxy-cis-11-cis-17-eicosadienoic acid) or dimorphecolic acid (also called 9-hydroxy-trans-10-cis-12-octadecadienoic acid).

[0045] They can also be obtained by hydrogenation of natural hydroxylated fatty acids such as those mentioned above, such as 12-hydroxystearic acid.

[0046] They can also be obtained by chemical or enzymatic hydration of a double bond of an unsaturated fatty acid. The unsaturated fatty acid can be, for example, myristoleic acid (C14), palmitoleic acid (C16), sapienic acid (C16), oleic acid (C18), elaidic acid (C18), trans-vaccenic acid (C18), linoleic acid (C18), linolelaidic acid (C18), alpha-linoleic acid (C18), gamma-linoleic acid (C18), di-homo-gama-linoleic acid (C20), arachidonic acid (C20), erucic acid (C20), eicosapentaenoic acid (C20), clupanodonic acid (C22) or docosahexaenoic acid (C22).

[0047] Hydroxy fatty acids useful in prevention include, but are not limited to, 5-, 6-, or 7-hydroxyeicosanoic acid; 8-, 9-, 10-, or 11-hydroxyoctadecanoic acid; 7-, 8-, or 9-hydroxyhexadecanoic acid; 11-, 12-, or 13-hydroxy docosanoic acid; 12-, or 13-hydroxy-5-docosenoic acid (all described in US5,380,894); 12,13,17-trihydroxy-9(Z)-octadecenoic acid (described in US5,852,196); 10-hydroxy stearic acid (described in US4,582,804); 12-hydroxystearic acid (described in patent FR2906530); 7,10-dihydroxy-8-octadecenoic acid (described in patent US5,900,496); ricinoleic acid (with the hydroxyl in position 12, described in patent FR2942628); 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, 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, 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, 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, 10,13-dihydroxy-myristic acid, 10,13-dihydroxy-pentadecylic acid, 10,13-dihydroxy-palmitic acid, 10,13-dihydroxy-margaric acid, 10,13-dihydroxy-stearic acid, 10,13-dihydroxy-nonadecylic acid, 10,13-dihydroxy-arachidic acid, 10,13-dihydroxy-heneicosanoic acid, 10,13-dihydroxy-behenic acid, 10,13-dihydroxy-tricosanoic acid, 10,13-dihydroxy-lignoceric acid, 10,13-dihydroxy-pentacosanoic acid, or 10,13-dihydroxy-cerotic acid, individually or a mixture thereof.

[0048] Preferably, the hydroxylated fatty acids are 10-hydroxystearic acid, 12-hydroxystearic acid, 13-hydroxy stearic acid, ricinoleic acid, 10-hydroxy-12-octadecenoic acid, 13-hydroxy-9-octadecenoic acid, individually or a mixture, in particular a mixture of two of them.

[0049] In some embodiments, the hydroxy fatty acids are in the form of a vegetable oil.

[0050] In certain embodiments, the hydroxylated fatty acids are in the form of triglycerides, in particular present in a vegetable oil.

[0051] The process of the present invention can advantageously be carried out from hydroxylated fatty acid esters. 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.

[0052] In some embodiments, a single hydroxy fatty acid (i.e., a single type of hydroxy fatty acid) is used in the method of the present invention. The use of a single hydroxy fatty acid allows a homopolymer-type estolide to be obtained.

[0053] Alternatively, at least two hydroxylated fatty acids (i.e. a mixture of at least two different hydroxylated fatty acids) are used in the process of the present invention. A mixture of at least two different hydroxylated fatty acids makes it possible to obtain a copolymer-type estolide. In particular, the hydroxylated fatty acids are a mixture of two different hydroxylated fatty acids.

[0054] In some embodiments, the hydroxy fatty acids comprise a mixture of saturated and unsaturated hydroxy fatty acids. Reaction conditions

[0055] 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.

[0056] 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.

[0057] The process can also take place in the presence of water, in particular less than 10% m / m of water in the reaction medium.

[0058] 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 for carrying out the process is chosen according to the starting product. The temperature is selected so that the starting product is in the liquid phase. Thus, the reaction is advantageously initiated at a temperature above the melting point of the starting product.

[0059] Optionally, an alcohol is added to the reaction medium to esterify the terminal acid function of the estolide. The alcohol can 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 can be evaluated according to the number of estolide desired to add the alcohol at the beginning of polymerization. The lower the acid number at the end of the reaction, the more the terminal acid function of the estolide is esterified. In general, the alcohol is added at a rate of 10 to 35% equivalent of the starting product. 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.

[0060] 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, polyglycerol, or 2-ethylhexanol, preferably 2-ethylhexanol.

[0061] 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 number of estolide desired to add the acid at the start of polymerization or be evaluated according to the residual acid number of the reaction medium. The lower the acid number, the higher the number of estolide, the greater the amount of acid to be added is small. 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.

[0062] 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.

[0063] The process of the present invention is typically a one-pot process.

[0064] 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. Estolides

[0065] The process of the present invention makes it possible to prepare estolides with a high estolide number (EN), in particular greater than or equal to 1.1, or even greater than 2. Typically, such a number of estolides is obtained very quickly, i.e. in less than 48 hours, even advantageously in 24 hours or less. This number of estolides is advantageously obtained at a low lipase concentration.

[0066] Advantageously, the estolide formed has an estolide number (EN) having a value of 1.5 to 2.0, or 2.0 to 2.5, or 2.5 to 3.0, or 3.0 to 3.5, or 3.5 to 4.0, or 4.0 to 4.5, or 4.5 to 5.0.

[0067] In some embodiments, the produced estolides have a number of EN estolides selected from 1.1 to 7.0 in increments of 0.1 (i.e., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,). Advantageously, EN is a value of 2.0 to 2.5, or 2.5 to 3.0, or 3.0 to 3.5, or 3.5 to 4.0, or 4.0 to 4.5, or 4.5 to 5.0, or 5.0 to 5.5, or 5.5 to 6.0, or 6.0 to 6.5, or 6.5 to 7.0. Advantageously, EN is a value between 2.0 and 7.0 or 3.0 and 6.5.

[0068] The EN index can be determined by NMR, from 1TA or by GPC / SEC from Mn (number molar mass).

[0069] The estolides prepared by the process of the present invention may have at least one, or at least two of the following characteristics:

[0070] - An acid number of less than 60, preferably between 10 and 55; - A melting point between -40°C and +10°C; - A viscosity between 50 and 650 cSt at 40°C measured according to ASTM D445 standard; - A viscosity between 10 and 60 cSt at 100°C measured according to the ASTM D445 standard; - A pour point of -25°C to -30°C or -40°C measured according to ASTM D97; - A pale yellow to amber color.

[0071] In certain embodiments, the estolide is of the homopolymer type. It is obtained from a single hydroxylated fatty acid as described above.

[0072] Alternatively, the estolide is of the copolymer type. It is obtained from at least two hydroxylated fatty acids (i.e. a mixture of at least two different hydroxylated fatty acids), in particular two different hydroxylated fatty acids. In particular, it is obtained from at least one saturated hydroxylated fatty acid and one unsaturated hydroxylated fatty acid.

[0073] The estolide may be functionalized by a hydrocarbon group on its terminal carboxylic acid function. In particular, the hydrocarbon group is an R3 group as described below. For example, it may have formula (V), formula (VI) or formula (VII) as described below. The process for obtaining such an estolide takes place in the presence of at least one hydroxylated fatty acid and an alcohol or in the presence of a hydroxylated fatty acid ester.

[0074] The estolide may be functionalized by a hydrocarbon group on its terminal alcohol function. In particular, the hydrocarbon group is an R2 group as described below. For example, it may have the formula (VI) as described below. The process for obtaining such an estolide takes place in the presence of an organic acid.

[0075] In particular, the process of the present invention allows the preparation of estolides (formula II) from hydroxylated fatty acids (formula I) according to the following scheme (1):

[0076] (1) n HO-CHRrQ-COOH + Lipase -> H-(O-CHRrQ-CO)n-OH

[0077] III

[0078] where

[0079] Ri is a hydrogen atom or a non-cyclic, saturated or unsaturated, linear or branched aliphatic group comprising from one to twelve carbon atoms (Cl to C12), optionally substituted by a hydroxyl group,

[0080] Q is a divalent, saturated or unsaturated, non-cyclic aliphatic group comprising from three to twelve carbon atoms (C3 to C12), optionally substituted by a hydroxyl group, and

[0081] n is a value greater than or equal to 2.1, preferably greater than or equal to 2.5.

[0082] The process of the invention also makes it possible to produce ester estolides, the terminal acid function of which is protected in the presence of a lipase as described above. These estolides advantageously have an estolide number (EN) greater than or equal to 1.1. In particular, the process allows the preparation of estolides (formula V) at from hydroxylated fatty acids (formula I) in the presence of an alcohol (formula IV) according to the following scheme (2):

[0083] (2) n HO-CHRrQ-COOH + HOR3 + Lipase -> H-(O-CHRrQ-CO)n-OR3

[0084] IIV V

[0085] where

[0086] Ri is a hydrogen atom or a non-cyclic, saturated or unsaturated, linear or branched aliphatic group comprising from one to twelve carbon atoms (Cl to C12), optionally substituted by a hydroxyl group,

[0087] R3 is a (saturated), linear or branched alkyl group comprising from one to twenty-two carbon atoms (Cl to C22), optionally substituted by one or more, in particular 1 to 7, hydroxyl groups, or a polyether alcohol radical (such as polyglycerol or polyalkylene glycol), the polyether alcohol radical being chosen so that the HOR3 molecule represents a polyether alcohol,

[0088] Q is 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

[0089] n is a value greater than or equal to 2.1, preferably greater than or equal to 2.5.

[0090] The process of the invention also makes it possible to produce estolide esters, the terminal acid function and the terminal hydroxyl function of which are protected in the presence of a lipase as described above. These estolides advantageously have an estolide number (EN) greater than or equal to 1.1. In particular, the process allows the preparation of estolides (formula VI) from hydroxylated fatty acids (formula I) in the presence of an organic acid (formula III) and an alcohol (formula IV) according to the following scheme (3):

[0091] (3) n HO-CHRrQ-COOH + R2-COOH + HOR3 + Lipase -> R2-CO-(O-CHRrQ-CO) n-0R3

[0092] IIIIIV VI

[0093] where

[0094] Ri is a hydrogen atom or a non-cyclic, saturated or unsaturated, linear or branched aliphatic group comprising from one to twelve carbon atoms (Cl to C12) optionally substituted by a hydroxyl group,

[0095] R2 is a saturated or unsaturated, linear or branched hydrocarbon group comprising from one to twenty-two carbon atoms (Cl to C22),

[0096] R3 is a linear or branched (saturated) alkyl group comprising from one to twenty-two carbon atoms (Cl to C22), optionally substituted by one or more, in particular 1 to 7, hydroxyl groups, or a polyether alcohol radical, the polyether alcohol radical being chosen so that the HOR3 molecule represents a polyether alcohol,

[0097] Q is a saturated or unsaturated divalent non-cyclic aliphatic group comprising three to twelve carbon atoms (C3 to C12), optionally substituted by a hydroxyl group, and

[0098] n is a value greater than or equal to 2.1, preferably greater than or equal to 2.5.

[0099] In particular, the process allows the preparation of estolides (formula VII) from hydroxylated fatty acid esters (formula VIII) according to the following scheme (4):

[0100] (4) n HO-CHRrQ-COOR4 + Lipase -> H-(O-CHR1-Q-CO)n-OR4

[0101] VIIIVII

[0102] where

[0103] Ri is a hydrogen atom 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,

[0104] R4 is a linear or branched (saturated) alkyl group comprising from one to twenty-two carbon atoms (Cl to C22), optionally substituted by one or more, in particular 1 to 7, hydroxyl groups, or a polyether alcohol radical, the polyether alcohol radical being chosen so that the HOR3 molecule represents a polyether alcohol,

[0105] Q is a saturated or unsaturated divalent non-cyclic aliphatic group comprising from three to twelve carbon atoms (C3 to C12), optionally substituted by a hydroxyl group, and

[0106] n is a value greater than or equal to 2.1, preferably greater than or equal to 2.5.

[0107] In schemes (1), (2), (3) and (4) above, for compounds of formulas I to VIII, the variables RB R2, R3, Q, and n can be more particularly defined as follows.

[0108] Par exemple, Ri peut être sélectionné parmi les groupes suivants : -CH3, -C2H5, -C3H7 , -c4h9, -c5Hh, -c6h13, -c7h15, -c8h17, -c9h19, -c10h21, -chh23, -c12h25, -ch=ch2, -CH=CH-CH3, -CH=CH-C2H5, -CH=CH-C3H7, -CH=CH-C4H9, -CH=CH-C5Hn, -ch=ch-c6h13, -ch=ch-c7h15, -ch=ch-c8h17, -ch=ch-c9h19, -ch=ch-c10h21, -ch2-ch=ch-ch3, -ch2-ch=ch-c2h5, -ch2-ch=ch-c3h7, -ch2-ch=ch-c4h9, -CH2-CH=CH-C5Hn, -CH2-CH=CH-C6H13, -CH2-CH=CH-C7H15, -CH2-CH=CH-C8H17, -ch2-ch=ch-c9h19, -c2h4-ch=ch-ch3, -c2h4-ch=ch-c2h5, -c2h4-ch=ch-c3h7, -C2H4-CH=CH-C4H9, -C2H4-CH=CH-C5Hn, -C2H4-CH=CH-C6H13, -C2H4-CH=CH-C7H 15, -C2H4-CH=CH-C8H17, -CH2-CH(OH)-CH3, -CH2-CH(OH)-C2H5, -CH2-CH(OH)-C3 H7, -CH2-CH(OH)-C4H9, -CH2-CH(OH)-C5Hn, -CH2-CH(OH)-C6H13, -CH2-CH(OH)-C 7H15, -CH2-CH(OH)-C8H17, -CH2-CH(OH)-C9H19, -CH2-CH(OH)-CioH21, -c2h4 -CH(OH)-CH3, -C2H4-CH(OH)-C2H5, -C2H4-CH(OH)-C3H7, -C2H4-CH(OH)-C4H9, -c2h 4-CH(OH)-C5Hn, -C2H4-CH(OH)-C6H13, -C2H4-CH(OH)-C7H15, -C2H4-CH(OH)-C8H17 et -C2H4-CH(OH)-C9H19.

[0109] For example, R2 may be selected from the following groups: a linear alkyl in Cl to C22 (saturated) such as -CH3, -C2H5, -C3H7, -C4H9, -C5Hn, -C6H13, -C7H15, -C8H17, -C9H19, -CioH2i,-ChH23, -Ci2H25, -Ci3H27, -C14H29, -Ci5H31, -Ci6H33, -Ci7H35, -Ci8H37, -Ci9 H39, -C20H4i,-C2iH43, -C22H45; a branched C2-C22 alkyl (saturated) of formula -CrH2r CH(CsH2s+i)CtH2t+i where r is an integer from 0 to 19, s and t are integers from 1 to 20 and the sum r+s+t ranges from 2 to 21; and a C2-C22 alkenyl (unsaturated) of formula -CuH2u -CH=CH2ou-CmH2m-(CH=CH-CH2)p-CqH2q+iWhere u is an integer from 0 to 20, m and q are integers from 0 to 19, and p is an integer from 1 to 3.

[0110] For example, R3 is selected from the following groups: a linear (saturated) alkyl such as -CH3, -C2H5, -C3H7, -C4H9, -C5Hn, -C6H13, -C7H15, -C8H17, -C9H19, -C10H 2i» "CnH23, -Ci2H25, -Ci3H27, -C14H29, -Ci5H31, -Ci6H33, -Ci7H35, -Ci8H37, -c19h39, -c20h41 ,-C2[H43, -C22H45; a branched (saturated) alkyl of formula -CrH2r-CH(CsH2s+i)CtH2t+i where r is an integer from 0 to 9, s and t are integers from 1 to 20 and the sum r+s+t varies from 2 to 22; said linear or branched alkyl being optionally substituted by 1 to 7 hydroxyls; a polyether alcohol radical (so that R3OH corresponds to the polyether alcohol) formed from one or more, in particular 1 or 2, of said linear or branched alkyls substituted by 2 to 7 hydroxyls as monomers. Preferably, R3 is selected from the following groups: methyl, ethyl, n-propyl, iso-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2-methyl-pentyl, 3-methyl-pentyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, heptyl, octyl, 2-ethylhexyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, iso-octadecyl, 2-butyloctyl, 2-octyldodecyl, 2-hexyldecyl, 2-octyldecyl, 2-hydroxyethyl, 2-hydroxypropyl, 2,3-bis(hydroxy)propyl, 3-hydroxy(2,2-bis(hydroxymethyl)propyl, 2,3,4-ter(hydroxy)butyl, 2,3,4,5-tetra(hydroxy)pentyl, 2,3,4,5,6-penta(hydroxy)hexyl, a radical of polyether alcohol formed from: ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,4-butanediol, pentamethylene glycol, hexamethylene glycol, 1,1,1 -tris(hydroxymethyl)methane, l,l,l-tris(hydroxymethyl)ethane, l,l,l-tris(hydroxymethyl)propane, glycerol, threitol, tetritol, erythritol, meso-erythritol, xylitol, ribitol, arabinitol, arabitol, pentitol, pentaerythritol, mannitol, allitol, altritol, hexitol, glucitol, sorbitol, dulcitol, volemitol,or a mixture thereof as monomer(s). Preferably, the polyether alcohol radical is a radical of polyglycerol (so that R30H is polyglycerol), polyerythritol (so that R30H is polyerythritol), polypentaerythritol (so that R30H is polypentaerythritol), polymannitol (so that R30H is polymannitol), a polyalkylene glycol (so that R30H is polyalkylene glycol) or a copolymer of alkylene glycols (so that R30H is the copolymer of alkylene glycols). Preferably, the polyether alcohol radical, is a polyglycerol radical, a polypentaerythritol radical, and a polyalkylene glycol radical (for example poly((CrC8)alkylene glycol), in particular poly((Ci-C6)alkylene glycol), preferably poly((Ci-C3)alkylene glycol) such as polyethylene glycol PEG or polypropylene glycol PPG) or a copolymer of alkylene glycols, in particular two alkylene glycols (for example (CrC8)alkylene glycols, in particular (Ci-C6)alkylene glycols, preferably (Ci-C3)alkylene glycols - this could be, for example, a copolymer of ethylene glycol and propylene glycol).

[0111] Q peut être sélectionné parmi les groupes divalents suivants : -C6Hi2-, -C7Hi4-, -C8Hi6 -, -c9h18-, -c10h20-, -cuh22-, -c12h24-, -ch=ch-c4h8-, -ch=ch-c5h10-, -ch=ch-c6 h12-, -ch=ch-c7h14-, -ch=ch-c8h16-, -ch=ch-c9h18-, -ch=ch-c10h20-, -ch2 -ch=ch-c3h6-, -ch2-ch=ch-c4h8-, -ch2-ch=ch-c5h10-, -ch2-ch=ch-c6h12-, -ch2-ch=ch-c7h14-, -ch2-ch=ch-c8h16-, -ch2-ch=ch-c9h18-, -c2h4-ch=ch-c2 h4-, -c2h4-ch=ch-c3h6-, -c2h4-ch=ch-c4h8-, -c2h4-ch=ch-c5h10-, -c2h4 -CH=CH-C6H12-, -C2H4-CH=CH-C7H14-, -C2H4-CH=CH-C8H16-, -CH2-CH(OH)-C4H8-, -CH2-CH(OH)-C5H10-, -CH2-CH(OH)-C6H12-, -CH2-CH(OH)-C7H14-, -CH2-CH(OH)-C8 H16-, -CH2-CH(OH)-C9H18-, -CH2-CH(OH)-CioH20-, -C2H4-CH(OH)-C3H6-, -c2h4 -CH(OH)-C4H8-, -C2H4-CH(OH)-C5H10-, -C2H4-CH(OH)-C6H12-, -C2H4-CH(OH)-C7H14 -, -C2H4-CH(OH)-C8H16- et -C2H4-CH(OH)-C9H18-,

[0112] n is a value selected from 2.1 to 8.0 by incrementing by 0.1 (i.e. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0). Advantageously, n is a value of 2.1 to 2.5, or 2.5 to 3.0, or 3.0 to 3.5, or 3.5 to 4.0, or 4.0 to 4.5, or 4.5 to 5.0, or 5.0 to 5.5, or 5.5 to 6.0, or 6.0 to 6.5, or 6.5 to 7.0, or 7.0 to 7.5, or 7.5 to 8.0. Advantageously, n is a value of 3.0 to 6.0 or 3.5 to 5.5.

[0113] In certain embodiments, the estolides prepared result from the reaction between a saturated hydroxy fatty acid and an unsaturated hydroxy fatty acid. EXAMPLES General Conditions

[0114] 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)

[0115] Novozym®435 lipase is a commercial enzyme derived from Candida antarctica and immobilized sold by Novozymes (Bagsvaerd, Denmark).

[0116] Lipases lpsA2, MAS1 and mutated MAS1 were produced by Proteus by Seqens. Methods

[0117] The acid number (AI) and the estolide number (EN) are calculated as described below.

[0118] The acid number (AI) is calculated as the mass of KOH (mg) required to neutralize 1g of acid or reaction sample.

[0119] 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 number of estolides (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 functionalized, EN is calculated according to the formula:

[0120] FN~( wLKOHjlIQQO j _ i

[0121] 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.

[0122] 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 (Gel Permeation Chromatography) analyses allowing the Mn to be determined. It has been previously verified that the EN determined by the 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 where a and b are coefficients and the origin ordinate). 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.

[0123] Viscosity is measured according to the ASTM D445 standard which consists of measuring the flow time of a volume of liquid contained in a calibrated glass capillary tube at a given temperature (40°C and 100°C for lubricants).

[0124] The pour point is measured according to ASTM D97 which consists of measuring the lowest temperature at which movement of a liquid sample is observed.

[0125] Example 1: Preparation of Lipases

[0126] 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. The cel- lulars are freeze-dried before being used in the protocols.

[0127] The protein sequence SEQ ID NO. 1 of MAS1 lipase is as follows:

[0128] ATATAATPAAEATSRGWNDYSCKPSAAHPRPVVLVHGTFGNSIDNWLVLAP YLVNRGYCVFSLDYGQLPGVPFFHGLGPIDKSAEQLDVFVDKVLDATGAPKA DLVGHSQGGMMPNYYLKFLGGADKVNALVGIAPDNHGTTLLGLTKLLPFFPG VEKFISDNTPGLADQVAGSPFITKLTAGGDTVPGVRYTVIATKYDQVVTPYRT QYLDGPNVRNVLLQDLCPVDLSEHVAIGTIDRIAFHEVANALDPARATPTTCA SVIG

[0129] The protein sequence SEQ ID NO. 9 of the mutated MAS1 lipase is as follows (the mutation is F153A):

[0130] ATAT AATPAAEATSRGWND YSCKPS AAHPRPVVLVHGTFGNSIDNWLVLAP YLVNRGYCVFSLDYGQLPGVPFFHGLGPIDKSAEQLDVFVDKVLDATGAPKA DLVGHSQGGMMPNYYLKFLGGADKVNALVGIAPDNHGTTLLGLTKLLPFAPG VEKFISDNTPGLADQVAGSPFITKLTAGGDTVPGVRYTVIATKYDQVVTPYRT QYLDGPNVRNVLLQDLCPVDLSEHVAIGTIDRIAFHEVANALDPARATPTTCA SVIG

[0131] These two enzymes are expressed with the signal peptide MLPWIRAARVPRTRSL-LAALLLALTALVAPA (SEQ ID NO. 10).

[0132] The protein sequence SEQ ID NO. 11 of lipase lpsA2 is as follows:

[0133] DS APSSGWND YSCKPS AAHPRPVVLVHGTLGNSVDNWLGLAPYLEHRGYC VFSLDYGQLSGVPFFHGLGPIDKSAEQLQVFVDKVLTATGATKADLVGHSQG GMMPRYYLKFLGGAGKVNALVGIAPNNHGTTLSGLTNLLPYFPGAEDLLSTA TPGLADQVVGSAFMAKLNAGGDTVAGVHYTVIATQYDEVVTPYRTAFLSGSD VHNVLLQDLCPLDLSEHVAIGLIDRIAFHEVTNALDPAHATRTTCASVFS

[0134] This enzyme is expressed with the signal peptide MLPWKRVLRPLTALLLTVA-VALVPAATAHA (SEQ ID NO. 12). Example 2: Comparative study of lipases

[0135] The formation of estolide from ricinoleic acid was carried out in parallel with Novozyme435 lipase (comparative), lpsA2 lipase (comparative), MAS1 lipase (according to the invention) and mutated MAS1 lipase (according to the invention). The reactions were carried out without solvent in a closed vial by mixing 1 g of ricinoleic acid in the presence of 5% w / w lipase at 50°C for the indicated time. The results are shown in Table 1 below.

[0136] [Tables2] Enzyme Novozym435® lpsA2 MAS1 MAS1 mutated EN (t=4h) 0.23 0.40 1.03 1.38 EN (t=24h) 0.28 1.21 2.71 2.74

[0137] Example 3: Preparation of estolide functionalized in the terminal acid position

[0138] An estolide was prepared from a fatty acid monoester (ethyl ricinoleate or methyl ricinoleate) to obtain an estolide esterified at the acid terminal position with an ethyl or methyl group. The reactions were carried out in an open vial by mixing 1 g of ricinoleic acid ester in the presence of 5% w / w lipase and 3% w / w water at 60°C for 24 hours. The results are shown in Table 2 below.

[0139] [Tables3] Enzyme Ricinoleic acid ester EN estolide at 24h (by GPC analysis) IA estolide at 24h lpsA2 methyl 0.03 7.8 lpsA2 ethyl 0.04 4.0 MAS1 methyl 2.60 5.0 MAS1 ethyl 2.60 4.8

[0140] Example 4: Preparation of estolide copolymer of ricinoleic acid and 12-hydroxystearic acid functionalized in the terminal acid position by 2-ethyl-1-hexanol

[0141] 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 and an EN of 3.3 by GPC analysis.

[0142] Example 5: Preparation of ricinoleic acid estolide with EN>6

[0143] In a three-necked flask equipped with a bubbler, 50g of ricinoleic acid are introduced followed of 1.25g of water and 2.5g of mutated MAS1 lipase. The reaction medium is stirred under nitrogen flow at 60°C for 96 hours. Titration of a sample of the reaction medium indicated an AI of 26 and an EN of 6.6 according to the AI.

[0144] Example 6: Preparation of 12-hvdroxv stearic acid estolide with EN>6

[0145] In a beaker, 30g of 12-hydroxy stearic acid are introduced followed by 0.81g of water and 0.96g of mutated MAS1 lipase. The reaction medium is stirred at 80°C for 1 hour, then 75°C for 4 hours, then 70°C for up to 24 hours. Titration of a sample of the reaction medium indicated an AI of 26.5 and an EN of 6.4 according to the AI.

Claims

Claims

1. Process for the preparation of estolides from at least one hydroxylated fatty acid or at least one hydroxylated fatty acid ester, characterized in that it is carried out in the presence of a lipase comprising the amino acid sequence SEQ ID NO. 1 ATATAATPAAEATSRGWNDYSCKPSAAHPRPVVLVHGTFGNSI DNWLVLAPYLVNRGYCVFSLDYGQLPGVPFFHGLGPIDKSAEQL DVFVDKVLDATGAPKADLVGHSQGGMMPNYYLKFLGGADKV NALVGIAPDNHGTTLLGLTKLLPFFPPGVEKFISDNTPGLADQVAG SPFITKLTAGGDTVPGVRYTVIATKYDQVVTPYRTQYLDGPNVR NVLLQDLCPVDLSEHVAIGTIDRIAFHEVANALDPARATPTTCAS VIG or an amino acid sequence comprising at least 83% identity with the sequence SEQ ID NO.

1.

2. A method according to claim 1, characterized in that the lipase has the amino acid sequence SEQ ID NO. 1 or a sequence having at least 85%, in particular at least 90%, especially at least 95% identity with the sequence SEQ ID NO.

1.

3. Method according to claim 1 or 2, characterized in that the amino acid sequence comprises the pentapeptide motif GHSQG (SEQ ID NO: 2), 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.

4. 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), the oligopeptide YLKFLGGA (SEQ ID NO: 7) and the tripeptide PAR (SEQ ID NO: 8), 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).

5. Method according to any one of claims 1 to 4, characterized in that the estolide produced has an estolide number (EN) greater than or equal to 1.1, in particular greater than or equal to 2.0, in particular greater than or equal to 3.

0.

6. Method according to any one of claims 1 to 5, characterized in which is conducted without solvent.

7. A method according to any one of claims 1 to 6, characterized in that the hydroxylated fatty acid has the following chemical formula (I): HO-CHRrQ-COOH (I) With Ri being a hydrogen atom or a non-cyclic, saturated or unsaturated, linear or branched aliphatic group comprising from one to twelve carbon atoms (Cl to C12), optionally substituted by a hydroxyl group, Q being a divalent saturated or unsaturated non-cyclic aliphatic group of three to twelve carbon atoms (C3 to C12), optionally substituted by a hydroxyl group.

8. A method according to any one of claims 1 to 7, characterized in that the hydroxylated fatty acid is selected from the group consisting of 10-hydroxystearic acid, 12-hydroxystearic acid, 13-hydroxystearic acid, ricinoleic acid, 10-hydroxy-12-octadecenoic acid, 13-hydroxy-9-octadecenoic acid, and a mixture thereof.

9. Process according to any one of claims 1 to 8, characterized in that the estolide is obtained from two different hydroxylated fatty acids, in particular from a saturated hydroxylated fatty acid and an unsaturated hydroxylated fatty acid.

10. A process according to any one of claims 1 to 9, characterized in that the reaction is initiated at a temperature above the melting point of the hydroxylated fatty acid.

11. Process according to any one of claims 1 to 10, characterized in that an alcohol is added to the reaction medium.

12. Process according to any one of claims 1 to 11, characterized in that an organic acid is added to the reaction medium.