WO2013001126A1

WO2013001126A1 - Acylation method for producing food and/or pharmaceutical compounds using fungal sterol esterases

Info

Publication number: WO2013001126A1
Application number: PCT/ES2012/070473
Authority: WO
Inventors: Víctor BARBA CEDILLO; Alicia PRIETO ORZANCO; Ángel T. MARTÍNEZ FERRER; María Jesús MARTÍNEZ FERNÁNDEZ
Original assignee: Consejo Superior De Investigaciones Científicas (Csic)
Priority date: 2011-06-29
Filing date: 2012-06-27
Publication date: 2013-01-03
Also published as: ES2395582B1; ES2395582A1

Abstract

The invention relates to a method for the acylation of free phytosterols or saturated derivatives thereof with an acylation agent selected from the group consisting of a free fatty acid and a fatty acid ester, catalysed by a sterol esterase enzyme, characterised in that said method comprises an acylation reaction in the presence of a sterol esterase produced by fungi of the genus Ophiostoma, preferably of the species O. piceae. The invention also relates to esters of phytosterols or of the saturated derivatives thereof produced by the above-mentioned method. The invention further relates to a product enriched by the esters of phytosterols or of the saturated derivatives of those of the invention, selected from a food, a food preparation, a dietary supplement and a medicament, and to the use of any of these products to reduce the cholesterol levels in blood plasma.

Description

Acylation procedure for obtaining compounds of food and / or pharmaceutical interest using fungal sterol esterases

SECTOR OF THE TECHNIQUE

The invention is directed mainly to the sector of the food industry, although also to the sector of the nutraceutical industry, recently called "medical nutraceuticals". Using the described invention it is possible to: i) synthesize esters of plant sterols (phytosterols) or saturated derivatives thereof (phytostanols) by acylation reactions of phytosterols or phytostanols respectively with fatty acids, both in the form of free acid and in the form of ester, ii) improve the properties of previously existing products, that is, of phytosterols or free phytostanols, by acylating them to incorporate them into food, iii) contributing to the prevention of atherosclerosis thanks to the regulatory role of cholesterol absorption of esters of phytosterols and phytostanol esters, and iv) introduce a biotechnological process and more specifically a biocatalyst (enzyme) for the synthesis of these compounds. STATE OF THE TECHNIQUE

The current pace of life with a tendency to sedentary lifestyle and the increase in the consumption of saturated fats has caused a large part of the population to have elevated serum cholesterol levels, facilitating the development of atherosclerosis, and thus increasing the chances of suffer ischemia To this we must add those people who have deficiencies at the level of cholesterol metabolism and suffer from hypercholesterolemia. According to the Spanish Heart Foundation, from January 1 to March 2, 2011, 2,839,348 deaths due to cardiovascular failure would have occurred worldwide. Although they do not specify how many would have been due to strokes or acute myocardial attacks, it is easy to assume that, in a high percentage of cases, deaths would have been caused by high cholesterol levels. It is for all this that excess cholesterol can be considered as a true pandemic that in the year 2002 caused about 4.4 million deaths according to the WHO.

Plant sterols or phytosterols, and by extension their saturated derivatives, also called phytostanols, have a chemical structure common to that of cholesterol, derived from the nucleus of cyclopentaneperhydrophenanthrene. However, studies conducted In the last decade they have shown their ability to lower levels of serum cholesterol of LDL (low density lipoprotein) type, decreasing the probability of developing atheroma plaques. Although its mechanism of action is not fully known, some studies show that it could reduce the absorption of cholesterol in the intestine, by displacing it from the digestive micelles.

Therefore, the consumption of phytosterols and phytostanols could dampen and help prevent high cholesterol levels in the population but not being synthesized by the body, they must be incorporated into the diet, by ingesting fruits and vegetables or products enriched in them (Chen et al. Cholesterol-lowering nutraceuticals and functional foods. Journal of Agricultural and Food Chemistry 56: 8761-8773. 2008). In fact, there are patents such as US 7,147,859 B2 (Bruno and Falci, 2006) in which different formulations with β-sitosterol, one of the most abundant phytosterols, among its components are described. However, one of the main problems for its incorporation in different foods, mainly dairy products or vegetable oils, has been its high melting point and low solubility. One solution is to esterify sterols and tin free with fatty acids of interest to increase their solubility (Thompson and Grundy. History and development of plant sterol and stanol esters for cholesterol-lowring purposes. The American Journal of Cardiology 96: 3-9. 2005 ; Villenueve et al. Lipase-catalyzed synthesis of cañadala phytosterols oléate esters as cholesterol lowering agents. Enzyme and Microbial Technology 37: 150-155. 2005). It has been shown to be more effective in reducing cholesterol absorption at the intestinal level with a diet supplemented with esterified phytosterols than with free phytosterols (Weber et al. Cholesterol-lowering food additives: lipase-catalysed preparation of phytosterol and phytostanol esters. Food Research International 35 : 177-181. 2002). One of the first enriched products that began to be marketed in 1995 under the Benecol ^® brand is a margarine that incorporates a stanol ester and whose market launch was a consequence of the patent created by the Finnish company Raisio. This patent was the starting point for the development of new products, so in US 6,087,353 (Stewart et al., 2000) the possibility of hydrogenating phytosterol esters is described in order to further improve their solubility and stability to incorporate them into various types of products, such as beverages, foods, nutraceuticals, etc. It should be noted that of the extensive studies carried out for the safety evaluation of plant sterols and tinplates, with various animal and even cellular models, over the past few years, none has revealed any adverse health effects. For this reason, they are considered as substance G AS (Generally Regarded As Safe) by both the US regulatory agencies (FDA) and the European ones (Thompson et al., Supra).

The synthesis reaction of these compounds is the key point to obtain them, which may be chemical or enzymatic. However, the high energy expenditure involved in chemical synthesis processes, which usually develop at high temperatures, together with the formation of atherogenic and cytotoxic products means that alternative catalysts are still being sought and among them enzymatic biocatalysts can play an important role. (Villeneuve et al., Supra).

Lipases (EC 3.1.1.3), especially those produced by microorganisms, are undoubtedly the ones most frequently used for biotechnological purposes in synthesis reactions.

There are other enzymes called sterol esterases (EC 3.1.1.13) that are also capable of mediating synthesis reactions under the right conditions. Among the most studied sterol esterases is that of human origin (Loomes and Senior. Bile salt activation of human cholesterol esterase does not require protein dimerisation. FEBS Letters 405: 369-372. 1997; Ikeda et al. Cholesterol esterase accelerates intestinal cholesterol absorption Biochimica et Biophysica Acta (BBA) - General Subjects 1571: 34-44. 2002; Brown et al. Plant sterol and stanol substrate specificity of pancreatic cholesterol esterase. The Journal of Nutritional Biochemistry 21: 736-740. 2010). There are not many sterol microbial esterases described, but some of the best known are the LIP2 and LIP3 isoforms produced by Candida rugosa yeast (Benjamin and Pandey. Candida rugosa lipases: Molecular biology and versatility in biotechnology. Yeast 14: 1069-1087. 1998; Tenkanen et al. Hydrolysis of steryl esters by a lipase (Lip 3) from Candida rugosa. Applied Microbiology and Biotechnology 60: 120-127. 2002; Dominguez de María et al. Understanding Candida rugosa lipases: An overview. Biotechnology Advances 24: 180 -196. 2003; López et al. Reactivity of mash Candida rugosa lipase isoenzymes (Lipl, Lip2, and Lip3) in aqueous and organic media. Influence of the isoenzymatic profile on the lipase performance in organic half. Biotechnol Progress 20: 65-73. 2004; Chang et al. Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of purified enzyme. J. Agrie. Food Chem. 54: 5831-5838. 2006; Ferrer et al. Recombinant Candida rugosa LIP2 expression in Pichia pastoris under the control of the AOX1 promoter. Biochemical Engineering Journal 46: 271-277. 2009; Yen et al. Site-specific saturation mutagenesis on residues 132 and 450 of Candida rugosa LIP2 enhances catalytic efficieney and alters substrate specificity in various chain lengths of triglycerides and esters. Journal of Agricultural and Food Chemistry 58: 10899-10905. 2010). In fact, there are enzymatic crudes marketed by Sigma (lipase type VII of C. rugosa) and Amano (Lipasa AY, Amano 30) with LIP3 activity that have been used repeatedly for the synthesis of different compounds (Bezbradica et al. The Candida rugosa lipase catalyzed synthesis of amyl isobutyrate in organic solvent and solvent-free system: a kinetic study. Journal of Molecular Catalysis B: Enzymatic 38: 11-16. 2006; Kim and Akoh. Modeling and optimization of lipase-catalyzed synthesis of phytosteryl esters of oleic acid by response surface methodology. Food Chemistry 102: 336-342. 2007). Other enzymatic crude oils of the Aspergillus fungus, which are poorly characterized, have been used for the synthesis of phytosteric esters (Toke et al. Production and application of novel sterol esterases from Aspergillus strains by solid state fermentation. Journal of the American Oil Chemists' Society 84 : 907-915, 2007) although productivity is low and long incubation times are required. In addition, an enzyme of the fungus Trichoderma sp. AS59 capable of synthesizing ester esters although little is mentioned about it in the published work (Maeda et al. Characterization of novel cholesterol esterase from Trichoderma sp AS59 with high ability to synthesize steryl esters. Journal of Bioscience and Bioengineering 105: 341-349. 2008).

Within the enzymatic reactions that are used for the synthesis of esters of plant sterols, these can proceed through: i) Transesterification as described in US Patent 2004/0105931 Al (Basheer and Plat, 2004), where commercial lipases are used , in the presence of stabilizers and / or immobilized, and at high reaction temperatures (60 ° C). In this case the reactions take place in situ so that the foods enriched in this type of compounds would be limited to those that among their constituents naturally contained triglycerides as "donor" molecules of the acyl group; that is, fatty acid. In particular, dairy derivatives such as butter, yogurts and foods would be susceptible to enrichment. cheeses or oils of diverse origin (olive, sunflower, rapeseed, etc). This is a problem in the case of those allergic to milk and its derivatives or for strict vegetarians (vegans) who could not benefit from this type of products. ii) By contrast, other works and patents describe the obtaining of these compounds by direct esterification of phytosterols with fatty acids, under different experimental conditions using commercial enzymatic preparations of C. rugosa as biocatalyst. Specifically, in relation to the use of this enzyme for the synthesis of this class of compounds, the reactions take place at temperatures between 30 and 60 ° C, with reaction times between 24 and 72 hours obtaining yields ranging from 40 and 90%, although these results depend on the acyl group donor, the type of solvent used, and the presence or absence of water in the reaction medium (Norinobu et al., 2003; Vu et al. Lipase-catalyzed production of phytosteryl esters and their crystallization behavior in corn oil. Food Research International 37: 175-180. 2004; Villeneuve et al., supra; Teixeira et al. Production of steryl esters from vegetable oil deodorizer distillates by enzymatic esterification. Industrial & Engineering Chemistry Research 50: 2865-2875. 2011).

Considering the results set forth in the works and patents mentioned, the main differentiating characteristics of our invention would be the ability of the sphrase esterase of the fungus Ophiostoma piceae, both native and recombinant, to catalyze with high performance acylation reactions of phytosterols and saturated derivatives of these with free fatty acids or in the form of an ester. Preferably, our invention allows to obtain a yield of about 90% for direct acylation reactions with fatty acids, for example, lauric acid in the presence of different organic solvents, in the presence and / or absence of water, at a temperature between 24 and 35 ° C, preferably at 28 ° C, in short reaction times of 4-24 hours, and using quantities of the order of milligrams of lyophilized crude without any prior stabilization and / or immobilization process in which the enzyme subject to the The invention is the only one with sterol esterase activity, unlike what happens in commercial preparations of C. rugosa, which contain a mixture of different isoenzymes with different specificities (some more sterol esterase type and others more lipases). Precisely, due to the difficulty in separating these isoenzymes from C. rugosa by chromatographic techniques, it would be advisable to obtain the different recombinant variants for their industrial application. However, to Unlike O. piceae, C. rugosa makes a non-universal use of the genetic code and this makes it difficult to express enzymes of this yeast in heterologous expression systems (Brocea et al. Design, total synthesis, and fuctional overexpression of the Candida rugosa lipl gene coding for a major industrial lipase Protein Sci. 7: 1415-1422. 1998). DESCRIPTION OF THE INVENTION

A first object of the present invention is a method of acylation of free phytosterols or saturated derivatives thereof, also called phytostanols, with an acylation agent selected from the group consisting of free fatty acid and fatty acid ester, catalyzed by a sterol esterase enzyme, characterized in that process because it comprises an acylation reaction in the presence of a biocatalyst. Preferably the biocatalyst is a sterol esterase produced by fungi of the genus Ophiosotoma.

In a preferred embodiment of the invention, the acylation reaction comprised in the process of the present invention is a direct acylation reaction where the phytosterol or phytostanol reacts with an acylation agent that is a free fatty acid in the presence of a sterol esterase produced. by fungi of the genus Ophiosotoma.

In another preferred embodiment of the invention, the acylation reaction comprised in the process of the present invention is a transesterification reaction where the phytosterol or phytostanol reacts with an acylation agent which is a fatty acid ester (FAE) selected from the group consisting of methyl ester, ethyl ester, n-propyl ester, isopropyl ester, butyl ester, as well as fatty acid esters with more complex alcohols such as sorbitol, glycerol or other alcohols that can be used in the food sector, in the presence of a Sterol esterase produced by fungi of the genus Ophiosotoma. Preferably, the acylating agent may be a fatty acid ester selected from the group consisting of methyl ester and ethyl ester.

In the present invention, the term "sterol esterase enzyme" refers to an amino acid sequence of the sterol esterase enzyme encoded by the sterol esterase gene. Said enzyme is extracellular and can be native when it is expressed in the original organism (although when it is extracellular it has lost the signal peptide and speaks of mature protein) or recombinant if the complete protein sequence or the mature protein sequence is expressed in another organism.

In a preferred embodiment of the invention, said sterol esterase gene can be obtained from fungi of the species O. piceae.

In another embodiment of the present invention, said sterol esterase gene can be obtained from a clone of a library in which the sterol esterase gene can be found in a phage, cosmid, plasmid, or artificial chromosome (BAC or YAC) employed to generate this library.

In another embodiment of the present invention, said sterol esterase gene can be obtained from a gene vector in which the sterol esterase gene sequence had previously been cloned and, likewise, any DNA fragment or cDNA (since the gene lacks of introns) from a process of directed evolution and / or directed mutagenesis of the sterol esterase gene of O. piceae independently of the host used.

In a preferred embodiment of the present invention, the sterol esterase enzyme is characterized by having high sequence identity (at least 40-95%, or 60-95%) with the sterol esterase gene sequence of O. piceae. In a more particular embodiment of the invention, said homology is greater in the substrate binding area of the enzyme, as is preferably the case with the enzymes of Candida rugosa LIP2 and LIP3.

In a preferred embodiment of the present invention, the sterol esterase enzyme is obtained from Candida rugosa, ie the LIP2 and / or LIP 3 isoenzymes. Candida rugosa enzymes with sterol esterase activity can be obtained from commercial preparations, as mentioned. previously, marketed by Sigma and Amano.

In the present invention, the term "sterol esterase gene" refers to a nucleotide sequence, DNA, which encodes the sterol esterase enzyme of O. piceae and identified by SEQ. ID No: 1 The native form of the enzyme sterol esterase is the complete amino acid sequence of the protein encoded by the sterol esterase gene of O. piceae, including the signal peptide (SEQ. ID No: 2). The mature form of the sterol esterase enzyme is the amino acid sequence encoded by the sterol esterase gene, not including the signal peptide (SEQ ID No: 3), and is identified with SEQ ID No: 4. Hereinafter we will talk about native enzyme as the mature form, without signal peptide, to differentiate it from recombinant forms.

In a particular embodiment, the mature amino acid sequence encoded by the sterol esterase gene is expressed in P. pastoris yeast using as a signal peptide the prepropeptide of the Saccharomyces cerevisiae α factor. The cloning process performed in the expression vector pPIC9; as well as, post-translational processing of said signal peptide results in a mature recombinant protein with a modified N-terminal end relative to that of the mature native protein, as will be discussed later. In a more particular embodiment of the present invention, the recombinant sterol esterase enzyme exhibits modifications in its amino acid sequence as a consequence of i) its heterologous expression in organisms commonly used for the industrial production of enzymes, as an organism selected from the group consisting of Escherichia coli, Saccharomyces cerevisiae, P. pastoris, Hansenula polymorpha, Yarrowia lipolytica, Aspergillus nidulans, Aspergillus niger and Trichoderma reesei and / or ii) of a process of directed mutagenesis and rational design, or directed evolution of the sterol esterase gene, in both cases followed of its expression in suitable hosts such as E. coli, S. cerevisiae or P. pastoris.

In the present invention the terms "heterologous expression" and "heterologous gene" refer to the introduction of a foreign gene (heterologous) into an organism in order to modify its genetic material and expression products. A method for the heterologous expression of the sterol esterase gene is protected in the present invention. Said method includes i) PC amplification of the sequence encoding the sterol esterase enzyme, ii) the introduction of the sterol esterase gene into a cloning and / or expression vector; and iii) its heterologous expression in any of the organisms used for the industrial production of enzymes mentioned above. In a particular embodiment of the present invention, said organism is a methylotrophic yeast, preferably P. pastoris. In a still more particular embodiment of the present invention, the recombinant form of the sterol esterase enzyme obtained by the heterologous expression of the sterol esterase gene, preferably from O. piceae, presents as a modification between 6-8 new amino acids at the N-terminal end. , added on the amino acid sequence of the mature native protein, due to i) the cloning process in the integrative plasmid (pPIC9) used to transform P. pastoris that adds 4 amino acids (YVEF) to the end of the protein that are part of the sequence of the plasmid multiple cloning site, specifically the SnaB \ (TACGTA) and EcoR \ (GAATTC) restriction targets, the latter used for cloning in said plasmid at the 5 ^' end of the gene. The Not \ enzyme (GCGGCCGC) was used for cloning by the 3 'end, and ii) the post-translational misprocessing of the recombinant protein to eliminate the signal peptide (pre-propeptide of the Saccharomyces cerevisiae α factor), which It is responsible for the incorporation into the sequence of two or four more amino acids (EA or EAEA). Therefore, two forms of recombinant protein can be obtained, one with 6 and the other with 8 amino acids, identified with SEQ ID No: 5 and SEQ ID No: 6, respectively.

In other particular embodiments of the present invention the restriction targets could be any that form part of the multiple cloning site of the vector used, and this will in turn depend on the producing organism. In any case, the chosen targets will not be part of the sequence of the sterol esterase gene and it will be ensured that the modifications arising at the N-terminal end of the protein, as a consequence of the cloning process, are the minimum possible, always assuming a correct processing of the signal peptide that is used.

In the present invention, the term "primer" or first refers to short nucleotide sequences that may comprise: i) the sequence of the signal peptide of the mature sterol esterase of O. piceae or the N-terminal sequence of the mature protein identified with SEQ ID No: 7 and ii) those based on the 3 'end of the native enzyme gene identified with SEQ ID No: 8; as well as iii) those derived from them if modified DNAs resulting from obtaining variants of the protein after a process of directed evolution and / or directed mutagenesis are used as template. It also refers to any type of primers that include the above characteristics along with any other modifications introduced in the protein sequence, such as histidine N or C terminal tails, etc.

In the present invention, "cloning vector" means those DNA molecules originating from a virus, bacteria, or cells of a higher organism into which another DNA fragment can be integrated, without losing the capacity for self-replication. The "vectors" introduce foreign DNA into a host cell, where it can reproduce in large quantities. Examples: plasmids, cosmids and artificial yeast chromosomes. In the present invention, "expression vector" is understood as those small plasmids containing a multiple cloning site flanked by one or two promoter sequences. These promoters are required to express the DNA fragments inserted at the multiple cloning site. Preferably, the acylation process of free phytosterols or saturated derivatives thereof of the present invention can be catalyzed by the said sterol esterase enzyme in its native form encoded by the sterol esterase gene of a fungus of the genus Ophiostoma, more preferably of the O. piceae species. In accordance with another preferred embodiment, the acylation process of free phytosterols or saturated derivatives thereof of the present invention can be catalyzed by a recombinant enzyme originally produced by a fungus of the genus Ophiostoma, more preferably of the species O piceae. Even more preferably, the recombinant enzyme used in the process of the present invention can be expressed in P. pastoris, more preferably the recombinant enzyme has a modified N-terminal end whose sequences are SEQ ID No: 5 and SEQ ID No: 6 The activity of the enzymes used in the process of the present invention are compared with the enzyme of Candida rugosa marketed by Sigma (lipase type VII). It is important to highlight that some lipase-type enzymes, although they can carry out direct esterification reactions, perform the synthesis of phytosteric esters preferably by means of transesterification reactions of the substrates because many of them present steric impediments that hinder the development of esterification reactions Direct (Kirk and Christensen. Upases from Candida Antarctica: unique biocatalysts from a unique origin. Organic Process Research & Development 6: 446-451. 2002). This can be verified in US 2004/0105931 Al (Basheer and Plat, 2004), cited above in which different marketed lipases from different origins (bacterial, fungal and even vegetable) are studied. However, unlike what is stated in this and other patents, the enzyme of O. piceae (native or recombinant) has the advantage that it can be used starting from freeze-dried crudes, without any previous stabilization process with additives and / or immobilization process, since it is very stable and functional in a very wide temperature range, preferably between 4-35 ° C for at least 24 h, and in different organic solvents. In addition, the O. piceae enzyme, either native or recombinant, can catalyze direct acylation reactions between phytosterols and / or its saturated derivatives, and a free fatty acid. This is done, as described with the commercial enzyme of C. rugosa (Norinobu et al., 2003; Vu et al., Supra; Villeneuve et al., Supra; Teixeira et al., Supra), by esterification direct from alcohol, alcohol meaning any sterol and / or stanol present in the mixture of phytosterols and / or phytostanols. It should be noted that the yields obtained with C. rugosa enzymatic preparations in the production of these compounds by direct acylation reactions are higher than in transesterification reactions (Villeneuve et al., Supra).

Examples of the present patent application include direct esterification reactions that have been performed with both the native and recombinant O. piceae sterol esterase, and with the commercialized enzyme of C. rugosa that contains sterol esterase activity (it has LIP3 ). The results obtained indicate that although the percentage of esterification achieved with the enzymes of O. piceae and C. rugosa is similar, in the case of phytosterols, smaller doses of enzyme and shorter reaction times with the enzyme are required. from O. piceae. These direct esterification reactions could probably also take place using other sterol microbial esterases that exhibit high sequence identity, preferably 40-60%, with that of O. piceae, particularly in the substrate binding zone, among which are the LIP2 and LIP3 of C. rugosa. Table 1 compares the levels of synthesis achieved by various enzymes in direct esterification reactions under similar conditions and the enzymes object of the present invention, as well as with the commercial preparation of C. rugosa used under identical conditions as those of O. Piceae

Table 1. Acylation percentages of sterols with fatty acids achieved by various enzymes under similar conditions.

Raw

Raw raw

Raw Enzyme Fixed - Reference stabilized ¹ Fixed ¹

stabilized ¹

Antarctic Candida Lipase ³ 12 67 14 24

Candida cylindracea Patent Lipase

2 0 11 48

(C. rugosa) ³ US

Chromobacterium viscosum ³ lipase ³ 1 63 11 67 2004/01059

Lipoprotein B of Pseudomonas sp. ^at 23 71 26 77 31 Al

Novozym 868 ^to 7 32 - -

Lipase type VII of C. rugosa "33 - - -

Esteral esterase native of 0. piceae "58 - - - Patent

Recombinant esteral esterase of 0. proposal

39 - - - piceae "

"Acylation% expressed as the percentage (w / w) of conversion of cholesterol to its fatty acid ester in reactions with n-hexane using a 1: 1.4 molar ratio stearic cholesterohacid and developed at 40 ° C, 16h.

b% acylation expressed as the percentage (mM) of conversion of phytosterols to their esterified form in reactions with isooctane using a 1: 1 molar ratio phytosterols: lauric acid and developed at 28 ° C, 15h.

¹ Stabilized enzymes were prepared by adding sorbitan mono-stearate or other sugar esters to the crude enzymes, according to W099 / 15689. Immobilized enzymes, stabilized or not, were prepared according to the same patent.

The acylation percentages achieved with the native and / or recombinant sterol esterases of O. piceae in reasonable reaction times exceed those cited in other patents and even exceed or equal those obtained under the same conditions with a commercial enzyme, understood as C enzyme enzyme. rough (table 1).

As described in patent application PCT / CA95 / 00555 and which includes US 6,087,353 (Stewart et al., 2000), phytosterols or saturated derivatives thereof, also called phytostanols, used in the process of the present invention they can be obtained from virtually any plant source, for example, soy, corn, sunflower, rapeseed, legumes, nuts, fruits and vegetables; as well as agricultural waste. With respect to the latter, and taking into account the industrial concept of biorefinery, lignocellulosic materials that are currently contemplated for the production of second generation ethanol, such as cereal straw, pruning waste, etc., could be used. , as well as the waste generated after bioethanol production and beer manufacturing, which would contain these types of compounds in the fat-soluble fraction. Even process water from the paper industry could be used as these compounds are also found in hardwood timber removals such as eucalyptus (Gutierrez et al. The biotechnological control of pitch in paper pulp manufacturing. Trends Biotechnol 19: 340-348 . 2001). Preferably, in the process of the invention, the phytosterols used as the starting product originate from soybeans.

Additionally, the phytosterols used in the process of the present invention can be any known phytosterol or phytosterol derivative, as well as mixtures thereof. Preferably, the process of the present invention comprises acylation of phytosterols selected from the group consisting of β-sitosterol, stigmasterol, campesterol, brasicasterol and any derivative or mixture thereof. More preferably, the phytosterols used in the process of the present invention are a mixture comprising, with respect to the total weight of the mixture, 55% β-sitosterol, 10% stigmasterol, 29% campesterol and 6% brasicasterol .

It is also a preferred embodiment of the present invention that the phytosterols used as the starting product comprise, with respect to the total weight of the mixture, 70% by weight of β-sitosterol, at least 10% by weight of campesterol and at least 10% by weight of stigmasterol, since this composition has proven to be highly effective in lowering plasma cholesterol levels due to some type of synergistic effect between the different phytosterols present as described in patent application PCT / CA95 / 00555 and which is included in US Patent 6,087,353 (Stewart et al., 2000).

Preferably, the process of the present invention comprises acylation of phytostanols selected from the group consisting of sitoestanol, campestanol, brasicastanol and any derivative or mixture thereof. More preferably, phytostanol used comprises a minimum of 95% of sitoestanol, compound also known as stigmastanol, and is a commercially available product.

In any case, the process of the present invention allows the synthesis of phytosteric fatty acid esters or saturated derivatives thereof, where the acylation agent is selected from the group consisting of free fatty acid and acid ester fatty, and can be saturated, monounsaturated or polyunsaturated, of variable chain length, preferably the fatty acid chain comprises between 6 and 18 carbon atoms. More specifically, the acylation agent is a saturated medium chain length (MCFA) acid, such as caproic (C6), caprylic (C8), capric (CIO) and lauric (C12), being especially preferably that the free fatty acid used in the process described in the present patent application be dodecanoic acid (lauric acid).

Lauric acid is a medium-chain fatty acid found naturally in tropical oils such as coconut oil and palm kernel oil, as well as in cow's and goat's milk. It is perfectly metabolizable by the body and its consumption in adequate amounts does not represent a risk of toxicity according to studies in rats (Fitzhugh et al. Oral toxicities of lauric acid and certain lauric acid derivatives. Toxicology and Applied Pharmacology 2: 59-67. 1960) . Considering the total cholesterol / HDL cholesterol ratio as a marker of the risk of cardiovascular disease, it seems that, unlike other fatty acids studied, lauric acid would decrease this ratio more as a result of the increase in HDL cholesterol or "good cholesterol.""(Mensink et al. Effects of dietary fatty acids and carbohydrates on the ratio of total serum to HDL cholesterol and on serum lipids and apolipoproteins: a meta-analysis of 60 controlled triáis. The American Journal of Clinical Nutrition 77: 1146-1155. 2003). In addition, studies carried out, both with the free and esterified form (specifically monolaurin), demonstrate its antimicrobial activity when added to human milk or infant formulations at concentrations between 5 and 45 mM as added, free or esterified, and according to the pathogen (Isaacs et al. Antimicrobial activity of lipids added to human milk, infant formula, and bovine milk. The Journal of Nutritional Biochemistry 6: 362-366. 1995). There are even commercial formulations of monolaurin (Lauricidin ^® ) in which this antimicrobial potential is exploited, in addition to its use as a biocide in "films" prepared for food packaging (Hoffman et al. Antimicrobial effects of corn zein films impregnated with nisin, lauric acid, and EDTA. Journal of Food Protection 64: 885-889. 2001; Dawson et al. Effect of lauric acid and nisin-impregnated soy-based films on the growth of Listeria monocytogenes on Turkey Bologna. Poult Sci 81: 721-726. 2002). To all these properties is added its antioxidant effect and the additional advantage of its low cost and long half-life that facilitates its use at the industrial level.

On the other hand, in another particularly preferred embodiment of the invention, the acylating agent may be a C18 monounsaturated free fatty acid or one of its esters, which may be selected from the group consisting of oleic acid and methyl oleate. It should be noted that the oleic acid esters obtained by the acylation process of the present invention will generally be more soluble than those of lauric acid which would facilitate their incorporation into foods and therefore their bioavailability. This would favor a greater effect in terms of reducing cholesterol absorption at the intestinal level. In addition, being oleic acid an unsaturated fatty acid of the omega-9 series (ω-9) exerts a much more beneficial action on the cardivascular system than lauric acid.

In the acylation process of free phytosterols or saturated derivatives thereof, also called phytostanols, with an acylation agent comprising an acylation reaction in the presence of a sterol esterase produced by fungi of the genus Ophiostoma, in particular by the species O Native or recombinant piceae, as described in the present patent application, the molar ratio of acylation phytosteric acid or acylation phytosteric acid may preferably comprise values between 1: 1 and 1: 6. In an even more preferred embodiment, this acylating agent is a free fatty acid and the process of the invention comprises a direct acylation reaction.

In accordance with another preferred embodiment of the present invention, the sterol esterase enzyme produced by fungi of the genus Ophiostoma, preferably of the species O. piceae, more preferably one of its natural or recombinant variants, can be used in the acylation process of Phytosterols or saturated derivatives thereof described in the present patent application in the form of enzymatic crude produced as a result of fungus culture. In accordance with another preferred embodiment of the present invention, the sterol esterase enzyme produced by fungi of the genus Ophiostoma, preferably of the species O. piceae, more preferably one of its native or recombinant variants, can be used in the acylation process of Phytosterols or saturated derivatives thereof described in the present patent application after being purified by a chromatographic method, preferably by hydrophobic interaction chromatography, specifically Hi-Trap Octyl FF (GE Healthcare).

Preferably, the sterol esterase enzyme used in the process of acylation of phytosterols or saturated derivatives thereof of the present invention, used both as enzymatic and purified crude, can be used in a dose comprising between 1.5 and 12 U / mL of reaction, more preferably between 3 and 6 U / mL.

The acylation procedure described in the present patent application provides the use of biocatalysts, sterol esterases, as lyophilized crude oils without any prior stabilization and / or immobilization process perfectly functional and stable, and in which the enzyme of interest is the majority and unique in its activity unlike what happens with commercial preparation. The use of "high" doses of biocatalyst corresponds to milligrams of lyophilized crude and is sufficient to obtain high percentages of acylation, preferably 90% when the process comprises a direct acylation reaction using a free fatty acid.

According to another preferred embodiment, the acylation process of phytosterols or saturated derivatives thereof with an acylating agent, preferably a free fatty acid, catalyzed by a sterol esterase enzyme produced by fungi of the genus Ophiostoma, preferably of the native or recombinant O. piceae species, as described in the present patent application, can be run in an organic or biphasic single phase system comprising a mixture of organic solvent and water. Preferably, when the system is biphasic it comprises 10% water.

According to an even more preferred embodiment, the organic solvent comprised in said single-phase or two-phase system, in particular with 10% water, can be selected from the group consisting of isooctane, n-hexane and toluene. The use of isooctane or n-hexane is optimal catalytically and environmentally compared to the use of toluene. In addition, the use of biphasic systems, in the presence of water, results in high percentages of acylation with isooctane and n-hexane, and are preferable to monophasics in terms of future prospects. In addition, these types of systems are attractive from an industrial point of view as they make separation and recovery processes much more efficient and demand less energy. According to another preferred embodiment of the present invention, the process of acylation of phytosterols or saturated derivatives thereof with an acylation agent, preferably a free fatty acid, catalyzed by a sterol esterase enzyme produced by fungi of the genus Ophiostoma, preferably of the native or recombinant O. piceae species, as described in the present patent application can take place between 24 and 35 ° C, more preferably between 27 and 29 ° C, it being especially preferred that the acylation reaction takes place at 28 ° C.

Thus, the process of the present invention provides an enzymatic acylation process that develops at low temperatures, preferably at 28 ° C, with the consequent energy savings. On the other hand, the process of the present invention allows obtaining high acylation percentages in short reaction times, preferably between 4-24 hours, with 4 hours the yields obtained are already around 50% depending on the enzyme and the solvent employed. In accordance with another preferred embodiment of the present invention, the acylation process of free phytosterols with an acylation agent catalyzed by a sterol esterase enzyme comprising an acylation reaction in the presence of a sterol esterase produced by fungi of the genus Ophiostoma, and in particular by the species O. piceae, native or recombinant, as described in the present patent application may comprise: a) the plant phytosterols used are selected from the group consisting of β-sitosterol, stigmasterol, campesterol, brasicasterol and any derivative or mixture thereof;

b) the acylating agent is selected from the group consisting of free fatty acid and fatty acid ester, is saturated or unsaturated, and of chain length between 6 and 18 carbon atoms, preferably being free fatty acid;

c) the sterol esterase used as a catalyst is an enzymatic crude produced as a result of the fungus culture;

d) the reaction takes place in an organic or biphasic single phase system comprising a mixture of organic solvent and water; Y

e) the acylation reaction takes place between 24 and 35 ° C.

According to another preferred embodiment, the acylation process of free phytosterols with an acylation agent catalyzed by a sterol esterase enzyme comprising an acylation reaction in the presence of a sterol esterase produced by fungi of the genus Ophiostoma, and in particular by the species Native or recombinant piceae, as described in the present patent application may comprise: a) the phytosterols used are a mixture comprising 55% β-sitosterol, 10% stigmasterol, 29% campesterol, and 6% of brasicasterol, expressed in weight with respect to the total of the mixture;

b) the acylation agent is lauric acid;

c) the proportion of lauric phytosteric acid comprises values between 1: 1 and 1: 6;

d) the sterol esterase used as a catalyst is an enzymatic crude produced as a result of the fungus culture;

e) an enzyme dose comprising between 1.5 and 12 U / mL of reaction is used;

f) the reaction proceeds in an organic single-phase system, with isooctane, n-hexane or toluene as solvents, or biphasic comprising a mixture of any of these organic solvents with 10% water; Y

g) the acylation reaction proceeds at 28 ° C.

In accordance with another preferred embodiment, the method of acylation of phytostanols of the present invention may comprise: a) the plant phytostanols used are selected from the group consisting of sitoestanol, campestanol, brasicastanol and any derivative or mixture thereof;

e) the acylation reaction takes place between 24 and 35 ° C,

Preferably, the phytosanol acylation process of the present invention may comprise:

a) the phytostanol used comprises a minimum of 95% of sitoestanol;

b) the acylating agent is selected from the group consisting of lauric acid, oleic acid and methyl oleate;

c) the proportion of phytostanic acylation agent comprises values between 1: 1 and 1: 6;

e) an enzyme dose comprising between 1.5 and 12 U / mL of reaction is used;

g) the acylation reaction proceeds at 28 ° C.

In accordance with another preferred embodiment of the present invention, in the process of the invention the substrates are in phytosterols molar proportions: acylating agent or phytostanols: acylating agent, comprised between 1: 1 and 1: 6, being preferred that the acylating agent is a free fatty acid; and the sterol esterases are applied to the synthesis reaction once they have dissolved in the organic medium of choice. For this, according to the solvent, the reaction media is preheated in water at 100 ° C for 5-10 minutes, preferably 10 minutes. Once at room temperature, in the case of two-phase systems, water will be added in a preferred percentage 10% with respect to the volume of organic solvent and finally the enzymes will be added. The reactions will preferably take place between 24-35 ° C, more preferably at a temperature of 28 ° C, with magnetic stirring at 1,200 rpm, to eliminate mass transfer barriers for the substrates (Klibanov. Improving enzymes by using them in organic solvents Nature 409: 241-246. 2001), for a preferred time between 4-96 hours, more preferably between 4 and 24 hours. The concentration of the sterol esterases used preferably will be between 1.5-12 U / mL of total reaction volume, more preferably between 3-6 U / mL of total reaction volume, achieving a synthesis percentage of about 90% in the Best of cases. Once the products of interest are obtained, the biocatalyst may be separated from the reaction mixture, for example by centrifugation processes, for reuse.

According to a preferred embodiment of the present invention, the acylation process of free phytosterols or saturated derivatives thereof with an acylating agent, preferably a free fatty acid, catalyzed by a sterol esterase enzyme produced by fungi of the genus Ophiostoma, and in particular by the species O. piceae, native or recombinant, as described in the present patent application may comprise an additional step of solid powder isolation of the phytosterol ester or saturated derivatives thereof.

Preferably, the phytosterol acylation process of the present invention may comprise:

a) the plant phytosterols used are selected from the group consisting of β-sitosterol, stigmasterol, campesterol, brasicasterol and any derivative or mixture thereof;

d) the reaction takes place in an organic or biphasic single phase system comprising a mixture of organic solvent and water;

e) the acylation reaction takes place between 24 and 35 ° C; Y

f) solid powder isolation of the phytosterol ester. According to another preferred embodiment, the phytosterol acylation process of the present invention may comprise:

a) the phytosterols used are a mixture comprising 55% of β-sitosterol, 10% of stigmasterol, 29% of campesterol, and 6% of brasicasterol, expressed by weight with respect to the total of the mixture;

b) the acylating agent is lauric acid;

e) an enzyme dose comprising between 1.5 and 12 U / mL of reaction is used;

f) the reaction proceeds in an organic single-phase system, with isooctane, n-hexane or toluene as solvents, or biphasic comprising a mixture of any of these organic solvents with 10% water;

g) the acylation reaction proceeds at 28 ° C; Y

h) solid powder insulation of the phytosterol ester.

In another preferred embodiment, the method of acylation of phytostanols of the present invention may comprise:

a) the plant phytostanols used are selected from the group consisting of sitoestanol, campestanol, brasicastanol and any derivative or mixture thereof;

b) the acylating agent is selected from the group consisting of free fatty acid and fatty acid ester, is saturated or unsaturated, and the chain length between 6 and 18 carbon atoms, preferably being free fatty acid;

e) the acylation reaction takes place between 24 and 35 ° C, and

f) solid powder insulation of the phytostanol ester.

In accordance with another preferred embodiment, the method of acylation of phytostanols of the present invention may comprise: a) the phytostanol used comprises a minimum of 95% of sitoestanol;

b) the acylating agent is selected from the group consisting of lauric acid, oleic acid and methyl oleate,

c) the proportion of phytostanol acylation agent comprises values between 1: 1 and 1: 6;

e) an enzyme dose comprising between 1.5 and 12 U / mL of reaction is used;

g) the acylation reaction proceeds at 28 ° C, and

h) solid powder insulation of the phytostanol ester.

Synthesized esters of phytosterols or saturated derivatives thereof, also called phytostanol esters, can be separated from the excess fatty acid used by different procedures, such as: i) by molecular distillation (Hirota et al. Purification of steryl esters from soybean oil deodorizer distillate, Journal of the American Oil Chemists' Society 80: 341-346. 2003) and ii) by deacidification facilitating the formation of fatty acid salts (Weber et al. Fatty acid steryl, stanyl, and steroid esters by esterification and transesterification in vacuo using Candida rugosa lipase as catalyst. Journal of Agricultural and Food Chemistry 49: 67-71. 2001). Even without removing excess fatty acids in the reaction, the solvent could be removed by evaporating in a rotary evaporator or using a lyophilizer. In any case, the process of the present invention preferably comprises an additional step of obtaining a product in the form of solid powder with organoleptic characteristics suitable for direct incorporation into food, beverages, drugs and nutraceuticals, so that the amount of residual solvent or volatile organic impurities are within the acceptable limits proposed by the GPC guide (Guide to Good Clinical Practice) of the International Harmonization Committee (ICH) or pharmacopoeias (Grodowska and Parczewski. Organic solvents in the pharmaceutical industry. Acta Poloniae pharmaceutica-drug Research 67: 3-12. 2010). It must be considered that up to 50% of the energy expenditure required for a chemical process comes from the purification of the products and the recycling of the solvents used (Clark and Tavener. Alternative solvents: shades of green. Organic Process Research & Development 11: 149-155. 2007).

Additionally, the present invention also relates to phytosterols asters or phytostanols asters obtained by the acylation process defined in the present patent application.

According to a further aspect, the present invention also relates to products enriched with phytosterols esters or saturated derivatives thereof obtained by the acylation process defined in the present patent application. Preferably these products can be selected from the group consisting of a food, a food preparation, a dietary supplement and a medicament.

More preferably, when the enriched product is a food product, it can be: i) a food belonging to the group of dairy products, ii) a food or product that does not belong to the group of milk products, being suitable for allergy sufferers to milk as for example a product derived from soybeans. In this regard, it should be noted that the administration of ester esters together with soy protein helps lower cholesterol levels further (Lin et al. I am protein enhancing the cholesterol-lowering effect of plant sterol esters in cholesterol-fed hamsters The Journal of Nutrition 134: 143-148, 2004), or iii) a product that comes from cereals or cereal mixtures such as pastry doughs and pastries.

More preferably, when the enriched product is a dietary supplement, for example food or vitamin supplement, or a medicament, this is a solid form suitable for oral administration, such as, for example, tablet, capsule, grajea, granule, etc.

In accordance with a further aspect, the present invention also relates to the use of phytosterols esters or saturated derivatives thereof obtained by the acylation process defined in the present patent application; and of the products enriched with phytosterols esters or of the saturated derivatives thereof, object of the present invention to obtain products of food and / or pharmaceutical interest. In accordance with another additional aspect, the present invention also relates to phytosterols esters or saturated derivatives thereof obtained by the acylation process defined in the present patent application; and to products enriched with phytosterols esters or saturated derivatives thereof, which are the object of the present invention for use in medicine, preferably to reduce blood plasma cholesterol levels.

In accordance with another additional aspect, the present invention also relates to a method for reducing blood plasma cholesterol levels comprising administering a therapeutically effective amount of phytosterols esters or saturated derivatives thereof obtained by the acylation process. defined in the present patent application; or administering a therapeutically effective amount of a product enriched with phytosterols esters or with the saturated derivatives thereof object of the present invention.

According to a preferred embodiment, the acylation process of free phytosterols or saturated derivatives thereof with fatty acids catalyzed by a sterol esterase enzyme produced by fungi of the genus Ophiostoma as described in the present patent application, is characterized in that the enzyme preparations can be produced in a culture medium for O. piceae, which includes a lipid compound as an inducer, and for Pichia pastoris for the production of the same enzyme in its recombinant variant. The sterol esterase produced in both systems is a majority enzyme and unique in its activity in the obtained crude enzymes. Crude can be subjected to a lyophilization process and stored for months while retaining its activity.

The sterol esterase used in the acylation process of phytosterols or saturated derivatives thereof with fatty acids or fatty acid esters of the present invention can be any recombinant enzyme encoded in origin by the O. piceae gene.

Preferably, the recombinant enzyme is obtained by a procedure that includes 3 steps described below: i) The amplification by PC (polymerase chain reaction) of the sequence encoding the enzyme, where said sequence corresponds to the complete (including the signal peptide) or mature (without signal peptide) sequence of the protein. The templates for the PCR reaction may be any DNA fragment or cDNA containing the sequence in question as the genomic DNA of the fungus preferably, or clones of a library generated from genomic DNA or cDNA in which the acid fragment The nucleic responsible for encoding the enzyme is found in any phage, cosmid, plasmid, or artificial chromosome (BAC or YAC) used to generate such a library. According to a further preferred embodiment, the template used for PCR could also be any vector in which the sequence had been previously cloned and, likewise, any DNA fragment or cDNA from a process of directed evolution and / or directed mutagenesis regardless of the host employed. Preferably, they are primers of the amplification primers containing the sequence of the SEQ signal peptide. ID No: 3 or N-terminal of the mature protein SEQ ID No: 9 and primers based on the 3 'end of the native enzyme gene (SEQ ID No: 8); or those derived from them if modified DNA or cDNA resulting from obtaining variants of the protein as a result of a process of directed evolution and / or directed mutagenesis are used as a template. Also any type of primers that include the above characteristics along with any other modifications introduced in the protein sequence, such as histidine N or C terminal tails, etc.

ii) The introduction of the mature gene or sequence that encodes the protein, modified or not, in any type of cloning and / or expression vector; understood as vector plasmids of modified bacterial origin or eukaryotic viral vectors.

iii) Its heterologous expression in any of the organisms used for the industrial production of enzymes.

The present invention has its origin in previous studies on the sterol esterase of O. piceae. After a screening between different fungi, the ascomycete O. piceae was chosen for producing an enzyme capable of hydrolyzing both ester esters and triglycerides and mixtures thereof derived from hardwood or coniferous wood (Calero-Rueda et al. Production, isolation and characterization of a sterol esterase from Ophiostoma piceae, Biochim, Biophys, Acta 1599 [1-2]: 28-35, 2002; Calero-Rueda et al. Hydrolysis of sterol esters by an esterase from Ophiostoma piceae: Application for pitch control in pulping of Eucalyptus globulus wood. Intern. J. Biotechnol. 6: 367-375. 2004). These studies gave rise to the international patent WO 02/075045 A1R1 (Calero-Rueda et al., 2002) for its application in pitch reduction, deposits that cause problems during the manufacture of paper pulp.

Due to the industrial interest aroused with the enzyme, it was decided to carry out its heterologous expression in one of the most used eukaryotic systems, P. pastorls methylotrophic yeast, in order to try to improve its production levels. Different patents reflect the improvements made in this expression system during the last years (Bollok et al. Recent patents on the Pichia pastorls expression system: expanding the toolbox for recombinant protein production. Recent Patents on Biotechnology 3: 192-201. 2009). This, together with the recent sequencing of the yeast genome (De Schutter et al. Genome sequence of the recombinant protein production host Pichia pastoris. Nature Biotechnology 27: 561-566. 2009), will make it possible in the future to improve strains through strategies of systems biology.

The maximum production of the sterol esterase of O. piceae, in a synthetic medium with glucose and mineral salts supplemented with 0.5% olive oil (Calero-Rueda et al., Supra), was obtained between 15 and 21 days of culture and activity levels were approximately 1.8 U / mL using p-nitrophenylbutyrate (pNPB) as a substrate. In the chosen expression system it is intended to increase activity levels, decrease production time and avoid induction with olive oil, which would have a very positive effect on the production of this enzyme at the industrial level. The mature sequence (without signal peptide) of the sterol esterase of O. piceae has been used to express it in P. pastoris yeast. The production of the recombinant enzyme is strongly regulated by the AOX1 promoter (alcohol oxidase 1) of the yeast that responds to the presence of methanol as an inducer. The recombinant protein produced is directed and secreted into the extracellular environment thanks to the incorporation at its N-terminal end of the pre-propeptide of S. cerevisiae α factor, used for the extracellular expression of many other proteins (Crepin et al. Production and characterization of the Talaromyces stipitatus feruloyl esterase FAEC in Pichia pastoris: identification of the nucleophilic serine Protein Expression and Purification 29: 176-184. 2003; Dámaso et al. Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris. Appl. Environ. Microbiol 69: 6064-6072. 2003; Brunel et al. High-level expression of Candida parapsilosis lipase / acyltransferase in Pichia pastoris. Journal of Biotechnology 111: 41-50. 2004). The fact that production is strongly regulated by the induction of the AOX1 promoter guarantees the obtaining of high levels of recombinant protein since the AOX1 gene is only overexpressed in the presence of methanol, and its product is the first enzyme in the catabolic pathway for its use in methylotrophic yeasts. There is also an AOX2 gene whose product does not represent more than 15% of the cellular alcohol oxidase activity, so that between both enzymes they can constitute almost 30% of the total soluble cell protein in cells growing in alcohol (Cregg et al. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 9: 1316-1323. 1989; Daly and Hearn. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production J. Mol. Recognit. 18: 119-138. 2005). Considering this, the levels of activity that have been obtained in Erlenmeyer with a strain Mut ⁺ (Methanol utilization plus) of P. pastoris as biofactories for the production of sterol esterase would be about 3 times the levels reached with O. piceae when employs a complex medium, and the third part if a minimum medium is used. However, production may increase further if, in addition to methanol, as a carbon source and inducer, a second source of carbon such as sorbitol is added in the culture medium, which does not interfere with induction (Inan and Meagher. Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris, Journal of Bioscience and Bioengineering 92: 585-589. 2001), in this case producing activity levels up to about 7 times higher when using a complex medium. In addition, it is important to note that the aforementioned productions would be achieved in just 4 days of cultivation compared to the 2-3 weeks needed if the native enzyme is produced, and that the volumes of medium that are handled on this scale for the production of the Recombinant enzyme are considerably inferior. The production of recombinant sterol esterase can be further enhanced by bioreactor scaling in perfectly controlled bioprocesses, in minimal media of perfectly defined composition, and in production times not exceeding 4 days, which would facilitate its possible industrial use, to unlike what happens with the different isoenzymes of C. rugosa from which only the scaling under these conditions has been described in the recombinant LIP2 (Ferrer et al., supra). The recombinant enzyme produced is mostly in the crude and can be concentrated by tangential ultrafiltration using a 5 kDa membrane and used for different applications, such as the one described in the present invention, after being lyophilized.

The purification of the native and recombinant enzymes was carried out according to the previously described method (Calero-Rueda et al., Supra) with minor modifications: the ultrafiltered liquid was equilibrated with 0.5 M ammonium sulfate and applied to a hydrophobic interaction cartridge Hi Trap Octyl Sepharose (GE Healthcare) balanced with the same salt concentration in 25 mM Tris-HCI, pH 7.0. The retained proteins were eluted with a decreasing linear gradient of salt in 50 minutes at a flow of 1 mL / min. Finally, the sterol esterase was eluted with a 0.2% (v / v) reduced Triton X-100 solution. Through this single step, it was possible to obtain fully purified proteins once dialyzed by ultrafiltration to remove the detergent.

The kinetic characterization of the recombinant enzyme in terms of hydrolysis of different types of esters (of p-nitrophenol, glycerol and cholesterol) showed that the affinity of the recombinant enzyme for most of the substrates is somewhat greater than in the case of the native enzyme. What is striking, and it is noteworthy, is that with any of the substrates tested the catalytic efficiency calculated for the recombinant enzyme is of the order of 8-10 times higher than that of the native enzyme. According to this, the recombinant enzyme is capable of catalyzing the hydrolysis of more molecules of each of the substrates per unit of time. As with the native enzyme, the greatest efficiencies correspond to the long chain esters and also these increase with the number of unsaturations of the substrate. So far, the synthesis capacity of both enzymes has not been characterized from a kinetic point of view due to the complexity of the reactions. In this case, the systems in which the reactions are carried out (monophasic or biphasic systems) could condition these results.

Initially, the catalytic improvement of the recombinant enzyme was attributed to its higher degree of N-glycosylation compared to that of the native enzyme (= 30% vs. 8%), but this does not appear to be the cause of the improvement in the recombinant enzyme Well, there are hardly any changes significant in their activity against different substrates after deglycosylation with endoglycosidase H. However, where differences between both enzymes have been found, which could explain this improvement is in the sequence of their N-terminal ends. Specifically, the recombinant enzyme has the modified N-terminal end with between 6-8 new amino acids added to the native protein sequence (SEQ ID No: 5 and SEQ ID No: 6) as a consequence of i) the cloning process in the integrative plasmid used to transform P. pastoris that adds 4 amino acids (YVEF) to the end of the protein that are part of the multiple cloning site of that one, and ii) the post-translational misprocessing of the recombinant protein to eliminate the peptide signal (pre-propeptide of S. cerevisiae α factor), because of which two (more) or four (more EAEA) amino acids are incorporated into the sequence. Therefore, there are two forms of recombinant protein, one with 6 and another with 8 amino acids. The poor processing of this signal peptide has also been described in other proteins expressed recombinantly in P. pastoris without drastically affecting the catalytic properties of the enzyme (Crepin et al., Supra; Dámaso et al., Supra; Brunel et al ., supra) and it seems because one of the enzymes responsible for it, specifically the STE13 enzyme, which mediates the elimination of EA repeats, would not be able to process, as a minority enzyme, a product (recombinant protein) that is being overexpressed (Brake. Secretion of heterologous proteins directed by the yeast a-factor leader Yeast Genetic Engineering. Biotechnology series. pages 269-280, 1989).

The existence of the same enzyme with differences in its N-terminal end as well as the different catalytic properties of each of the forms has been described in the literature (Mandrich et al. Role of the N Terminus in Enzyme Activity, Stability and Specificity in Thermophilic Esterases Belonging to the HSL Family, Journal of Molecular Biology 345: 501- 512. 2005; Sayari et al. N-terminal peptide of Rhizopus oryzae lipase is important for its catalytic properties. FEBS Letters 579: 976-982. 2005; Niu et al. Secretion of pro and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins. Molecular Biotechnology 32: 73-81. 2006; Yu et al. Rhizopus chinensis lipase: Gene cloning, expression in Pichia pastoris and properties. Journal of Molecular Catalysis B: Enzymatic 57: 304-311. 2009; Peña-Montes et al. Differences in biocatalytic behavior between two variants of Stcl esterase from Aspergillus nidulans and its potential use in biocatalysis. Journal of Mol Ecular Catalysis B: Enzymatic 61: 225-234. 2009), and it has even been proposed how such an extreme would interact not only with the substrate of a lipidic nature but also with the rest of the molecule of enzyme (Frikha et al. Structural homologies, importance for catalysis and lipid binding of the N-terminal peptide of a fungal and a pancreatic lipase. Protein and Peptide Letters 17: 254-259. 2010). Therefore, we assume that in the case of recombinant sterol esterase this change could be involved in the improvement of its kinetic constants. This could be verified by sedimentation rate studies performed using analytical ultracentrifugation to compare the native and recombinant enzymes (Fig. 1). These studies have shown that while in aqueous solution (25 mM sodium phosphate, pH 7.0) the native enzyme is highly aggregated, the recombinant is in monomeric and dimeric form. If the proteins are in an aqueous solution in the presence of detergent, in this case Genapol X-100 at 1% (v / v), both are mostly in monomeric form. If the studies are carried out with deglycosylated recombinant enzyme, to avoid the possible "solubilizing" effect that carbohydrates bound to the protein might have (since the percentage of these is much higher than in the native one), it is observed that the recombinant enzyme is it is found in a monomeric and dimeric form in aqueous solution, the form compatible with the monomer appearing in the presence of the detergent. Therefore, it is conclusive that the modification of the N-terminal end of the recombinant protein is influencing the state of aggregation of the protein and this makes its kinetic properties improve. There are works that show the different activity of a protein, specifically lipases, according to their state of aggregation. When the protein is added, pseudo quaternary structures are formed as a result of the high hydrophobicity in amino acids exposed to the medium. Although there is a disparity in the effects caused, it seems to be demonstrated that the increase in the state of aggregation is accompanied by a loss of activity that may become total, as the case may be, as a consequence of the blocking of the substrate binding sites (Rúa et al Thermoalkalophilic lipase of Bacillus thermocatenulatus Large-scale production, purification and properties: aggregation behavior and its effect on activity Journal of Biotechnology 56: 89-102 1997, Palomo et al. General trend of lipase to self-assembling giving bimolecular aggregates greatly modifies the enzyme functionality Biomacromolecules 4: 1-6, 2003; Ferrer et al., supra). BRIEF DESCRIPTION OF THE FIGURES

Fig. 1. Study of the state of aggregation of native and recombinant enzymes by analytical ultracentrifugation. Profiles obtained with the native and recombinant enzyme in aqueous solution at pH 7.0 are shown in sedimentation velocity experiments.

Fig. 2A. Acylation of soy phytosterols with lauric acid by direct esterification reaction. Gas chromatogram illustrative of the control reaction with a phytosterols: 1: 1 fatty acid in isooctane ratio after 48 hours.

Fig. 2B. Acylation of soy phytosterols with lauric acid by direct esterification reaction. Gas chromatogram illustrative of the synthesis process in reactions with a 1: 1 ratio after 48 h with any of the enzymes used in the present invention.

Fig. 3. Effect of different phytosterols: lauric acid on acylation by direct esterification of soy phytosterols with the acid in two-phase isooctane / water systems after 48 h of reaction.

Fig. 4. Effect of the enzyme dose on acylation by direct esterification of soy phytosterols with lauric acid in two-phase isooctane / water systems using excess acid after 48 h of reaction. Activity values refer to the activity obtained using p-nitrophenyl butyrate as substrate at a final concentration of 1.5 mM.

Fig. 5. Effect of the organic solvent used in acylation by direct esterification of soy phytosterols with lauric acid in biphasic systems using excess acid after 48 h of reaction.

Fig. 6. Effect of different ratios lauric acid in the acylation by direct esterification of commercial sitoestanol with the acid in two-phase isooctane / water systems after 48 h of reaction. Fig. 7. Effect of different ratios oleic sitoestanohácido in the acylation by direct esterification of commercial sitoestanol with the acid in two-phase isooctane / water systems after 48 h of reaction.

Fig. 8. Effect of different ratios of methyl sitoestanoholeato in the acylation of commercial sitoestanol by means of transesterificación reactions in biphasic systems isooctano / water after 48 h of reaction. EXAMPLES

Example 1

Acylation of phytosterols with lauric acid in two-phase isooctane / water systems. Effect of the phytosterols: lauric acid.

According to bibliographic data, phytosterol molecules, due to strong steric impediments, are difficult to acylate substrates for a large majority of lipases or sterol esterases by direct esterification reactions (Kirk et al., Supra), and are usually resorted for this reason to the development of transesterification reactions.

Unpurified enzyme preparations, also called raw, of the native and recombinant sterol esterase were concentrated by tangential ultrafiltration through a 5 kDa membrane, frozen at -80 ° C and subjected to a lyophilization process for 48 hours. The enzymatic activity against pNPB before and after the lyophilization process did not change significantly, keeping an average activity close to 80% for the native enzyme, and 70-100% for the recombinant enzyme depending on the batch and medium of production. The lyophiles were maintained at 4 ° C for more than 6 months showing great stability over time without hardly losing activity. A commercial preparation (Sigma) with sterol esterase activity from C. rugosa was also used for comparisons.

For the acylation of phytosterols with lauric acid a commercial mixture of these (Sigma), from soybeans, containing 55% of β-sitosterol, 10% of stigmasterol, was used. 29% of campesterol and 6% of brasicasterol as could be verified by gas chromatography coupled to mass spectrometry.

Different molar ratios lauric phytosteric acid were tested, specifically ratios from 1: 1 to 1: 6. Specifically, the base reaction was the one in which the concentration of phytosterols was 10 mM, estimated as an average of the molecular weights of the species present, and in which the concentration of lauric acid was also 10 mM. The reactions took place in a biphasic organic solvent / water system, specifically isooctane / water, where the amount of water corresponded to 10% of the amount of solvent used. The reactions were carried out in glass tubes with screw cap and teflon gasket containing magnets for homogenization of the mixture during the reaction. Directly on the tubes the necessary amounts of each of the substrates and the organic solvent were added and vortexed. After heating the tubes at 100 ° C for 5-10 minutes, preferably 10 minutes, to promote dissolution of the components, and allowing them to cool to room temperature, the appropriate amount of milliQ water was added. Immediately afterwards the different enzymes were added at a concentration of 6 U / mL reaction, activity measured against p-nitrophenyl butyrate at a final concentration of 1.5 mM. An enzyme-free control was established and the synthesis reactions developed at 28 ° C and agitation of 1,200 rpm for a maximum of 96 hours. Samples of the reactions were obtained at different incubation times. These were diluted 10 times with isooctane, centrifuged at 7,500 rpm at room temperature for 10-15 minutes, after which 200 μί of the supernatant was taken and diluted in half with 0.5 mM cholesteryl oleate in isooctane (internal standard) .

The samples were analyzed by gas chromatography using GC 7890A (Agilent Technologies) equipment, equipped with split / splitless injector and backflush with restrictor, with a Supelco SPB-1 column (5m x 250μιη x 0.25μιη) and ionization detector call (FID). The volume of sample to be injected was 1 μί. Injector and detector temperatures were set at 350 ° C. The column temperature was maintained at 115 ° C for 1 minute, then increased to 170 ° C at a rate of 10 ° C / min, and finally a ramp of 20 ° C / min to 350 ° C was programmed, maintaining at this temperature 1 minute. The race time was about 17 minutes. As carrier gas helium was used at a 20 psi pressure Data analysis was performed with the GC-ChemStation Rev.B.04.02 (96) software from Agilent Technologies (2001-2009). The quantification of the substrates and reaction products was carried out based on their response factors with respect to the cholesteryl oleate used as an internal standard. The calibration lines showed correlation coefficients of 0.99.

Figure 2 shows, by way of example, the gas chromatography monitoring of a control reaction, without enzyme, and another in which enzyme is used after 48 hours of incubation at 28 ° C. In both reactions the phytosterols: lauric acid ratio was 1: 1 and, where appropriate, the amount of enzyme of 6 U / mL. The peak at 1.55 minutes corresponds to lauric acid, the peaks at 10.75; 11.00; 11.15; and 11.35 minutes correspond to the phytosterols brasicasterol, campesterol, stigmasterol and β-sitosterol respectively, the peaks with retention times of 14.40; 14.55; 14.65; and 14.80 minutes correspond to the phytosterols esters of lauric acid, namely, brasicasteryl laurate, campesteryl laurate, stigmasteryl laurate and β-sitosteryl laurate; finally, the peak at 15.6 min corresponds to cholesteryl oleate (internal standard).

As can be deduced from the analysis of the samples by gas chromatography, the native sterol esters (native OPE) and recombinant (recombinant OPE), as well as the commercialized C. rugosa (CRL), are capable of performing fatty acid acylation with any of the phytosterols in the mixture. The quantification with respect to the percentage of synthesis achieved, calculated according to the initial amount of phytosterols, shows that with the three enzymes high acylation percentages are achieved in short reaction times. This percentage is higher as the excess lauric acid in the reaction increases (Fig. 3). However, among the enzymes used there are differences showing better levels of acylation of the native and recombinant enzymes of O. piceae than the commercial ones at low excesses of the acid.

Using excess of one of the substrates is a recurring strategy in the synthesis or acylation reactions to optimize the yields of obtaining asters. Although reactions in which the substrates appear in a 1: 1 ratio may seem ideal from an economic point of view and even for the subsequent purification of the products, on certain occasions, such as the one in question, it is advantageous to use such excess (Villeneuve et al., supra) because it reduces the reaction time necessary to obtain high percentages of acylation. In principle, synthesized phytosterols asters can be separated from excess fatty acid by molecular distillation (Hirota et al., Supra), or by deacidification facilitating the formation of fatty acid salts (Weber et al., Supra). The reaction mixture can also be directly treated in a rotary evaporator or in a lyophilizer to remove the organic solvent and obtain a product with acceptable organoleptic properties in the presence of free fatty acid.

Example 2

Acylation of phytosterols with lauric acid in two-phase isooctane / water systems. Effect of enzyme dose.

In order to verify the effect of the dose of the different enzymes on the synthesis efficiency of phytosterols esters of lauric acid, acylation reactions were carried out in two-phase isooctane / water systems, using an excess of lauric acid.

The reactions were prepared as indicated in example 1. Dose of each of the enzymes between 1.5 and 12 U / mL, activity against pNPB, were added to the reaction tubes and incubated between 4-48 hours at 28 ° C with magnetic stirring at 1,200 rpm. At different time intervals samples were obtained that were processed and analyzed as mentioned in example 1.

The quantifications obtained after the analyzes performed by gas chromatography (Fig. 4) show that at low doses of enzyme (1.5 U / mL) the percentage of acylation achieved with the enzymatic preparation of C. rugosa (CL) is lower than that obtained with Ophiostoma enzymes (native OPE and recombinant OPE). It is important to note that when high doses of enzyme are used, the same results are obtained with the three enzyme preparations tested. The enzyme doses that have been used correspond to amounts of solid in the order of milligrams. It would also be possible to recover after the end of the reaction by filtration or centrifugation. However, for the realization of the following example it was decided to continue using doses of 6 U / mL of total reaction of each of the enzymes, since this value is within the range in which the best acylation percentages are achieved and allows some savings in biocatalyst.

Example 3

Acylation of phytosterols with lauric acid in two-phase organic solvent / water systems and organic single-phase systems. Effect of the organic solvent used.

The efficacy of the native and recombinant sterol esterases of O. plceae, as well as of the commercialized enzyme of C. rugosa in acylation reactions of phytosterols with lauric acid in biphasic solvent / water systems was tested using different organic solvents to verify their effect on The result of the reaction. In the literature it is reflected how the use of different solvents can affect the performance of the reaction as a result of its different polarity, which determines the "esterifying" activity of lipases and sterol esterases (Hirata et al. Lipase-catalyzed transesterification in organic solvent : effects of water and solvent, thermal stability and some applications. Journal of Biotechnology 14: 157-167. 1990; Grodowska et al., supra). Even by means of the so-called "solvent engineering" changes in the catalytic activity, stability and selectivity of the enzyme can be produced without the need to modify it by means of directed mutagenesis techniques, directed evolution or phage display (Klibanov., Supra). Isooctane, n-hexane and toluene were the chosen solvents. All of them have been used for the synthesis of other compounds of interest and continue today being used in the pharmaceutical industry under strict usage standards (Grodowska et al., Supra). In fact, toluene, in 2005, was ranked number 7 in the ranking of the 10 most used solvents in GlaxoSmithKIine Pharmaceuticals (GSK) (Constable et al. Perspective on solvent use in the pharmaceutical industry. Organic Process Research & Development 11: 133-137, 2007), while n-hexane is one of the few organic solvents accepted in the food industry (Li et al. Lipase-catalyzed synthesis of conjugated linoleyl b-sitosterol and its cholesterol-lowering properties in mice Journal of Agricultural and Food Chemistry 58: 1898-1902. 2010). All of them have adequate water / solvent separability when used in biphasic systems (Constable et al., Supra).

Acylation reactions were prepared as indicated in example 1 using an excess of lauric acid with the different solvents. The reactions are They developed in glass tubes with screw cap and Teflon gasket, the volume of water being 10% of the volume used of organic solvent. As indicated in Example 1, after adding the substrates and solvents, and vortexing, the mixtures were heated in a 100 ° C water bath for 5-10 minutes, preferably 10 minutes, to favor dissolution of those. , although in the case of toluene it would not have been necessary. Once it was cooled to room temperature, between 22 and 25 ° C, the necessary volume of milliQ water was added, and the enzymes were added at a concentration of 6 U / mL. The reactions developed at 28 ° C, 1,200 rpm for 4-48 hours. At different time intervals, samples of the different reactions that were processed and analyzed were obtained as set forth in Example 1 above.

Gas chromatography analyzes of the samples obtained from the different reactions show the ability of the enzymes tested to acylate the phytosterols in the presence of any of the organic solvents. However, with the three enzymes, the best acylation percentages are achieved when isooctane is used as a solvent, then n-hexane, and finally in toluene. Specifically, the acylation percentages achieved after 48 hours of reaction in isooctane were 85, 81 and 84% for the enzymes O. piceae native (OPE native), O. piceae recombinant (OPE recombinant), and C. rugosa commercial (CL ) respectively; in n-hexane of 85, 76 and 78% respectively; and finally, in toluene of 6, 4 and 14% respectively (Fig. 5).

In the same way, these reactions can be developed in single-phase systems with each of the three organic solvents studied. The results of esterification with the native enzyme of O. piceae and the commercial enzyme of C. rugosa were similar to those obtained in biphasic systems when using solvents with a higher partition coefficient (logP). However, the yields obtained with the recombinant sterol esterase of O. piceae were lower.

Example 4

Acylation of sitoestanol with lauric acid in two-phase isooctane / water systems. Effect of the relationship lauric sitoestanohácido. Tinols are the saturated derivatives of sterols and are present in varying proportions in natural phytosterols extracts. The activity of the native esterase of O. piceae (OPE) and of the enzyme of C. rugosa (CL) in the synthesis of sitoestanyl laurate has been evaluated using different molar ratios lauric sitoestanohacid, specifically ratios from 1: 1 to 1: 6. Specifically, the base reaction was assigned in which the concentration of sitostanol and lauric acid was 10 mM. These reactions were carried out in two-phase isooctane / water systems, with a water content of less than 10% of the amount of solvent used, following the same methodology described in example 1, using an enzyme dose of 3 U / mL of reaction. An enzyme-free control was established and the synthesis reactions were carried out at 28 ° C and agitation of 1,200 rpm for a maximum of 48 hours, same reaction conditions as for the synthesis of phytosterols asters described in Example 1. Obtained samples of reactions at different incubation times. These were diluted 10 times with isooctane, centrifuged at 7,500 rpm at room temperature for 10-15 minutes, after which 200 μί of the supernatant was taken and diluted in half with 0.5 mM cholesteryl oleate in isooctane (internal standard) .

The samples were analyzed by gas chromatography using the same equipment and conditions described in the previous examples, with the exception that the injection volume was 2 μί.

Chromatographic analyzes (Fig. 6) reveal that, under the three conditions tested, with the OPE enzyme, high levels of esterification of lauric acid are reached within 48 hours, while using CRL the maximum is esterified. 20% of the substrate in the same period. Specifically, for OPE, with a molar excess of 3 for lauric acid with respect to sitostanol, 80% esterification was reached after 24 h of reaction. The addition of a molar excess of 6 did not improve yields at 24 h or at 48 h. Example 5

Acylation of sitoestanol with oleic acid in two-phase isooctane / water systems. Effect of the ratio sitoestanol: oleic acid. In order to check if the length of the fatty acid chain could influence the effectiveness of the esterase in the esterification reaction, a long chain unsaturated fatty acid, specifically oleic acid (C18: 1), was used as the agent acylation Using the same conditions and methodology detailed in the previous example, the ability of the native OPE enzyme to synthesize sitoestanyl oleate was studied. Simultaneously, reactions were carried out under identical conditions using the commercial enzyme of C. rugosa CRL and the results obtained were compared with both enzymes, by analyzing gas chromatography samples taken at different reaction times in all the conditions tested.

The results of these reactions (Fig. 7) indicate that the sterol esterase of O. piceae is more efficient than the commercial CRL enzyme in the esterification of oleic acid in all the conditions evaluated. The degree of esterification of this substrate, values between 88-92% in 48 hours, is comparable to that achieved using lauric acid. On the contrary, CRL esterifies oleic acid more efficiently than lauric acid, although the maximum amount of sitoestanyl oleate formed does not exceed 67% in 48 hours.

As in the previous example, in reactions catalyzed by OPE, a molar excess of 3 fatty acid produced the maximum yield of 92% at 24 h of reaction and the addition of a molar excess of 6 decreased the esterification yields at 24 at 48 h.

Example 6

Acylation of sitoestanol with methyl oleate in two-phase isooctane / water systems. Effect of the ratio sitoestanol: methyl oleate.

As mentioned in example 1, most lipases and sterol esterases have difficulty catalyzing the direct esterification of phytosterols, which is why transesterification reactions are usually used to obtain the esters. All the examples presented so far in the present application illustrate conditions of direct esterification. In this case the synthesis of sitoestanil oleate by enzymatic transesterification is described, using the native OPE and CRL enzymes as catalysts, and methyl oleate as a donor of the acyl group. Transesterification reactions were prepared using methyl sytostandholeate molar ratios between 1: 1 and 1: 6 using the same conditions described in the previous examples, 3 U / mL enzyme, 28 ° C, 1200 rpm.

The results of these reactions (Fig. 8) indicate that OPE sterol esterase is more effective than CRL in this type of reaction, under any of the conditions evaluated. The degree of esterification of the sitoestanol by this procedure was slightly lower than in the direct esterification reactions, both for OPE and for CRL. Using OPE as a catalyst, a% acylation of between 65-87% and with CRL between 50-74% at 48h was achieved. Reactions with OPE required lower molar excesses of methyl oleate, finding similar results with 1: 3 and 1: 6 ratios, around 87% esterification, while with CRL the maximum esterification percentage of 74% was achieved with a 1: 6 ratio

As in other examples, in reactions catalyzed by OPE, a molar excess of donor of the acyl group of 3 produced 78% esterification at 24 h of reaction, while CRL only esterified 24% of sitoestanol under the same conditions and a 33% with an excess of 6 times with respect to the amount of sitoestanol.

Claims

1. Acylation procedure of free phytosterols or saturated derivatives thereof with an acylation agent selected from the group consisting of free fatty acid and fatty acid ester, catalyzed by a sterol esterase enzyme, said process characterized in that it comprises an acylation reaction in the presence of a sterol esterase produced by fungi of the genus Ophiostoma.

2. Acylation process of phytosterols or saturated derivatives thereof according to claim 1, wherein the acylation reaction is a direct acylation reaction with an acylation agent which is a free fatty acid.

3. Method of acylation of phytosterols or saturated derivatives thereof according to any one of claims 1 or 2, wherein the sterol esterase is produced by fungi of the species O. piceae.

4. Method of acylation of phytosterols or saturated derivatives thereof according to claim 3, wherein the sterol esterase is a recombinant enzyme encoded by the O. piceae gene.

5. Method of acylation of phytosterols or saturated derivatives thereof according to claim 4, wherein the recombinant sterol esterase is expressed in an organism selected from the group consisting of E. coli, S. cerevisiae, P. pastoris, H. polymorpha , Y. lipolytica, A. nidulans, A. niger and T. reesei.

6. Method of acylation of phytosterols or saturated derivatives thereof according to claim 5, wherein the recombinant sterol esterase has a modified N-terminal end whose sequences are SEQ. ID No: 5 and SEQ. ID No: 6.

7. Procedure for acylation of phytosterols or saturated derivatives thereof according to any one of claims 1 to 6, wherein the phytosterols or phytostanols used come from any plant source, lignocellulosic residue or industrial manufacturing residue of ethanol, beer or paper.

8. Method of acylation of phytosterols or saturated derivatives thereof according to claim 7, wherein the phytosterols or saturated derivatives of these employees come from soybeans.

9. Method of acylation of phytosterols according to any one of claims 1 to 6, wherein the plant phytosterols used are selected from the group consisting of β-sitosterol, stigmasterol, campesterol, brasicasterol and any derivative or mixture thereof.

10. A method of acylation of phytosterols according to claim 9, wherein the mixture of phytosterols used comprises 55% of β-sitosterol, 10% of stigmasterol, 29% of campesterol and 6% of brasicasterol expressed by weight with respect to the total of the mixture.

11. Method of acylation of phytosterols or saturated derivatives thereof according to any one of claims 1 to 6, wherein the acylation agent is selected from the group consisting of free fatty acid and fatty acid ester, is saturated or unsaturated, and chain length between 6 and 18 carbon atoms.

12. Method of acylation of phytosterols or saturated derivatives thereof according to claim 11, wherein the acylation agent is lauric acid.

13. Acylation process of phytosterols or saturated derivatives thereof according to claim 11, wherein the acylation agent is selected from the group consisting of oleic acid and methyl oleate.

14. Method of acylation of phytosterols or saturated derivatives thereof according to any one of claims 1 to 6 or 11 to 13, wherein the molar ratio of phytosteric acid of acylation or saturated derivative of phytosteric acid of acylation comprises values between 1: 1 and 1: 6

15. Method of acylation of phytosterols or saturated derivatives thereof according to any one of claims 1 to 6, wherein the sterol esterase used as a catalyst is an enzymatic crude produced as a result of fungus culture.

16. Acylation process of phytosterols or saturated derivatives thereof according to any one of claims 1 to 6, wherein the sterol esterase used as catalyst has been purified by a chromatographic method.

17. Acylation process of phytosterols or saturated derivatives thereof according to any one of claims 1 to 6 or 15 to 16, wherein an enzyme dose comprising between 1.5 and 12 U / mL of reaction is used.

18. Acylation process of phytosterols or saturated derivatives thereof according to any one of claims 1 to 6, wherein the reaction takes place in an organic or biphasic single phase system comprising a mixture of organic solvent and water.

19. Acylation process of phytosterols or saturated derivatives thereof according to claim 18, wherein the biphasic system comprises 10% water.

20. Acylation process of phytosterols or saturated derivatives thereof according to any one of claims 18 or 19, wherein the organic solvent is selected from the group consisting of isooctane, n-hexane and toluene.

21. Acylation process of phytosterols or saturated derivatives thereof according to any one of claims 1 to 6, characterized in that the acylation reaction takes place between 24 and 35 ° C.

22. Method of acylation of phytosterols according to any one of claims 1 to 6, wherein:

b) the acylation agent is selected from the group consisting of free fatty acid and fatty acid ester, is saturated or unsaturated, and of chain length between 6 and 18 carbon atoms;

e) the acylation reaction takes place between 24 and 35 ° C.

23. Method of acylation of phytosterols according to any one of claims 1 to 6, wherein: a) the phytosterols used are a mixture comprising 55% of β-sitosterol, 10% of stigmasterol, 29% of campesterol, and 6% of brasicasterol, expressed in weight respect to the total of the mixture;

b) the acylation agent is lauric acid;

e) an enzyme dose comprising between 1.5 and 12 U / mL of reaction is used;

g) the acylation reaction proceeds at 28 ° C.

24. Method of acylation of phytostanols according to any one of claims 1 to 6, wherein: a) the plant phytostanols used are selected from the group consisting of sitoestanol, campestanol, brasicastanol and any derivative or mixture thereof;

b) the acylation agent is selected from the group consisting of free fatty acid and fatty acid ester, is saturated or unsaturated, and of chain length between 6 and 18 carbon atoms; c) the sterol esterase used as a catalyst is an enzymatic crude produced as a result of the fungus culture;

e) the acylation reaction takes place between 24 and 35 ° C.

25. Method of acylation of phytostanols according to any one of claims 1 to 6, wherein:

a) the phytostanol used comprises a minimum of 95% of sitoestanol;

c) the proportion of phytostanol acylation agent comprises values between 1: 1 and 1: 6; d) the sterol esterase used as a catalyst is an enzymatic crude produced as a result of the fungus culture;

e) an enzyme dose comprising between 1.5 and 12 U / mL of reaction is used;

g) the acylation reaction proceeds at 28 ° C.

26. Acylation process of phytosterols or saturated derivatives thereof according to any one of claims 1 to 6, wherein additionally it comprises a solid powder isolation step of the phytosterol ester or phytostanol ester.

27. A phytosterol acylation process according to any one of claims 22 or 23, wherein the method further comprises an isolation step in the solid powder form of the phytosterol ester.

28. Method of acylation of phytostanols according to any one of claims 24 or 25, wherein the process further comprises an isolation step in the form of solid powder of the phytostanol ester.

29. Phytosterols esters obtained by a method as defined in any one of claims 1 to 23 or 26 to 27.

30. Phytostanol esters obtained by a method as defined in any one of claims 1 to 8, 11 to 21, 24 to 26 or 28.

31. Product enriched with phytosterols esters or saturated derivatives thereof as defined in claim 29 or 30, characterized in that said product is selected from the group consisting of a food, a food preparation, a dietary supplement and a medicament .

32. Product enriched with phytosterols esters or saturated derivatives thereof according to claim 31, characterized in that it is a food product selected from the group consisting of dairy product, product derived from soybeans, cereal and cereal mix.

33. Product enriched with phytosterols esters or saturated derivatives thereof according to claim 31, characterized in that it is a dietary supplement or medicament in a solid form suitable for oral administration.

34. Use of phytosterols esters or saturated derivatives thereof as defined in claims 29 and 30 respectively, or of a product enriched with phytosterols esters or saturated derivatives thereof as defined in the claims. 31 to 33 to obtain products of food and / or pharmaceutical interest.

35. Esters of phytosterols or saturated derivatives thereof as defined in claims 29 and 30 respectively, or of a product enriched with phytosterols esters or saturated derivatives thereof as defined in claims 31 to 33 to reduce blood plasma cholesterol levels.