INTERESTERIFICATION OF PHOSPHOLIPIDS

Abstract: Improved process for enzymatic interesterification of a phospholipid with a fatty acid or ester is obtained by using an enzyme immobilized by adsorption on a particulate macroporous carrier.

Description: INTERESTERIFICATION OF PHOSPHOLIPIDS TECHNICAL FIELD This invention relates to a process for exchanging acyl groups in a phospholipid by enzymatic ester exchange with a fatty acid ester or free fatty acid. BACKGROUND ART Phospholipids (glycerophospholipids), such as phosphatidyl choline, consist of glycerol esterified with 2 fatty acyl groups and one phosphate or esterified phosphate group. For some applications of the phospholipid it is desirable to exchange the acyl groups in the phospholipid, e.g. in order to modify the emulsification properties, to increase the stability against oxidation or to improve the physiological or nutritional value of the phospholipid. JP-A 63-185,391 discloses a process whereby phospholipid and fatty acid (or ester) are treated with a suitable enzyme to obtain 10-20% exchange of fatty acid in 24-48 hours. Use of a carrier such as celite in the process is suggested. It is the object of this invention to provide an improved enzymatic interesterification process for phospholipids such that a high degree of incorporation of a desired fatty acid into phospholipids can be obtained in a short time using an enzyme catalyst that can be easily separated after reaction and reused and is useful for fixed-bed reactors. STATEMENT OF THE INVENTION Surprisingly, we have found that such an improved process is obtained by using an enzyme immobilized by adsorption on a particulate macroporous carrier. Accordingly, the invention provides a process for exchanging acyl groups in a phospholipid by enzymatic ester exchange with a fatty acid ester or free fatty acid, characterized in that the enzyme is immobilized on a particulate macroporous carrier. Immobilization The enzyme used in the invention is immobilized on a particulate macroporous carrier. The enzyme may be simply adsorbed on the carrier, or it may be attached to the carrier by cross-linking with glutaraldehyde or other cross- linking agent known in the art. A preferred carrier type is weakly basic anion exchange resin, e.g. of acrylic, polystyrene or phenol-formaldehyde type. Examples of commercial products are Lewatit ® E 1999/85 (product of Bayer, West Germany) and Duolite ® ES-568 (Rohm & Haas). Another preferred carrier type is an adsorbent (non-ionic) carrier, e.g. of the phenol-formaldehyde type, acrylic type or polypropylene type. Examples of commercial products are Lewatit E2001/85 (acrylic, product of Bayer) and Accurel EP-100 (polypropylene, product of AKZO). Another preferred immobilization method uses an inorganic support material, and the enzyme is preferably attached to the support by adsorption or covalent coupling. Such support materials and immobilization techniques are described in K. Mosbach (ed.): Methods in Enzymology, 44, "Immobilized Enzymes" (Academic Press, 1976). A preferred inorganic support material is macroporous silica or silicate carriers e.g. macroporous silica carriers from Grace Chemicals described in Biocatalyst Supports SG BC 1E/June 1987 in which more than 90% of the particles have particle sizes between 100 and 1000 μm, wherein more than 80% of the pores in the particles exhibit a diameter between 5 and 45 times the diameter of the enzyme globules. The immobilized enzymes useful for interesterification of phospholipids typically are loaded with 20,000 - 200,000 LU per g (dry weight) of catalyst (LU, Lipase Unit is defined in US 4,810,414). Enzyme The enzyme to be used may be a lipase of animal, plant or microbial origin. For reasons of economy, a microbially produced lipase is preferred, e.g. a bacterial or fungal lipase. Some examples of suitable enzymes are lipases derived from the following organisms: - Positionally specific lipase from Rhizomucor (also designated Mucor), especially R. miehei (M. miehei), commercially available as Lipozyme™ (Novo Nordisk a/s). - Positionally specific lipase from Humicola, especially H. lanυginosa (also designated Thermomyces lanuginosus), see US 4,810,414, EP 305,216. - Positionally non-specific lipase from Candida rugosa (also termed C. cylindraceae, the lipase being available as Lipase OF (Meito Sangyo)). - Positionally non-specific lipase from Pseudomonas cepacia (WO 89/01032). Other enzymes are those indicated in JP-A 63-185,391, incorporated herein by reference, at col. 6-7. Process conditions The amount of water in the reaction system should be controlled, since a certain water activity is required to activate the immobilized enzyme, but too high water content may cause too much undesired hydrolysis of the phospholipid. It should be noted though, that the expected high value of a phospholipid containing a desired fatty acid may allow a high degree of hydrolysis during the interesterification process. Preferably, the amount of water in the reaction system should correspond to 5-15% of the dry weight of the immobilized lipase, or 0.05-1.5%, preferably 0.1-1.0% by weight of the total reaction system. Surprisingly, it was found that such a low water content is sufficient. In a batch system the immobilized enzyme may be hydrated before reaction, preferably to 5 - 15% water by weight. In a continuous column system, water may be introduced by hydrating the catalyst as above, and further having some water dissolved in the substrate. The interesterifying process should be carried out under conditions in which both the phospholipid and the fatty acid or fatty acyl ester are miscible in a fluid phase, e.g. solubilized in an organic solvent like hexane, heptane, petroleum ether or chlorinated hydrocarbons that also allows the enzyme catalyst to be active. Alternatively, the phospholipid may be solubilized directly in the fatty acid or fatty acyl ester. The process temperature should be chosen after considering thermostability of the immobilized enzyme. In many cases 20-60°C will be suitable. For very thermostable enzymes temperatures as high as 80°C may be used. The process may be carried out as a batch reaction, where the ingredients are stirred gently throughout the reaction period. After the reaction the reaction products is separated from the immobilized enzyme simply by decanting or filtration. The amount of immobilized enzyme in the reaction mixture will typically be 1 - 10% w/w, and the reaction time will generally be 0.5 - 72 hours, preferably 0.5 - 24 hours. Alternatively, the process may be carried out continuously by letting the substrate mixture (and solvent, if used) pass through a fixed bed column of immobilized enzyme. The residence time will typically be 1 - 12 hours. In processes where polyunsaturated fatty acids are to be incorporated into phospholipids, it may be essential to protect the fatty acids from oxidation. This can be alone by running the reaction under a blanket of an appropriate non-oxidizing gas like nitrogen, helium or argon. Phospholipid The process of the invention may be applied to any desired kind of phospholipid containing fatty acyl ester groups. Examples of such naturally occurring phospholipids are phosphatidic acid, phosphatidyl choline, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl ethanolamine and diphosphatidyl glycerol. Synthetic phospholipids with various hydroxy- compounds esterified to the phosphate group may also be processed. Acyl group to be incorporated The process of the invention may be used to incorporate any desired fatty acid into phospholipid. Some examples of fatty acids that may be of particular interest are: - Long-chain (CJ3-C22) polyunsaturated fatty acid, such as linoleic, arachidonic, alpha-linolenic, eicosapentaenoic, docosahexaenoic or gamma- linolenic acids. These may be incorporated to improve the physiological or nutritional value of the phospholipid. - Medium-chain (CgC 12 ) or long chain (C-^C- j g) saturated fatty acids. These may be incorporated to modify emulsification properties, to modify the physiological value or to improve oxidation stability of the phospholipid. Fatty acid or ester According to the invention, the acyl groups to be incorporated into the phospholipid may be provided as free fatty acids or esters thereof. The ester may particularly be a short-chain (C1-C3) alkyl ester, especially a methyl ester or ethyl ester. Alternatively, the ester may be a triglyceride, especially a naturally occurring one. EXAMPLES The following immobilized lipases were prepared for use in the examples: Rhizomucor miehei lipase produced by cultivating a transformed Aspergillus oryzae was immobilized on Duolite ® ES-568 N. The load was 99,200 LU per g (dry weight) of catalyst. Humicola lanuginosa lipase produced by cultivating a transformed Aspergillus oryzae was immobilized on a macroporous silica carrier (Grace 6, product of Grace Chemicals). The load was 166,500 LU per g (dry weight) of catalyst. Candida cylindracea lipase (Lipase-OF from Meito Sangyo) was immobilized on Accurel EP-100. The load was 34,200 LU per g (dry weight) of catalyst. EXAMPLE 1 As phospholipid was used the commercial product Epikuron 200 from Lucas Meyer GmbH. This is a fractionated soybean lecithin containing min. 95% phosphatidyl choline, max. 4% lyso-phosphatidyl choline and a moisture and oil content of max. 3%. 5.5 g of Epikuron 200 was mixed with 12.0 g of decanoic acid and 90 ml of petroleum ether (b.p. 80-100°C). In this mixture the molar ratio of decanoic acid to phosphatidyl choline is approx. 10:1. Each of the immobilized R. miehei lipase and immobilized H. lanuginosa lipase corresponding to a dry weight of 125 mg was weighed into a vial. The lipases were humidified overnight at room temperature to a water content of 10% w/w for the R. miehei lipase and 11.3% w/w for the H. lanuginosa lipase. 1.5 ml of the above mixture was added to the immobilized lipase. Gentle stirring was then carried out at 40°C for 24 hours. Then the substrate was separated from the enzyme catalyst. As a reference 1.5 ml of substrate was stirred at 40°C for 24 hours without lipase. The composition of fatty acids in the phosphatidyl choline was assayed as follows: Phosphatidyl choline was separated from fatty acids and lyso- phosphatidyl choline by thin layer chromatography on Silica gel 60 plates (Merck art. 5721) using CHCI 3 : CH3OH : H 2 0 (65 : 25 : 4, v/v/v) as solvent. After elution the plates were dried, and bands visualized by iodine vapors. The band corresponding to phosphatidyl choline was scraped off. The fatty acids esterified in the phosphatidyl choline in the scrape-off were methylated and the fatty acyl methyl esters were determined and quantitated by gas chromatography. The results were: Phosphatidyl choline in the reference sample contained no decanoic acid. Phosphatidyl choline treated with R. miehei lipase contained decanoic acid in an amount of 67% (w/w) of the total amount of fatty acids. Phosphatidyl choline treated with H. lanuginosa lipase contained decanoic acid in an amount of 43% (w/w) of the total amount of fatty acids. In both enzyme catalyzed experiments, approx. half of the phosphatidyl choline in the reaction substrate had become hydrolyzed (estimated from the thin-layer chromatograms) during the reactions. EXAMPLE 2 As Example 1 but using 15.9 g myristic acid instead of 12.0 g lauric acid. The molar ratio of myristic acid to phosphatidyl choline is approx. 10:1. The results were: Phosphatidyl choline in the reference sample contained virtually no myristic acid (less than 0.5% (w/w)). Phosphatidyl choline treated with R. miehei lipase or H. lanuginosa lipase contained myristic acid in an amount of 73% (w/w) and 56% (w/w), respectively, of the total amount of fatty acids. As in Example 1 approx. half of the phosphatidyl choline in the enzyme catalyzed experiments were estimated to have become hydrolyzed. When soluble R. miehei lipase was used for interesterification using the above substrate and the same amount of lipase (measured in LU) as above, essentially as described in JP-A 63-185,391 i.e. in a reaction mixture with 10% water, we observed that after 24 hours significantly more than half of the originally present phosphatidyl choline had become hydrolyzed and the residual phosphatidyl choline contained only 38% (w/w) myristic acid. This illustrates the improvement of this invention compared to JP-A 63-185,391. EXAMPLE 3 5.5 g of Epikuron 200 was mixed with 14.0 g lauric acid and 90 ml of petroleum ether (b.p. 80-100°C). In this mixture the molar ratio of lauric acid to phosphatidyl choline is approx. 10:1. Each of the immobilized R. miehei, H. lanuginosa, and C. cylindracea lipases corresponding to a dry weight of 125 mg was weighed into a vial. The lipases were humidified overnight at room temperature to a water content of 10% w/w for the R. miehei and C. cylindracea lipases and 11.3% w/w for the H. lanuginosa lipase. Reaction and analysis was then carried out as in Example 1. The results were: Phosphatidyl choline in the reference sample contained lauric acid in an amount of 1.6% (w/w) of the total amount of fatty acids. Phosphatidyl choline treated with R. miehei, C. cylindracea, or H. lanuginosa lipases contained lauric acid in an amount of 51% (w/w), 30% (w/w), and 40% (w/w), respectively, of the total amounts of fatty acids. As in Example 1 approx. half of the phosphatidyl choline in the enzyme catalyzed experiments were estimated to have become hydrolyzed. EXAMPLE 4 As previous examples using the R.miehei lipase and the following substrate: 0.1833 g Epikuron 200 0.6433 g alpha-linolenic acid 3 ml petroleum ether (b.p. 80-100°C) In this substrate the molar ratio of alpha-linolenic acid to phosphatidyl choline is approx. 10:1. In order to avoid excessive oxidation of the alpha-linolenic acid the reaction was carried out under a blanket of argon. The results were: Phosphatidyl choline in the reference sample and the R.miehei lipase treated sample contained C13.3 fatty acids in amounts of 7% (w/w) and 42% (w/w), respectively, of the total amount of fatty acids. As in previous examples approx. half of the phosphatidyl choline in the enzyme catalyzed experiment was estimated to have become hydrolyzed. EXAMPLE 5 As previous examples using the R. miehei lipase and H. lanuginosa lipase and using the following substrate: 211 mg egg yolk phophatidyl choline (grade 1 , from Lipid Products) 0.57 g lauric acid 3.63 ml petroleum ether (b.p. 80-100°C) The results were: Phosphatidyl choline in the reference sample contained virtually no lauric acid. Phosphatidyl choline treated with miehei or H. lanuginosa lipases contained lauric acid in an amount of 53% (w/w) and 43% (w/w), respectively, of the total amount of fatty acids. As in previous examples approx. half of the phosphatidyl choline in the enzyme catalyzed experiments were estimated to have become hydrolyzed. EXAMPLE 6 A substrate containing 50 mg lauric acid and 10 mg phosphatidyl ethanolamine (grade 1, from Lipid Products) per ml petroleum ether (b.p. 80- 100°C) was made. Immobilized R. miehei lipase corresponding to a dry weight of 125 mg was weighed into a vial. The lipase was humidified overnight to a water content of 5% (w/w). 1.5 ml of the above substrate was added to the lipase and stirred gently at 40°C for 12 hours. Analysis of reaction products were performed as in previous examples. The results were: Phosphatidyl ethanolamine in the reference sample contained virtually no lauric acid (less than 0.5% (w/w)). Phosphatidyl ethanolamine treated with lipase contained lauric acid in an amount of 40% (w/w) of the total amount of fatty acids. Estimated from the thin layer chromatograms approx. half of the phosphatidyl ethanolamine in the reaction substrate had become hydrolyzed during the reaction. EXAMPLE 7 As Example 5 but using phosphatidyl glycerol (grade 1, from Lipid Products) instead of phosphatidyl ethanolamine and using a reaction time of only 30 minutes. The results were: Phosphatidyl glycerol in the reference sample contained virtually no lauric acid (less than 0.5% (w/w)). Phosphatidyl glycerol treated with lipase contained lauric acid in an amount of 22% (w/w) of the total amount of fatty acids. On the thin layer chromatogram with enzyme treated reaction products only a low amount of lysophosphatidyl glycerol was apparent. However, a new band of an unidentified compound had appeared. This compound may have been made by esterification of lauric acid to one of the free hydroxy groups in phosphatidyl glycerol. The fatty acid composition of the unidentified compound was analyzed and found to contain 39% (w/w) lauric acid.

Claims: CLAIMS 1. A process for exchanging acyl groups in a phospholipid by enzymatic ester exchange with a fatty acid ester or free fatty acid, characterized in that the enzyme is immobilized on a particulate macroporous carrier. 2. A process according to Claim 1, wherein the macroporous carrier is a weakly basic anion exchange resin, an adsorbent carrier, silica or a silicate carrier. 3. A process according to one of the preceding claims, wherein the amount of water in the reaction system is 0.05-1.5% (by weight), preferably 0.1- 1.0%. 4. A process according to any of the preceding claims, wherein the immobilized enzyme has a water content prior to contact with the phospholipid in the range 5-15% (by weight). 5. A process according to any of Claims 1 - 4, wherein the enzyme is a lipase, preferably a microbially produced lipase. 6. A process according to Claim 5, wherein the lipase is a positionally specific lipase preferably derived from Humicola (especially H. lanuginosa) or Rhizomucor (especially ft miehei). 7. A process according to Claim 5, wherein the lipase is a positionally nonspecific lipase preferably derived from Candida (especially C. cylindracea), or Pseudomonas (especially P. cepacia). 8. A process according to any of Claims 1 - 7, wherein the phospho¬ lipid is phosphatidyl choline, phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidic acid, diphosphatidyl glycerol, synthetic phospholipids containing esterified fatty acids, or a mixture of 5 two or more of these. 9. A process according to any of Claims 1 - 8, wherein the fatty acid is a Ci8-C 2 2 polyunsaturated fatty acid, preferably eicosapentaenoic, docosa- hexaenoic, gamma-linolenic, alpha-linolenic, linoleic, or arachidonic acid. 10. A process according to any of Claims 1 - 8, wherein the fatty acid is 10 a C 6 -C 18 saturated fatty acid. 11. A process according to any of Claims 1 - 10, wherein the ester is a C- j -C-3 alkyl ester, preferably a methyl or ethyl ester. 12. A process according to any of Claims 1 - 10, wherein the fatty acid ester is a triglyceride. 15 13. A process according to any of Claims 1 - 12, wherein the temperature is 20 - 80°C. 14. A process according to any of Claims 1 - 13, carried out in the presence of an organic solvent, preferably hexane, heptane, petroleum ether or a chlorinated hydrocarbon. 20 15. A continuous process according to any of Claims 1 - 14, wherein a mixture of substrate and, optionally, solvent is passed through a fixed bed of immobilized enzyme with a residence time of 1 - 12 hours. 16. A batch process according to any of Claims 1 - 14, wherein a mixture of immobilized enzyme, substrate and, optionally, solvent is stirred for 0.5 - 72 hours, preferably 0.5 - 24 hours, and the immobilized enzyme is later separated from the mixture. 5 17. A process according to Claim 16, wherein the amount of the immobilized enzyme in the reaction mixture is 1-10% w/w. 18. A process according to any preceding claim, wherein at most 95%, preferably at most 90% and most preferably at most 75% of the initially present phospholipids become hydrolyzed. 10 19. A process according to any preceding claim, wherein at least 5%, preferably at least 10% and most preferably at least 20% of the acyl groups of the phospholipid are replaced.

Inventors: PEDERSEN KIM BRINT, PEDERSEN, KIM, BRINT
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1990
Main classification: C12P9
IPC codes: C12N9, C12N11, C12P7, C12P9, C12P13