JPH05501497A - Transesterification of phospholipids - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Transesterification of phospholipids Technical field The present invention uses enzymatic transesterification to convert acyl groups in phospholipids into free fatty acid esters. The present invention relates to a method of replacing fatty acids with fatty acids or free fatty acids.

foreground technology Phospholipids (glycerophospholipids), such as phosphatidylcholine, are composed of two fatty acids. Glycerol esterified with a syl group and one phosphate or ester conversion of phosphate groups or acyl groups in phospholipids to improve their nutritional value. Replacement is recommended.

JP-A No. 63-185.391 discloses that phospholipids and fatty acids (or esters) The process involves treatment with appropriate enzymes for 24-48 hours to obtain 10-20% exchange of fatty acids. Disclose. In the process, the use of carriers such as celitization has been proposed.

The object of the present invention is to be able to be easily separated and reused after the reaction, and to be suitable for fixed bed reactors. Achieving a high degree of incorporation of desired fatty acids into phospholipids in a short time using useful catalysts provides an improved enzymatic transesterification process for phospholipids that There are many things.

Disclosure of invention It has been found that this can be obtained using an immobilized enzyme. Therefore, the present invention provides enzymatic Transesterification is used to exchange acyl groups in phospholipids with free esters or free fatty acids. In this process, the enzyme is transferred to a particulate macroporous carrier. It is characterized by being fixed at the top.

quadruped fire The enzyme used in the present invention is immobilized on a granular macroporous carrier. The enzyme is It may be simply adsorbed onto a carrier, or glutaraldehyde or is bound to the carrier by crosslinking using another crosslinking agent.

Preferred carrier types are e.g. acrylic, polystyrene or phenol-formal. It is a dehyde type weakly basic anion exchange resin. An example of a commercial product is Revachit (L ewatit) (registered trademark) E1999/85 (Bayer product, West Germany) and Shuolide Duolite (registered trademark) BS-568 (ROHM and Haas).

Other preferred carrier types are e.g. phenol formaldehyde type, acrylic adsorbent (non-ionic) carrier of type or polyprene type. An example of a commercial product is Revati Lewatit 2001/85 (acrylic, Bayer product) and Accurel E P-100 (polypropylene, manufactured by Akzo) product).

Other preferred immobilization methods are those using inorganic support materials, and the enzyme is preferably Preferably, it is bound to the support by adsorption or covalent bonding. Such support materials and and immobilization techniques are described by K. Mosbach (supplementary study): Methoras in En zymology, 44'Immobilized Enzymes J ( Academic Press, 1976).

Preferred inorganic support materials include macroporous silica or silicate supports, e.g. Biocatalyst 5upports SC4BCIE/6/1987 This is macroporous silica sold by Press Chimical Co., Ltd., which is listed in In this case, more than 90% of the particles have a particle size in the range of 100 to 1000 pm, and this In this case, 80% or more of the particle diameters are 5 to 45 times the diameter of the enzymatic method.

The immobilized enzyme useful for transesterification of phospholipids is 20 per gram (dry weight) of catalyst. ．． 000 to 200.000 L U adsorbed (LU1 lipase alone has a US special 4.810.414) Plan M The enzymes to be used are lipases of animal, vegetable or microbial origin. economic theory In contrast, microorganism-produced lipases, such as bacterial or fungal lipases, are preferred.

Some examples of suitable enzymes include Neelizomucor (R), a lipase of the following origin disclosure: hizomucor (also called Mucor), especially R, Maihe miehri (M, m1ehei) free-length regiospecific lipase, Riboza Novo Nordisk A/S as Lipozyme (registered trademark) commercially available.

- Humicola, especially H, Lanuginosa (Thermomyces la) Originated from Thermosyces lanuginosus (also called Thermosyces lanuginosus) regiospecific lipase, US Patent 4,810,414, European Patent 305 ．． See 216.

- Candida mugosa (C, Cylindracea) C., Cylindraceae); Pase is available as Lipase OF (Polysaccharide Sangyo Co., Ltd.).

- derived from Pseudomonas cepacia Regiononspecific lipase (W 089101032).

Other enzymes are listed in columns 6 to 7 of JP-A-63-185-391, which are listed for reference. The enzyme described.

1 Sanko Gokumesaku The amount of water in the reaction system should be controlled. This is because a certain water activity As required for immobilized enzymes, too high a water content will result in too much phospholipid content. This is because it causes many undesirable hydrolysis. However, please note the following It should be. That is, the expected high value phosphorus containing the desired fatty acids. Lipids will tolerate a high degree of hydrolysis during the transesterification process.

Preferably, the amount of water in the reaction system is 5-15% of the dryness of the immobilized lipase or 0.5-1.5%, preferably 0.1-1.0% of the reaction system should be reached. Surprise Surprisingly, it has been found that such a low water content is sufficient.

In batch systems, the immobilized enzyme is preferably hydrated to 5-15% by weight before the reaction. will be done. In a continuous column system, water serves to hydrate the catalyst as described above. It can also be introduced by dissolving some water in the matrix.

The transesterification process processes both phospholipids and fatty acids or interfat esters. be mixed in the flow state, e.g. hexane, which also activates the enzyme catalyst. under conditions such as dissolution in butane, petroleum ether, or chlorinated hydrocarbons. It should be. Alternatively, phospholipids can be dissolved directly in fatty acids or fatty acid esters. It will be.

The process temperature should be chosen taking into account the thermal stability of the immobilized enzyme. of In this case, a temperature of 20 to 60°C would be appropriate.

Temperatures as high as 80°C can be used for highly thermostable enzymes.

The process can be carried out as a batch reaction, where the components are mixed at different temperatures during the reaction time. is stirred. After the reaction, the reaction product is simply decanted or filtered to remove the immobilized enzyme. separated from

The amount of immobilized enzyme in the reaction mixture is typically 1-10% w/w, The reaction time is generally 0.5 to 72 hours, preferably 0.5 to 24 hours.

Separately, the process involves immobilizing the substrate mixture (plus solvent if used) to immobilize the enzyme. It is essential to protect continuous fatty acids from oxidation by introducing them into bed columns. It is. This is a suitable non-oxidizing gas such as nitrogen, helium or argon. This can be achieved by carrying out the reaction while covered with

Elili The process of the invention can be applied to any desired type of phospholipid with fatty acyl ester groups. can do. Examples of such natural phospholipids are phosphatidyl serine, Phosphatidylglycerol, phosphatidyl inositol, phosphatidyl Ethanolamine and diphosphatylglycerol. Phosphate Synthetic phospholipids with various hydroxy compounds esterified to the groups are also treated. can be managed.

Be Asil The method of the invention can be used to introduce desired fatty acids into phospholipids.

Some examples of fatty acids of particular interest are: -Long chain (C, ~C2□) polyunsaturated fatty acids, such as lilunic acid, arachidonic acid, α- lylunic acid, eicosapene citric acid, docosahexaenoic acid, or γ-lylunic acid. Ru. These are introduced to improve the physiological or nutritional value of phospholipids. .

Single chain (C,~C,z) or long chain (C+□~C1,) saturated fatty acids.

These include modifying the emulsifying properties of phospholipids, modifying their physiological value, or improving their oxidative stability. Introduced for.

is Essur According to the invention, the acyl group to be introduced into the phospholipid is free or its ester. given as. Esters are especially short chain (C1-C3) alkyl esters, especially methyl ester or ethyl ester. Separately, esters are triglycerides , especially natural triglycerides.

Example The following immobilized lipase was prepared for use in the examples: transformed Aspergillus Obtained by culturing Aspergillus oryzae Rhizomucor n+1ehei Fixed on a macroporous silica carrier (Booth 6, product of Grace Chemicals) was made into The adsorption was 166.560 LU per gram of catalyst (dry weight). Ta.

Candida cylindracea (Candida cylindracea) Pase 6F (Polysaccharide Sangyo) was immobilized on Accurel EP-100. Adsorption is a catalyst It was 34,200 LU per 1 g (dry weight).

Example 1 As the phospholipid, Epiclon 200 manufactured by Lucas Bayer was used. this contains a minimum of 95% phosphatidylcholine and a maximum of 4% phosphatidylcholine. Fractionated soy lecithin containing phosphorus and moisture and up to 3% oil content . 5.5 g of Epicron 200, 12.0 g of decanoic acid and 90 ml of stone Mixed with oil ether (boiling point 80-100°C).

The molar ratio of decanoic acid to phosphatidylcholine in this mixture is approximately 10 to l. It is.

Immobilized R, Maihei (R.

n+1ehei) Lipase and immobilized H, lanuginos a) Each of the lipases was weighed into a vial. R Myhei Lipase Lipase Ze (R, Mon 1ehei) Up to 1o% W/W water content and H , 11.3% w/ for H. Ianuginosa lipase Humidified overnight at room temperature to a moisture content of w.

1.5 ml of the above mixture was added to the immobilized lipase. Then at 40℃ for 24 hours Stir gently. The substrate was then 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 phosphatidylcholine was analyzed as follows: Zircholine is decomposed from fatty acid and then used as a solvent C) ICh: C1hOH: Silica gel 60 plate using H2O (65:25:4, v/v/v) fat by thin layer chromatography method with Separated from acid. After elution, the plate was dried and the bands were removed using iodine fumes. Visualized. The band corresponding to phosphatidylcholine was peeled off.

The esterified fatty acids in the phosphbutidylcholine during exfoliation are then methylated. Fatty acyl methyl esters were measured and quantified by gas chromatography. Ta.

The results were as follows: The phosphatidylcholine in the reference sample did not contain decanoic acid.

The phosphatidylcholine treated with R. miehei is completely free of fatty acids. It contained decanoic acid in an amount of 67% (W/W) I.

H, lanuginosa phosphatidium treated with lipase Lucorin contained desanoic acid at 43% (w/w) I of the total fatty acids.

In both enzyme-catalyzed experiments, approximately half of the phosphatidylcholine in the reaction substrate was , was hydrolyzed during the reaction (estimated from thin layer chromatography method)■ -Also Proceed as in Example 1, but instead of 12.0 g of lauric acid, 15.9 g of myristic acid Acid was used. The molar ratio of myristic acid to phosphatidylcholine is approximately 10: It is 1.

R, Maihei (n+1ehei) Phosphatidylcholine treated with lipase are 73% (w/w) and 56% (w/w) of the fatty acid content, respectively. Contains myristic acid.

As described in Example 1 (approximately half of the phosphatidylcholine in enzyme catalyzed experiments) was presumed to have been hydrolyzed.

ester species using the substrate and the same amount of lipase (measured in LU) as described above. When using soluble R, miehei lipase, in essence As described in JP-A-63-185.391, i.e. reaction mixture with 10% water At the center, we found that after 24 hours, the amount of phosphatidylcholine initially present Significantly more than half of the amount is hydrolyzed and the remaining phosphatidylcholine was observed to contain 38% (W/W) myristic acid. this This demonstrates the improvement achieved by the present invention compared to JP-A-63-185, 391. be.

廿B 5.5g Epicron 200, 14.0g lauric acid and 90ml stone Mixed with oil ether (boiling point 80-100°C).

In this mixture, the molar ratio of lauric acid to phosphatidylcholine is approximately The ratio was 10 to 1.

Immobilized R, miehei, H, Ranugi equivalent to a dry weight of 125 ■ lanuginosa and C, Cylindracea a) Each of the lipases was weighed into a vial.

10% w/ for R, Mayhei lipase and C, Cylindracea lipase w and H, Il for lanuginosalivase, up to a water content of 3% w/w; The lipase was allowed to humidify overnight at room temperature.

The reaction and analysis were then carried out as in Example 1.

The results were as follows. : Phosphatidylcholine in the reference sample was 1.6% (W/ It had lauric acid in an amount of W).

R, Mayhei, C, Cylindracea, or H, lanuginolipase-treated Osphatidylcholine accounts for 51% (w/w) and 30% (w/w) of the total amount of fatty acids. , and lauric acid in amounts of 40% (W/W), respectively.

As in Example 1, about half of the phosphatidylcholine in enzyme catalyzed experiments is , it was assumed that it had been hydrolyzed.

Example 4 Two reactions were carried out as in the previous example using R, Mayhei lipase and the following substrates: 0.1833g of Ebicuron 2000.6433g of α-lylunic acid In this substrate, 3 ml of petroleum ether (boiling point 80-100°C) The molar ratio of tidylcholine to alpha-lylunic acid is approximately 10:1.

The reaction was carried out under an argon blanket to avoid over-oxidation of α-lylunic acid. .

The results were as follows: Phosphatidylcholine in the reference sample does not substantially contain lauric acid. I hadn't.

Phosphatidylcholine treated with R, Mayhei or H, Lanuginosa, respectively. Lauric acid at 53% (W/W) and 43% (W/W) of the total amount of fatty acids. It contained.

As in the previous example, the approx. It was estimated that half of it was hydrolyzed.

Example 6 50 ■ lauric acid per 1n+1 petroleum ether (boiling point 80-100°C) and 10■ phosphatidylethanolamine (manufactured by Ribinodo Products, Inc., A substrate with grade 1) was prepared.

Weigh the immobilized R, Mayhei lipase equivalent to a dry weight of 125 μm and place it in a vial. I put it in. The lipase was humidified overnight to a moisture content of 5% (W/W). 1.5ml of the above substrate was added to the lipase and then gently stirred at 40°C for 12 hours.

Analysis of the reaction products was performed as in the previous example.

The results were as follows: The phosphatidylethanolamine in the reference sample was substantially lauric acid ( 0.5% (W/W)).

Phosphatidylethanolamine treated with lipase accounts for 40% of the total amount of fatty acids. It contained lauric acid in an amount of (W/W).

Phosphatidylethanolamine in the reaction substrate from thin layer chromatography It was estimated that about half of the amount was hydrolyzed during the reaction.

Example 7 Phosphatidylglycerol (replacement of phosphatidylethanolamine) Example 5, using Pinodo Products, grade 1) and a reaction time of only 30 minutes. I did the same.

The results were as follows: The phosphatidylcholine in the reference example was substantially lauric acid (0.5% (% )).

Phosphatidylglycerol treated with lipase is 22% (W) of the total amount of fatty acids. /W) in an amount of lauric acid.

Thin layer chromatography of the enzyme-treated reaction product revealed that only a small A large amount of sophosphatidylglycerol was observed. However, unidentified compounds A new band was observed. This compound is associated with phosphatidylglycerol. produced by esterification of lauric acid to free hydroxyl groups in Will. The fatty acid composition of the unidentified compound was analyzed, and 39% (W/W) of lauric acid was found to contain.

Submission of translation of written amendment (Article 184-8 of the Patent Law) February 28, 1992

Claims (19)

[Claims] 1. Using enzymatic transesterification, acyl groups in phospholipids are converted into free fatty acid esters or is a method of exchanging free fatty acids, in which the enzyme is immobilized on a granular macroporous carrier. The method, characterized in that: 2. Macroporous carrier is weak base anion exchange resin, absorbent carrier, silica or 2. The method of claim 1, wherein the carrier is a silicate carrier. 3. The amount of water in the reaction system is 0.05 to 1.5% (by weight), preferably 0.1 to 1 ．． 3. The method according to claim 1 or 2, wherein the amount is 0%. 4. The water content before the immobilized enzyme comes into contact with the phospholipid is in the range of 5 to 15% (by weight). The method according to any one of claims 1 to 3, comprising: 5. Claims 1 to 3, wherein the enzyme is a lipase, preferably a lipase produced by a microorganism. The method described in any of Section 4. 6. Lipases are present in Humicola, especially in H. lanuginosa (H. lanuginosa). lauginosa) or Rhizomucor (especially R. a regiospecific lipase preferably derived from R. miehei; The method according to claim 5. 7. Lipase can be used in Candida (especially C. cylindracea). ．． cylindracea), or Pseudomonas ) (especially regiospecific, preferably derived from P. cepacia) 6. The method according to claim 5, which is a sexual lipase. 8. Phospholipids include phosphatidylcholine, phosphatidylglycerol, and phosphatidylcholine. Fattylinositol, phophatidylethanolamine, phosphatidylcetate Phosphorus, phosphatidic acid, diphosphatidylglycerol, esterified fat Synthetic phospholipids containing acids, or mixtures of two or more of these. The method according to any one of items 1 to 7. 9. Fatty acid is C18-C22 polyunsaturated fatty acid, preferably eicosapentaenoic acid , docosahexaenoic acid, γ-linolenic acid, α-linolenic acid, linolenic acid or ara The method according to any one of claims 1 to 8, which is chydonic acid. 10. Any of claims 1 to 8, wherein the fatty acid is a C6 to C18 saturated fatty acid. The method described in 11. The ester is a C1-C3 alkyl ester, preferably methyl or ethyl ester. 11. The method according to any one of claims 1 to 10, wherein the ester is a ester. 12. Claims 1 to 10, wherein the fatty acid ester is a triglyceride. The method described in any of the following. 13. The temperature is 20 to 80°C, according to any one of claims 1 to 12. Method. 14. Organic solvent, preferably hexane, heptane, petroleum ether or chlorinated carbon 14. The method according to any one of claims 1 to 13, which is carried out in the presence of hydrogen. 15. Immobilization of the mixture of substrate and optional solvent with residence times of 1 to 12 hours 15. A method according to any of claims 1 to 14, wherein the enzyme is passed through a fixed bed. 16. The mixture of immobilized enzyme, substrate and optional solvent was incubated for 0.5 to 72 hours. Preferably stirring for 0.5 to 24 hours and then separating the immobilized enzyme from the mixture thereafter. The method according to any one of claims 1 to 14. 17. The claimed amount of immobilized enzyme in the reaction mixture is 1-10% (w/w). The method according to scope item 16. 18. At most 95%, preferably at most 90% and even most of the phospholipids initially present. Any of claims 1 to 17, preferably at most 75% hydrolyzed. The method described in. 19. at least 5%, preferably at least 10%, of the acyl groups of the phospholipids; Most preferably at least 20% is substituted with The method described in any of the following.