WO2022023924A1

WO2022023924A1 - Polyester derivatives of unsaturated fatty acids and process for their production

Info

Publication number: WO2022023924A1
Application number: PCT/IB2021/056707
Authority: WO
Inventors: Antonino BIUNDO; Silvio CURIA
Original assignee: Biundo Antonino
Priority date: 2020-07-27
Filing date: 2021-07-26
Publication date: 2022-02-03
Also published as: IT202000018148A1

Abstract

The present invention relates to a polyester consisting of hydroxyacid monomer units wherein the hydroxyacid monomer unit is 10-hydroxy stearic acid (10-HSA) having a hydroxyl group in position C-10 involved in the ester bond, wherein said hydroxyacid monomer units are present in a number ranging from 10 to 100 and/or the polyester has a number average molecular weight ranging from 2,800 to 28,000 Da, said number average molecular weight being determined by means of the SEC method. The invention also comprises the process for the preparation and use of said polyester.

Description

POLYESTER DERIVATIVES OF UNSATURATED FATTY ACIDS AND

PROCESS FOR THEIR PRODUCTION

DESCRIPTION

The present invention relates to polyester derivatives of unsaturated fatty acids and a process for their production.

Plastic materials are extremely important in everyday life and have the capacity of finding applications in various fields. Approximately 300 million tons of plastics are produced every year and it is expected that by 2050 that number could triple. At the moment, only 1% of the plastics produced comes from renewable resources. Research for replacing monomers of a fossil origin with monomers deriving from renewable material attracts interest from both public and private sectors. Renewable plastics are produced for limiting oil consumption and for forming sustainable, degradable and/or recyclable materials, with positive economic, social and environmental impacts. A renewable material should ideally not interfere with the food chain and should therefore use raw materials that do not damage the human and animal food chain. In this way, the industrial production of polymers deriving from waste represents the most suitable strategy for obtaining innovative products with high added value for different markets, avoiding the use of raw material destined for food and being able to give a second life to waste material, creating the possibility, within an optimized process, of a reduced production cost of bio-derived polymers.

In particular, synthetic polyesters find applications on the market thanks to their superior properties, such as a high hardness, high mechanical strength and water resistance, unlike other synthetic polymers. Currently, bio-derived polyesters, composed of renewable raw materials, find various applications. Examples of bio derived polyesters are, in particular, polylactic acid (PLA), and polyhydroxyalkanoates (PHAs).

Many renewable monomers are produced by the fermentation of sugars or by the action of recombinant enzymes for the formation of molecules with certain functional groups. The lactic acid present in PLA, for example, is produced by the fermentation of sugars by various organisms, which are able to form one or more stereoisomers of this hydroxy acid.

The hydroxy acid lactic acid is subsequently polymerized chemically or enzymatically for the formation of PLA with different characteristics.

Lipid materials, including vegetable or animal oils and fats, have always been used for nutritional requirements. Furthermore, lipid materials of different origins can be used for the production of various compounds, including lubricants and hydraulic fluids. Lipid materials are composed of long-chain saturated and unsaturated fatty acids, which are esterified with glycerol to form triglycerides. In vegetable oils, including sunflower, rapeseed and com oil, the presence of a high concentration of unsaturated fatty acids in the triglyceride molecules allows the characteristic liquid state of the material, unlike animal fats which contain low concentrations of unsaturated chains, resulting in a solid state. The double bonds present in unsaturated fatty acids impart a greater reactivity to these compounds, which allows the modification of these molecules and, therefore, the formation of other compounds of greater importance in the industrial field.

After their use in nutritional requirements, including the preparation of food, the lipid materials, must be recovered in order to reduce relative environmental problems, including the obstmction of urban aqueducts. The exhausted lipid materials, called Used Cooking Oils (UCOs) or used vegetable oil, can be regenerated by removing solid particles and reducing the degree of humidity and acidity. UCOs, through technological processes, can be converted into different materials, including biodiesel and coatings.

Enzymes are natural catalysts that allow the transformation of certain molecules in aqueous environments, at temperatures lower than those used with conventional catalysts and with a high specificity. The use of enzymes in the industrial field can therefore improve production processes making them more sustainable than conventional processes.

There are various enzymes in nature which are capable of adding a water molecule to a double bond, carrying out the reaction called hydration. These enzymes are called hydratases. Of these enzymes, the fumarase class is especially used in the industrial field for the production of L-malic acid. Hydratases have a high regio- and stereo-selectivity that allows the production of specific products.

Many patents and patent applications disclose the capacity of oleate hydratases (OAH, EC 4.2.1.53) of using the double bond in position D9 of unsaturated fatty acids.

This enzyme family was identified and classified as OAH first in a Pseudomonas strain, later reclassified as Elizabethkingia meningoseptica. These enzymes can be produced recombinantly in host organisms, including Escherichia coli, and in certain cases the versatility of these enzymes has been identified on different substrates containing double bonds.

Specifically, the use of oleic acid as a substrate allows the production of free hydroxy acid 10-hydroxy stearic acid (10-HSA) (Figure 1). The biotransformation of oleic acid into 10-HSA has very often been reported in the state of the art.

The whole-cell bioconversion of recombinant E. coli expressing the oleic acid OAH enzyme in 10-HSA has also been demonstrated using the OAH enzyme Stenotrophomonas maltophilia at pH 6.5 and 35°C with 0.05% Tween40. Studies on purified enzymes have identified the dependence of the OAH enzyme Macrococcus caseolyticus for the FAD cofactor and the versatility of the enzyme on different substrates, including myristoleic acid, palmitoleic acid, forming the respective 10-hydroxy acids with a high specificity.

Furthermore, the purified enzyme of E. meningoseptica has been characterized by its instability, if not properly immobilized. The document WO2016151115 describes the transformation of unsaturated fatty acids into hydroxy acids and the enzymatic synthesis of oligomers of hydroxy acid esters (estolides) bound to themselves or to other fatty acids.

The document WO2019092137 similarly describes the enzymatic formation of estolides from fatty acids.

Estolides are oligomers of hydroxy acid esters bound to themselves or to other fatty acids and can be produced both chemically and enzymatically. Candida antarctica lipase B, commercially known as Novozym-435, has been used for the production of estolides from stearic acid and methylricinoleic acid.

The use of lipases lacking 1,3 steric specificity has been identified as being favourable for the formation of estolides, due to the possibility of these lipases of acting on secondary alcohols. The length of the estolides showed the production of oligomers with a maximum number of four repeats.

The production of estolides can also occur in nature under certain conditions.

Numerous studies on estolides have been carried out with castor-oil-derived 12-hydroxystearic acid (12-HSA), which naturally contains ricinoleic acid (12- hydroxyctadec-9-enoic acid) in high concentrations in the triglyceride structure (JPS 6416591 and JPS6416592).

These studies mainly produce these oligomers which can be used as biodegradable lubricants, functional fluids and as components in cosmetics, inks and coating formulations (coatings).

Polyhydroxyalkanoates (PHAs) are considered as being high-molecular- weight estolides. PHAs are among the few completely compostable polyesters in marine environments, unlike many polyesters of a fossil nature and PLA, which can only be degraded under particular conditions of industrial composting and anaerobic digestion.

Many studies have been carried out for the synthetic formation of estolides from vegetable oils using metal and biological catalysts but the production of long- chain and possibly high-molecular- weight synthetic PHAs from these oils is not documented, to date. The possibility of being able to use long-chain PHAs obtained from lipid materials of a different nature would represent a significant step forward in the field of the plastic industry. These products can in fact be used alone or in combination with other polymers and/or additives, possibly also of a natural and sustainable origin, for improving the characteristics of the finished product and obtaining materials for different applications. Furthermore, the susceptibility of the ester bonds to hydrolysis would allow the production of biodegradable materials.

The objective of the present invention is to solve the above-mentioned problems of the prior art, by providing polyesters deriving from unsaturated fatty acids and a process for their production, which have the following advantages.

The transformation of unsaturated fatty acids into esters has not yet allowed the production of high-molecular-weight materials with characteristics that can allow the use of these materials in different applications. In addition, PHAs have been identified as interesting materials for use in the production of finished materials for various sectors, including aquaculture, agriculture, packaging and biomedical products. PHAs are produced by bacterial cells as intracellular granules. Subsequent forced purification steps of the polymer granule are necessary for eliminating the impurities that may derive from cell residues.

PHAs of a natural origin contain closely spaced ester groups. In the various PHAs, the hydroxyl group of the hydroxy acid is almost always in position C3 (PHB-3-hydroxybutyric acid, PHV-3 -hydroxy valeric acid).

The production of these hydroxy acids takes place through the use of enzymes. Furthermore, the polymerization, specifically the polycondensation of these hydroxy acids using the carboxylic acid and the hydroxyl present in the CIO position of the hydroxy acids can take place through metal or biological catalysts (enzymes). The production of polymers can also be effected through a specific mechanical stirring that creates frictional forces that allow the production of high- molecular-weight polymers related to the most appropriate use of the material.

Finally, the production of synthetic PHAs reduces the production and purification cost of the material, due to the fact that the purification of synthetic polymers is better known and studied than the purification of biological polymers.

Structurally, the presence of a longer carbon chain than that present in PHAs of a natural origin can allow more interesting characteristics and can be used as such or as mixtures in different formulations to replace polymers of a fossil nature.

The above and other objectives and advantages of the invention, as will emerge from the following description, are achieved with polyester derivatives of unsaturated fatty acids and with a process for their production such as those described in the respective independent claims. Preferred embodiments and non trivial variants of the present invention form the object of the dependent claims.

It should be understood that all of the enclosed claims form an integral part of the present description.

The present invention will be better described by some preferred embodiments, provided by way of non-limiting example, with reference to the attached drawings, in which:

Figure 1 is the hydration reaction of oleic acid catalyzed by the oleate hydratase enzyme to produce 10-hydroxystearic acid (10-HSA);

Figure 2 is a schematic representation of an explanatory process of the present invention for the production of biopolymers from various kinds of lipid material; and

Figure 3 is the structure of poly( 10-hydroxystearic) acid (poly (10-HSA)) produced by the polymerization reaction of 10-hydroxystearic acid.

With reference to the figures, a preferred embodiment of the present invention is illustrated and described. It is immediately obvious that innumerable variations and modifications (for example relating to form, dimensions, various colours and parts with an equivalent functionality) can be applied to that described without departing from the protection scope of the invention as appears from the enclosed claims.

The present invention is based on synthetically produced polyhydroxyalkanoates (PHAs) and the process for their production. The synthetic PHAs produced are polymers formed by hydroxylated fatty acids wherein these hydroxylated fatty acids are produced by the action of the oleate hydratase enzyme on substrates containing unsaturated fatty acids with a double bond in position cis A 9. This process involves the hydrolysis of triglycerides containing unsaturated fatty acids, followed by hydration of the mixture of free fatty acids (FFAs) with a biocatalyst produced by whole cells, which can be, but not limited to, recombinant bacterial expressing the oleate hydratase of interest and, after the subsequent separation of free hydroxy acids (HFFAs) from free fatty acids (FFAs), chemical or enzymatic polymerization in anhydrous environments.

The representation of the process of the invention is schematically illustrated in Figure 2.

The lipid mixtures can be optionally pretreated to remove solid particles, as in the case of used vegetable oils. Furthermore, for all types of lipid mixtures, the pretreatment for the release of free fatty acids (FFAs) and glycerol can take place through various processes. The FFA mixture can be produced from various types of lipid mixtures, including, but not limited to, used vegetable oils. The triglycerides present in the lipid mixtures, through saponification reactions with the addition of sodium hydroxide or potassium hydroxide, produce free fatty acids (FFAs) after subsequent acidification. Another method takes place through the action of enzymes of the carboxyl-hydrolase class, including lipase, for the release of FFAs.

The mixture containing FFAs, or their salts, glycerol and inorganic salts can be used as such or it can be purified by the saponification reaction through a liquid- liquid extraction with organic solvent, including, but not limited to, ethyl acetate or chloroform. After the subsequent removal of the solvent, the mixture of FFAs, or their salts, or the mixture of FFAs, or their salts, containing glycerol and inorganic salts can be used as a substrate for the hydration reaction using the oleate hydratase enzyme produced recombinantly in a host bacterial species, such as Escherichia coli in this case. The permeability of resting cells, used for improving the transfer of substrate and product, can occur through the addition of detergents, including, but not limited to, Tween20, or through the lyophilization of resting cells.

Part of the cells is removed by centrifugation.

The product, together with the FFAs or their salts, present in the mixture, is extracted from the reaction through a liquid-liquid extraction with organic solvent, including, but not exclusively, ethyl acetate, producing a second aqueous phase containing glycerol and inorganic salts.

A subsequent hydrogenation reaction can lead to the reduction of double bonds present in other unreacted compounds for the uniformity of the mixture.

The free hydroxy acids (HFFAs) produced by the reaction can be purified from the FFA mixture through various methods, including: solubilization in organic solvent, such as hexane, and left for 18 hours at 25°C. In this way, the HFFAs produced by the reaction are crystallized; separation by silica gel column chromatography of HFFA from FFA, previously produced.

Said chromatographic separation can be carried out starting from FFA, FFA salts, FFA esters, preferably FFA methyl ester, thus respectively obtaining HFFA, HFFA salts, HFFA esters, preferably HFFA methyl ester.

After the subsequent removal of the solvent, the monomers can be polymerized forming the bio-derived polymer ("biopolymer") poly( 10-hydroxy stearic) acid (poly (10-HSA)) (Figure 3) by means of a metal catalyst or lipase in an anhydrous environment.

The poly(lO-HSA) is characterized by a glass transition temperature (Tg) ranging from -160 to 30°C and a melting point (Tm) ranging from -60 to 150°C. The Young's modulus at room temperature ideally ranges from 1 to 10² MPa.

The last two steps of the invention, polymerization and the polymer are the most interesting aspects from a commercial point of view as they are not commercially available and can improve the use of bio-derived materials. In this way it is possible to produce polyesters (estolides or polyestolides) with a long aliphatic chain and above all with a much higher number of monomer repetitions than that present in other patents or publications.

In one embodiment, the lipid material is pretreated for the removal of solid particles. This pretreatment can be effected through filtration, decanting, centrifugation. In this case the material resulting from the separation is the raw material for the saponification process of the lipid material.

In an embodiment, the lipid material can come from the extraction of oils from oilseeds and used as such as raw material of the saponification process of the lipid material.

In the preferred case, the lipid material derives from used vegetable oil regenerated by a filtration and decanting action in which the solid particles are removed, and the humidity and acidity are controlled within certain parameters (0.1-0.2% and up to 4 mg KOH/g, respectively).

In the preferred case, the lipid material contains high quantities of unsaturated fatty acids, containing a C-C double bond in cis A 9 position, including, but not limited to, oleic acid, linoleic acid and linolenic acid, determined by the iodine number assay having values higher than 80 g I2/IOO g of oil and gas chromatography, in order to identify specific components.

In an embodiment, the lipid material removed from all of the solid particles is subjected to hydrolyzation by enzymes of the carboxyl esterase class, in particular lipases of the classification EC 3.1.1.3. This class of enzymes can derive from various organisms, including, but not limited to, Bacillus , Candida , Geothricum, Pseudomonas, Alcaligenes, Thermomyces, Aspergillus, Rhizopus, Mucor.

In the preferred case, the lipid material, removed from all of the solid particles, is subjected to hydrolyzation by saponification using concentrated sodium hydroxide or concentrated potassium hydroxide (5M), wherein the triglycerides are hydrolyzed to form, after acidification at a pH equal to or lower than 6, free fatty acids (FFA), or their salts, and glycerol.

In an embodiment, the free fatty acids or their salts can be separated by liquid-liquid extraction using an organic solvent, including ethyl acetate or chloroform, which allows the formation of an aqueous phase, containing glycerol and inorganic salts, and an organic phase containing FFAs or their salts.

In the preferred case, the mixture containing FFAs or their salts, glycerol and inorganic salts, having pH 6, can be used in the subsequent reactions as such without subsequent purification steps.

In an embodiment, the oleate hydratase enzyme is used for converting the double bond present in unsaturated fatty acids to cis D9 position. This class of enzymes can derive from various organisms, including, but not limited to, Pseudomonas, Rhodococcus, Flavobacterium, Enterococcus, Lysinibacillus, Lactobacillus, Stenotrophomonas, Elizabethkingia. The oleate hydratases deriving from these organisms are known in the state of the art. Other oleate hydratases, however, can be discovered by technical personnel and persons experienced in the screening of microorganisms using the reaction in Figure 1, wherein oleic acid is converted to 10-HSA, in test tubes using methods that can test thousands of microorganisms at the same moment. Furthermore, methods using computational techniques can be used for identifying the sequence homology of oleate hydratase using known sequences of enzymes containing this enzymatic activity.

The preferred case uses the oleate hydratase of the classification EC 4.2.1.53. In particular, the oleate hydratase enzyme of Elizabethkingia meningoseptica with protein sequence as indicated in Seq. 01 (UniProt C7DFJ6) with a DNA sequence in Seq. 02 (GenBank GQ144652.1).

The preferred enzyme for this reaction is the enzyme with the sequence Seq. 01 or a fragment of this polypeptide wherein said fragment has a specific catalytic activity for oleate hydratase containing at least 75% or 80%, preferably at least 85%, 90% or 95%, more preferably over 95% or 97% and even more preferably at least 98% or 99% identical to Seq. 01.

In an embodiment, the enzyme can be expressed and purified from host bacterial cells through various methods including ion chromatography and size exclusion.

In some embodiments, the enzyme may have sequences called "tags" that allow purification by affinity chromatography.

In the preferred case, the enzyme can be expressed and produced in bacterial cells. The cells can be used as such without extracting the enzyme and without its subsequent purification. In this case, the cells are said to be quiescent.

In an embodiment, the enzymatic hydration reaction can take place in a medium containing water or buffer solution and free fatty acids (FFAs) or their salts. If FFAs are used, two liquid phases will be formed. The oily phase and the aqueous phase. The two phases must be mixed uniformly in order to form a stable emulsion to have a reaction that takes place rapidly over time.

In the preferred case, the bacterial cells, freeze-dried or not, containing the oleate hydratase enzyme produced recombinantly can be immobilized or inserted on a carrier, which, after each subsequent reaction, will allow the biological catalyst to be recycled for subsequent reactions. The aqueous phase and oil phase ratio can vary significantly.

In an embodiment, the addition of solvents can be effected for improving the solubilization of the reagents and products. A skilled person in the field will be able to choose the best solvent for this step.

In an embodiment, at the end of the reaction, the reaction product, containing free hydroxy acids (HFFAs) or their salts and FFAs or their salts can be recovered by liquid-liquid extraction with organic solvent, including, but not limited to, ethyl acetate, chloroform, hexane, heptane, dodecane, hexadecane. In the preferred case, the bacterial cells can be recycled by filtration of the reaction mixture on a filter or by magnetic beads or packed in a column. The oily mixture, containing HFFAs, including 10-HSA, or their salts and FFAs or their salts can be recovered by liquid-liquid extraction with organic solvent, including, but not limited to, ethyl acetate, chloroform, hexane, heptane, dodecane, hexadecane.

In an embodiment, the unsaturations present in the FFAs or their salts and the HFFAs or their salts can be reduced to C-C single bonds by a hydrogenation reaction in the presence of various catalysts, including, but not limited to, Adams catalyst and Palladium on carbon.

In an embodiment, the HFFAs or their salts and the FFAs or their salts can be used as such for subsequent steps containing unsaturated molecules.

In an embodiment, the HFFAs or their salts can be purified from the mixture through crystallization, extraction, or chromatography with the free acid form, salt, or in the ester form, preferably a low-molecular-weight alcohol-bonded ester, such as methyl ester or ethyl ester of HFFA.

In the preferred case, the mixture of HFFAs and FFAs can be esterified to produce methyl esters. In order to separate the HFFA methyl esters from the FFA methyl esters, the solution is separated by chromatography.

In the present invention, the production of biopolymers, polyhydroxyalkanoates (PHAs), and specifically poly(lO-hydroxystearic) acid, are produced through the polycondensation reaction of the HFFAs and their short-chain alcohol esters, such as methyl and ethyl, but not limited to these. The catalyst can be an acidic metal catalyst, including but not limited to titanium (IV) butoxide and dibutyltin oxide, or enzymes, such as those classified as carboxyl esterases, including but not limited to lipases lacking the 1,3 activity, but not only, capable of activating secondary alcohols. The reactions must take place in anhydrous environments. The reaction steps are subject to temperature and pressure variations in order to improve the removal of by-products, including water and short-chain alcohols, but not only.

In the case of esters of short-chain alcohols, the preferred way of producing the esters is through the use of lipase for enzymatic conversion, including, but not limited to, Novozym-435, Lipase B of Candida antarctica.

In the preferred case, the HFFAs, and specifically 10-HSA, or their esters, are used for the polycondensation reaction in an anhydrous environment to form the "Biopolymer" . The reaction can be produced at temperatures higher than 100°C, preferably ranging from 140°C to 220°C, preferably ranging from 160°C to 200°C for a period of time not exceeding 72 hours, preferably from 2 hours to 50 hours, more preferably from 24 hours to 40 hours.

The term "bio-derived polymer" used in this context is a carbon polymeric material in which the monomers derive from biological material that can be transformed through chemical and/or enzymatic reactions to form monomers which are also bio-derived.

The term polyester used in this context is a polymer containing repeats of monomers bound through an ester bond.

The term "lipid material" used in this context, represents all material containing high quantities of triglycerides of a biological origin, both animal, vegetable, fungal, and bacterial, with high levels of esterified glycerol or free fatty acids. The concentration of fatty acids present in the mixture can be identified through various methods, including gas chromatography, a method that separates and quantifies the concentrations of these molecules. Triglycerides are chemicals produced by the esterification of three fatty acids (saturated and/or unsaturated) to a glycerol molecule. The lipid material can come from various sources. Virgin vegetable oils, including but not limited to oils deriving from sunflower, olive, soybean, canola, safflower, com can be used. Used vegetable oils deriving from pretreatment for removing solid particles can be used. The concentration of unsaturated fatty acids is preferably greater than 50%.

The term "enzyme" used in this context refers to a protein containing a catalytic activity given by the specific position of different amino acids within its stmcture to transform a specific substance (substrate) into the reaction product.

The term "polymerization" used in this context refers to the combining of certain molecules (monomers) for the formation of stmctures called polymers that contain a high repetition of monomers. Polymerization in this case is a polycondensation given by the combining of different monomers and the production of water or alcohols.

The present invention therefore relates to a polyester consisting of hydroxy acid monomer units wherein the hydroxy acid monomer unit is 10- hydroxystearic acid (10-HSA) having a hydroxy group in position C-10 involved in the ester bond, wherein said hydroxyacid monomer units are present in a number ranging from 10 to 100 and/or the polyester has a number average molecular weight ranging from 2,800 to 28,000 Da, said number average molecular weight being determined by the SEC method.

The hydroxyacid monomer units therefore form ester bonds with both the carboxyl function and with the hydroxyl function carried by the carbon atom in position 10 with respect to the carbon atom that forms the carboxyl group.

The term unsaturated fatty acids with a double bond in cis D 9 position comprises myristoleic acid, palmitoleic acid, oleic acid, gadoleic acid, linoleic acid, rumenic acid, alpha-linolenic acid and gamma-linolenic and steridonic acid.

According to a preferred embodiment, the polyester consists of hydroxyacid monomer units wherein the hydroxyacid monomer unit is 10-hydroxystearic acid (10-HSA) having a hydroxy group in position C-10 involved in the ester bond, wherein said hydroxyacid monomer units are present in a number ranging from 10 to 20 and/or the polyester has a number average molecular weight ranging from 2,800 to 5,700 Da, said number average molecular weight being determined by the SEC method.

In the present patent application, the term average molecular weight refers to a number average molecular weight. It was determined by the SEC method, using the TOSOH EcoSEC HLC-8320GPC system (Japan), equipped with an EcoSEC RI detector and three PSS PFG 5 pm columns (microguard, 100 A and 300 A, USA). Poly(ethylene glycol) (PEG) standards were used for the calibration and toluene as the internal standard.

According to a preferred embodiment, the polyester has a glass transition temperature (Tg) ranging from -160 to 30°C and/or or a melting point (Tm) ranging from -60 to 150°C and/or a Young's modulus at room temperature ranging from 1 to 10² MPa.

The glass transition temperature and melting point are measured by DSC analysis, effected using a Mettler Toledo DSC 820 module. Samples (5-10 mg) were prepared in 100 ml aluminum crucibles. The samples were heated from 30 to 170°C (or 160°C), then cooled to -60°C (or -80°C), and then heated again to 170°C (or 160°C) at a heating/cooling rate of 10°C min ¹ under a stream of nitrogen (50 ml min ¹). The data obtained from the second heating phase were used for the analyzes.

The Young's modulus is measured with a Mettler Toledo DMA 1 module equipped with stainless steel voltage clamps. The active length was set at 10 mm. The tests were carried out at room temperature by straining the sample with a force of 1 N m ¹ until breakage. Strength data vs. time and displacement vs. time were collected and converted into stress vs. deformation using the STARe software. Each sample was tested at least in triplicate.

According to a more preferred embodiment, the polyester has a glass transition temperature (Tg) ranging from -160°C to -80°C and/or a melting point (Tm) ranging from -40°C to 20°C and/or a Young's modulus at room temperature ranging from 1 to 50 MPa.

According to a more preferred embodiment, the polyester consisting of 10 hydroxyacid monomer units is preferred wherein the hydroxyacid monomer unit is 10-hydroxy stearic acid (10-HSA).

A further object relates to a process for the production of polyester comprising the following steps: a) hydrolysis of triglycerides containing unsaturated fatty acids with a double bond in cis A 9 position to give a mixture comprising unsaturated fatty acids; b) hydration of the mixture comprising unsaturated fatty acids by the oleate hydratase enzyme to give a mixture of a fatty acid having a hydroxyl group in position C-10 which is 10-hydroxy stearic acid (10-HSA) and non-hydro xylated fatty acids; c) separation of fatty acids having a hydroxyl group in position C-10, from non-hydroxylated fatty acids; d) chemical or enzymatic polymerization of fatty acids having a hydroxyl group in position C-10.

According to a preferred embodiment, the process wherein the fatty acid having a hydroxyl group in position C-10 is 10-hydroxy stearic acid (10-HSA), is preferred.

According to a preferred embodiment, the process wherein the enzyme of step b) is an oleate hydratase of the classification EC 4.2.1.53 or Elizabethkingia meningoseptica oleate hydratase with an amino acid sequence as specified in Seq. 01 (UniProt C7DLJ6) and/or with a DNA sequence in Seq. 02 (GenBank GQ144652.1), is preferred.

According to a preferred embodiment, in step c) of the process, the separation of 10-hydroxy stearic acid (10-HSA) from the non-hydroxylated fatty acids takes place by precipitation of 10-hydroxy stearic acid (10-HSA) from a solution in hexane at a temperature ranging from -30°C to -20°C, wherein the non- hydroxylated fatty acids remain solubilized in the hexane solution. The suspension thus obtained of 10-hydroxy stearic acid (10-HSA) precipitated in hexane is filtered and the 10-hydroxystearic acid (10-HSA) remains on the filter as a solid whereas the hexane solution containing the non-hydroxylated fatty acids is collected in the filtrate.

According to a preferred embodiment, in step d) of the process, the polymerization reaction is carried out in the presence of catalytic quantities of Titanium (IV) butoxide, dibutyltin oxide or Lipase B of Candida antarctica.

According to a more preferred embodiment, in step d) of the process, the polymerization reaction is carried out in the presence of catalytic quantities of Titanium (IV) butoxide or dibutyltin oxide and at a temperature ranging from 160°C to 200°C and for a period of time ranging from 24 hours to 72 hours. The polymerization reaction is more preferably carried out at a temperature ranging from 170°C to 190°C.

According to a preferred embodiment, the mixture of HFFAs and FFAs obtained in step b) is esterified to produce methyl esters. In order to separate the HFFA methyl esters from the FFA methyl esters in step c), the solution is separated by chromatography.

In the case of esters of short-chain alcohols (methyl or ethyl), the preferred way of producing the esters is through the use of lipase for enzymatic conversion, including, but not limited to, Novozym-435, Lipase B of Candida antarctica.

A further object relates to a polyester that can be obtained from the process described above.

Another object relates to the use of polyester consisting of hydroxy acid monomer units wherein the hydroxyacid monomer unit is 10-hydroxystearic acid (10-HSA) having a hydroxy group in position C-10 involved in the ester bond, wherein said hydroxyacid monomer units are present in a number ranging from 10 to 100 and/or the polyester has a number average molecular weight ranging from 2,800 to 28,000 Da, said number average molecular weight being determined by the SEC method, for solid and flexible packaging or for horticulture, agriculture or aquaculture or for the production of consumer goods or as a thickener or dispersant for applications in cosmetics.

The invention will be further illustrated by the following non-limiting examples, provided for purely descriptive and illustrative purposes, but absolutely non-limiting of the present invention.

Example 1

Production of the whole-cell biocatalyst.

About 5g of WCW are produced from 500 ml of culture and stored at

20°C.

Example 2

Preparation of free fatty acids or their salts.

Saponification allowed the production of FFA salts and glycerol. Acidification with concentrated HC1 and subsequent extraction made it possible to extract FFAs with a yield of 93%

Example 3

Biocatalytic hydration reaction.

After 24 hours of reaction at 37°C, the mixture containing FFAs and HFFAs was separated by liquid-liquid extraction with a recovery yield of 65%. Example 4 Reduction reaction.

The uniformity of the mixture was guaranteed by the reduction reaction through hydrogenation for 3 hours. After the reaction, the catalyst was removed by Celite® 545 and the product recovered by ethyl acetate with a recovery yield of 57%.

Example 5

Esterification reaction.

The conversion takes place with a yield of 95%

Example 6

Poly condensation reaction.

The polycondensation reaction produces biopolymers having a Tg ranging from -160 to 30°C and a melting point (Tm) ranging from -60 to 150°C. They show a Young's Modulus at room temperature ranging from 1 to 10² MPa.

The biopolymers can be used for:

- Solid and flexible packaging

- Horticulture and agriculture

- Regular consumer goods

- Aquaculture

- as thickeners or dispersants for cosmetic applications.

With respect to the materials and methods used in the present invention, reference is made to the following.

The entire DNA manipulation is conducted by means of standard methods. (Sambrook et al 1989).

The visualization of the chemical compounds (FFAs and HFFAs) is allowed through immersion in a 5% solution of ammonium molybdate and 0.2% of cerium (III) sulfate in 100 ml of 17.6% sulfuric acid and heating to 200°C until the formation of blue spots.

Preparation of the whole-cell biocatalyst.

The BL21 (DE3) strain of Escherichia coli is used as host expressing the enzyme. The pET28a(+) plasmid containing the gene encoding the oleate hydratase enzyme Elizabethkingia meningoseptica is inserted by chemical transformation in the strain of E. coli BL21 (DE3). The strain is then plated on Petri plates containing the medium Lysogeny Broth (LB)-agar with 40 pg mL-1 kanamycin.

A single colony is inoculated in 30 mL of 2xYT medium with 40 pg mL-1 of kanamycin. The culture is incubated at 37°C and 200 rpm overnight. The culture is then diluted in 500 ml of 2xYT to an ODeoo 0.02 and incubated at 37°C and 200 rpm. Upon reaching an ODeoo of 0.5-0.8, the expression of the enzyme is induced at 20°C and 150 rpm with 0.5 mM i s o p ro p y 1 - b - D - 1 hi o g a 1 ac to side (IPTG). After induction for 20 hours, the cells are recovered by centrifugation at 4,000 rpm at 4°C for 20 minutes. The cell pellet is then washed with 50 ml of 50 mM NaCitrate buffer pH 6. The cell suspension is subsequently centrifuged under the same conditions indicated above. The cell pellet that forms the whole-cell biocatalyst is stored at - 20°C until further use.

Preparation of free fatty acids.

The used vegetable oils (UCOs) are regenerated through a pre-treatment. The final lipid material has the following characteristics: humidity index lower than 3%, unsaponifiability lower than 3%, insoluble products lower than 3%, maximum acidity 5% and absence of foreign bodies. The resulting lipid material is used for the saponification reaction to extract the free fatty acids (LLAs) from the triglycerides. The reaction takes place by mixing 27% v/v of lipidic material in 5M KOH at 90°C and 420 rpm with reflux for 24 hours. The reaction is then acidified at room temperature.

If the mixture is used as such, the pH of the mixture is acidified to pH 6 to form the salts of the LLAs.

Should the LLAs need to be purified, a pH of 1-2 is reached in order to separate the LLAs. A saturated solution of NaCl is added for improving the separation of the emulsion. The mixture of free fatty acids (LLAs) is separated by adding 2 volumes of chloroform in a separating funnel. The organic fraction containing LLAs is recovered and the chloroform evaporated and recycled by means of a rotavapor. The LLA mixture is then stored at -20°C.

Biocatalytic hydration reaction. The presence of unsaturated fatty acids in the mixture, such as oleic acid, allows the mixture of free fatty acids (FFAs) to take place in liquid form at room temperature. The biocatalytic reaction is prepared in a flask (1 L) containing 50 mM pH 6 of NaCitrate buffer with 1.1% (v/v) of FFAs and 1% (m/v) (WCW) of whole cell biocatalyst containing the enzyme EmOAH.

Should the cells be lyophilized, no detergent is added, otherwise 0.05% of Tween20 is added in order to increase the permeability of the cell membrane. The reaction takes place at 37°C and 200 rpm for 24 hours. At the final time, the reaction is mixed with 2 volumes of ethyl acetate to extract the product and the unreacted FFAs, or their salts. The organic phase is recovered and, after a dehydration phase with sodium sulfate, the solvent is evaporated.

Reduction reaction.

The FFAs, or their salts, and the HFFAs, or their salts, which are recovered after the reaction, are reduced using 10% (mole/mole) platinium oxide (PtC ) (Adam's catalyst) for hydrogenation at 50°C for 3 hours using 13.3% (v/v) of the FFA mixture in EtOH. The catalyst is then removed by column chromatography using silica gel 60 with a particle-size distribution of 40-63 pm and 230-400 ASTM and Celite® 545 using ethyl acetate as eluent.

Esterification reaction.

The esterification of the mixture containing HFFAs and FFAs took place with 4% of concentrated HC1 in methanol at 80°C.

Separation by column chromatography.

Silica gel column chromatography was carried out for separating the methyl esters of the HFFAs. The elution of the product takes place using a mixture of organic solvents.

Poly condensation reaction.

The polycondensation for forming the polyester is carried out following a three-step condensation method at 180°C. The methyl esters of the purified HFFAs are added to a 2-necked flask and mixed by means of a magnetic stirrer at 50 rpm, releasing inert gas into the system for 15 minutes. The system is then heated to the final temperature continuing to release inert gas. The starting point of the polymerization is determined by reaching the temperature set. For the initial phase, the flow of argon is left for 3 hours. After this period of time, the flow is closed and the vacuum is activated (500 mbars) for 2 hours. The pressure is then brought to 0.8 mbars and is left constant for the appropriate period of time (19 hours for a reaction of 24 hours). Reactions of 72 hours are also tested. After the time necessary for the reaction and subsequent cooling of the reaction, the reaction product is dissolved in 10 ml of dichloromethane and purified by precipitation in 200 ml of methanol, filtered and dried at 10² mbars at room temperature.

Example 7

5 grams of used food oil were reacted with 10 mL of a solution of KOH at 20% in ethanol for 1 hour at 80°C. The reaction was then acidified with 5 mL of sulfuric acid at 20%. The free fatty acids (FFAs) were extracted with 30 ml of ethyl acetate. The organic solvent was then removed from the FFA mixture using a rotavapor. 93% of the FFAs was recovered from the reaction mixture. 4.5 g of FFAs were mixed with 5 g of Escherichia coli whole-cell biocatalyst containing the recombinant oleate hydratase enzyme of Elizabethkingia meningoseptica (prepared as described in the previous Example 6) in 500 mL of sodium citrate buffer 50 mM pH 6. 12.5 mL of Tween 20 16.4 mM were added to the reaction mixture. The reaction was incubated at 37°C and 200 rpm for 24 hours. The reaction products were extracted with 500 ml of ethyl acetate, which was subsequently removed by means of a rotavapor with a reaction yield of 80%.

The product thus obtained was treated to reduce the unsaturations still present to single bonds by means of a hydrogenation reaction using 10% mole/mole of Pt0₂ (Adams catalyst) in a suspension of 25 mL of ethanol in the presence of 1 bar of hydrogen at 50°C for 3 hours. The catalyst was filtered on a celite bed and the solvent was evaporated by means of a rotavapor with a yield of 65%. 2.5g of a mixture of fatty acids and hydroxyacids (FFAs and HFFAs) were esterified at 80°C for 24 hours with 25 mL of methanol in the presence of 1.2 mL of HC1 37% as catalyst. The reaction mixture was then separated from the methanol by means of a rotavapor and subsequently extracted using ethyl acetate for removing the presence of water with a yield of 70%. The mixture of methyl esters of FFAs and HFFAs (FAMEs and HFAMEs, respectively) was purified by means of a chromatographic column using a mixture of hexane: dichloromethane:ethyl acetate 8:2:1 as elution solvent. The methyl ester product of 10-hydroxy stearic acid was purified with a purity higher than 95%. 600 mg of product were transferred to a three-necked flask into which the dibutyltin oxide catalyst was introduced in a concentration of 2% mole/mole. In order to remove the presence of air and oxygen from the reaction environment, a flow of argon inert gas was added for 15 minuts, followed by the creation of vacuum by means of a pump. The procedure was repeated for a total of 3 times. The reaction was then heated to 180°C with a flow of argon inert gas. The polymerization time was calculated by reaching the temperature of 180°C. The rection mixture was kept for the first 2 hours under a flow of argon. The pressure was then kept at 500 mbars for 1 hour and the pressure was subsequently kept at a value ranging from 0.8 to 7 mbars for 21 hours. The reaction product was resuspended in about 5 ml of dichloromethane, which was subsequently evaporated by means of a rotavapor. The polyester thus obtained, with a conversion of 95%, was analyzed to determine its molecular weight by means of SEC chromatography analysis, showing an average molecular weight of 3,118 Da, which indicates the presence of 11 units, with a polydispersion index (PDI) of 24.68. The polymer, which is a viscous liquid at room temperature was also tested to identify the melting point (Tm) by means of DSC analysis, showing a melting point of -35.88 °C.

Some preferred embodiments of the present invention have been illustrated and described above: numerous variants and modifications, functionally equivalent to the previous ones, which fall within the protection scope of the invention as specified in the enclosed claims, will obviously appear immediately evident to skilled persons in the field.

Claims

1. A polyester consisting of hydroxyacid monomer units wherein the hydroxyacid monomer unit is 10-hydroxy stearic acid (10-HSA) having a hydroxyl group in position C-10 involved in the ester bond, wherein said hydroxyacid monomer units are present in a number ranging from 10 to 100 and/or the polyester has a number average molecular weight ranging from 2,800 to 28,000 Da, said number average molecular weight being determined by means of the SEC method.

2. The polyester according to claim 1, having a glass transition temperature (Tg) ranging from -160 to 30°C and/or a melting point (Tm) ranging from -60 to 150°C and/or a Young's modulus at room temperature ranging from 1 to 10² MPa, said Tg and Tm being determined by means of the DSC method.

3. The polyester according to any of claims 1 to 2, wherein said hydroxyacid monomer units are present in a number ranging from 10 to 20 and/or the polyester has a number average molecular weight ranging from 2,800 to 5,600 Da.

4. A process for the production of the polyester according to any of claims 1 to 3, comprising the following steps: a) hydrolysis of triglycerides containing unsaturated fatty acids with a double bond in cis A 9 position to give a mixture comprising unsaturated fatty acids; b) hydration of the mixture comprising unsaturated fatty acids by the oleate hydratase enzyme to give a mixture of a fatty acid having a hydroxyl group in position C-10 which is 10-hydroxy stearic acid (10-HSA) and non-hydro xylated fatty acids; c) separation of the fatty acids having a hydroxyl group in position C-10, from the non-hydroxylated fatty acids; d) chemical or enzymatic polymerization of the fatty acids having a hydroxyl group in position C-10, wherein said fatty acids can be fatty acids as such, or their salts or esters of short-chain alcohols, such as methyl or ethyl, preferably methyl.

5. The process according to claim 4, wherein the enzyme of step b) is an oleate hydratase of the classification EC 4.2.1.53 or oleate hydratase of Elizabethkingia meningoseptica with an amino acid sequence as specified in Seq. 01 (UniProt C7DLJ6) and/or with a DNA sequence in Seq. 02 (GenBank GQ144652.1).

6. The process according to any of claims 4 to 5, wherein in step c), the separation of the methyl esters of the fatty acids having a hydroxyl group in position C-10, from the methyl esters of the non-hydroxylated fatty acids takes place by chromatographic separation of the fatty acids having a hydroxyl group in position C-10 from a solution in organic solvent.

7. The process according to any of claims 4 to 6, wherein in step d), the polymerization reaction is carried out in the presence of catalytic quantities of a metal catalyst including Titanium (IV) butoxide or dibutyltin oxide or biocatalysts, such as for example Lipase B of Candida antarctica.

8. The process according to any of claims 4 to 7, wherein in step d), the polymerization reaction is carried out in the presence of catalytic quantities of Titanium (IV) butoxide or dibutyltin oxide and at a temperature ranging from 160°C to 200°C and for a period of time not exceeding 72 hours.

9. The process according to any of claims 4 to 8, wherein the triglycerides containing the unsaturated fatty acids with a double bond in cis D9 position are obtained starting from used vegetable oils (UCO).

10. A polyester that can be obtained from the process according to any of claims 4 to 9.

11. Use of the polyester according to any of claims 1 to 4 or according to claim 10, for solid and flexible packaging or for horticulture, agriculture or aquaculture or for the production of consumer goods or as a thickener or dispersant for applications in cosmetics.