JPH05236974A - Production of highly unsaturated fatty acid-containing phospholipid - Google Patents

Abstract

PURPOSE:To selectively introduce a fatty acid into the Sn2-position by adding glycerol in relation to the synthesis of a phospholipid. CONSTITUTION:The objective method for producing a phospholipid having high physiological activity with hardly any denaturation and decomposition of a highly unsaturated fatty acid is to add glycerol to a reaction system and thereby advance the synthesis with an enzyme in obtaining the phospholipid having the highly unsaturated fatty acid at the Sn2-position from a lyso type phospholipid having an acyl group at the Sn1-position and a highly unsaturated fatty acid or its ester by enzymic reaction of an enzyme phospholipase A2.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a phospholipid containing a highly unsaturated fatty acid at the Sn2 position by an enzymatic reaction.

[0002]

2. Description of the Related Art Various physiological activities of phospholipids have recently become clear. For example, eicosapentaenoic acid-containing phospholipid is known to have higher physiological activity than triglyceride or ethyl ester at the same amount of fatty acid. Also, in fish feed, phospholipids are essential nutrients for certain larvae. Further, it has been revealed that the position of fatty acid in the phospholipid has a great influence on its activity expression. It is known that phospholipids having an unsaturated fatty acid at the Sn2 position have a high addition effect in fish feeds. In humans, most phospholipids have unsaturated fatty acids at the Sn2 position, and highly unsaturated fatty acids cut out from the Sn2 position of phospholipids by phospholipase A ₂ serve as substrates for prostaglandins, for example. It is widely known that it contributes to the expression of various physiological activities.

At the 62nd Japan Biochemical Congress, Matsumoto et al. Reported that phosphadylcholine having docosahexaenoic acid at Sn2 position showed a strong inhibitory effect on the conversion of arachidonic acid to leukotriene. Among them, it is also reported that phosphatidylcholine having oleic acid and docosahexaenoic acid at Sn1 position and Sn2 position, respectively, has significantly higher inhibitory activity than phosphatidylcholine having docosahexaenoic acid at both positions.

As described above, it has been revealed that not only the composition of the fatty acid but also the position occupied by the fatty acid is important for the expression of the physiological activity of the phospholipid. Phospholipids are widespread in nature, but today medical and
Most of the extraction sources of phospholipids used in the fields of food and the like are soybean and egg yolk. The fatty acid composition is characterized in that soybean phospholipids contain a large amount of linoleic acid, and egg yolk phospholipids contain a large amount of palmitic acid and oleic acid. In each case, highly unsaturated fatty acids having three or more double bonds are Very little is included.

On the other hand, phospholipids in animal tissues generally contain a large amount of highly unsaturated fatty acids, but their phospholipid content is low and, unlike accumulated fat, they are strongly bound to proteins, It is not easy to extract this. An attempt to obtain a phospholipid having a desired fatty acid composition by an enzymatic reaction has been reported so far. Yoshimoto et al. Introduced eicosapentaenoic acid into phosphatidylcholine by using a polyethylene glycol-modified lipase (BIOTECHNOLOGY LETTERS VOL.8, pp771-776, 1986).
In addition, Toya et al. Are transesterifying soybean phospholipid and fish oil with lipase (abstract of the 44th Annual Meeting of the Japanese Society of Nutrition and Food Science in 1990). In addition, Yagi et al. Reported on the acyl group exchange reaction by lipase between phospholipids and fatty acids (JOURNAL OF FERMENTATION AND BIOE
NGINEERING VOL.69, pp23-25, 1990).

On the other hand, the patent also proposes a method in which a phospholipid and an arbitrary fatty acid or fatty acid ester are used to change the composition of the fatty acid by transesterification by an enzyme (JP-A-63-105686, JP-A-SHO-106686). 63-185391, JP-A
2-35093).

[0007]

However, in any of the above-mentioned reported examples or prior patents, the introduced fatty acid randomly enters Sn1 position or Sn2 position of phospholipid,
Alternatively, the Sn1 position is preferentially entered, and the fatty acid is not selectively introduced into the Sn2 position. Further, there are problems such as low transesterification rate, or hydrolysis of phospholipids during the reaction to form lyso-type phospholipids, which lowers the recovery rate of phospholipids. It is clear that there are difficulties.

A method of using phospholipase A ₂ for solving the above problems and introducing a polyunsaturated fatty acid at the Sn2 position was examined. Phospholipase A ₂ is an enzyme that acts only on the Sn2 position of phospholipids, and a method for producing a lyso-type phospholipid excellent in emulsifying property by hydrolysis from phospholipids using this has been issued many patents, It has been put to practical use (Japanese Patent Laid-Open Nos. 62-262998 and 63-279753).
No. 3, JP-A-63-134042, JP-A-63-209742, JP-A-2
-113891).

It is widely known that this enzyme phospholipase A ₂ performs not only hydrolysis but also the reverse reaction, and synthesis is described in, for example, Handbook of Oil and Fat Chemistry edited by Japan Oil Chemistry Association (February 1990). "For example, when phospholipase A ₂ is used, the product is 1-acylglycerophosphocholine + fatty acid, and a desired fatty acid can be introduced at the 2-position."

There are very few reports on methods for synthesizing phospholipids from lyso-type phospholipids and fatty acids using this enzyme, although there are very few reports. P.PERNAS et al. Synthesize phosphatidylcholine from lysophosphatidylcholine and oleic acid using various phospholipase A ₂ under the condition that water is not added and it is in a nonpolar organic solvent (BIOCHEMICAL AND BIOP).
HYSICAL RESEARCH COMMUNICATION Vol.168, pp644-650,1
990). An Na et al. Synthesize phosphatidylcholine from lysophosphatidylcholine and highly unsaturated fatty acids using porcine pancreatic phospholipase A ₂ using an emulsifier AOT (JOURNAL OF AMERICAN OIL CHEMIST'S SOCI
ETY VOL.67, pp766-770, 1990).

However, the phosphatidylcholine synthesis rate in the former case was 6.5% when the snake venom phospholipase A ₂ was used.
Is the highest value, and when bovine or porcine pancreatic phospholipase A ₂ is used, the synthesis rate is less than half of that. Even in the latter case, the synthesis rate of phosphatidylcholine is about 6%, and the synthesis rate is extremely low in all cases.

[0012]

[Means for Solving the Problems] In order to solve the above technical problems, the inventors of the present invention have developed an enzyme phospholipase A ₂ from a lyso-type phospholipid having an acyl group at the Sn1 position and a highly unsaturated fatty acid or its ester. When a phospholipid having a highly unsaturated fatty acid at the Sn2 position is obtained by an enzymatic reaction, glycerol was added to the reaction system to develop a method for producing a highly unsaturated fatty acid-containing phospholipid.

Since lyso-type phospholipids have only one acyl group, they are highly hydrophilic and have extremely low solubility in nonpolar organic solvents. In the assay system for the activity of lysophospholipase, which is an enzyme that cleaves the acyl group of lysophospholipids, it is customary to operate in a completely aqueous system. However, as a result of attempting the synthesis in such an aqueous system, the synthetic reaction did not proceed at all. It was considered that the reaction proceeded only in the decomposition direction due to the presence of excess water. Based on this result,
In the search for a substance capable of uniformly dissolving lysophospholipid and not inhibiting the synthetic reaction by the enzyme, it was found that glycerol is a substance which is excellent and suitable for this.

As a raw material for the lyso-type phospholipid, a lyso-type phospholipid having an acyl group at the Sn1 position is used. Lyso-type phospholipids produced from soybean phospholipids by enzymatic decomposition with phospholipase A ₂ are distributed at a relatively low cost and with stable quality, and can be fractionated / purified and used as needed. it can. Alternatively, a specific fatty acid may also be required at the Sn1 position for the expression of the target physiological activity, and it is necessary to use a lyso-type phospholipid that matches the structure of the final product.

Phospholipase A ₂ is present in large amounts in venoms such as snake venom, bee venom and scorpion venom and in the pancreas of animals, as well as in various organisms such as various organs of animals, plants and bacteria. Of these, those extracted and purified from the pancreas of animals are relatively inexpensive and easy to use.

Polyunsaturated fatty acids are those having 18 or more carbon atoms,
A fatty acid having three or more unsaturated bonds, specifically, α-linolenic acid, γ-linolenic acid, arachidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid,
It shows fatty acids such as docosatrienoic acid, docosatetraenoic acid, docosapentaenoic acid and docosahexaenoic acid.

[0017]

【Example】

Example 1 Lyso-type phospholipid (lysophosphatidylcholine content 99% or more) 110 mg was well dissolved in 5 ml glycerol, and fish oil fatty acid 180 mg and phospholipase A ₂ solution (porcine pancreatic phoslipase A ₂ 60 mg 0.5 ml containing 6 mM calcium chloride 200 mM). Dissolved in Tris-HCl buffer (pH 8.0))
Was added, the mixture was tightly stoppered, and well stirred with a tube mixer, and then reacted at 25 ° C. for 48 hours while stirring at 1500 rpm with a stirrer. After completion of the reaction, the lipid was extracted by partitioning with Folch solvent (chloroform-methanol (2: 1 v / v) solution added with 0.2 volume of water). The extracted lipid components were separated into phospholipid fractions using Sep-Pak silicic acid cartridge (Waters Associates, Inc.), developed and separated by thin layer chromatography-FID detection method (Iatroscan), and lipids were separated. The composition ratio was determined. As a result, the phosphatidylcholine synthesis rate (the weight ratio of phosphatidylcholine in the total phospholipids after the reaction) was 13.8%. Table 1 shows the fatty acid composition of lysophosphatidylcholine and fish oil fatty acid as substrates and the fatty acid composition of Sn1 position and Sn2 position of the synthesized phosphatidylcholine.

[0018]

【table 1 】

The fatty acid composition at the Sn2 position of the produced phosphatidylcholine was 1% for eicosapentaenoic acid (C20-5).
The content was 6.0% and the content of docosahexaenoic acid (C22-6) was 9.5%, which was almost the same as the composition of the substrate fish oil fatty acid.

[0020]

Example 2 A reaction was performed in the same manner as in Example 1 except that the fatty acid was changed to a highly unsaturated fatty acid concentrated fatty acid (the total amount of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was 65% or more of the total). went. As a result, the phosphatidylcholine synthesis rate was 14.8%. Table 2 shows the fatty acid composition of lysophosphatidylcholine and polyunsaturated fatty acid-enriched fatty acid as raw materials.

[0021]

[Table 2]

When the composition of fatty acids at the Sn2 position of the phosphatidylcholine produced was analyzed, EPA (C20-5),
DHA (C22-6) was 43.2% and 25.3%, respectively.
%, Which was almost the same as the composition of the substrate fatty acid.

[0023]

[Example 3] The reaction was performed in the same manner as in Example 1 except that the fatty acid was changed to EPA (purity 99% or more). As a result, the phosphatidylcholine synthesis rate was 15.9%, and when the composition of fatty acids at the Sn2 position of the phosphatidylcholine produced was analyzed, most of them were EPA.

[0024]

As described above, the present invention relates to the synthesis of phospholipids, in which the addition of glycerol promotes the enzymatic synthesis, resulting in less denaturation / degradation of highly unsaturated fatty acids as compared with the chemical synthesis method. A highly physiologically active phospholipid having a highly unsaturated fatty acid at the Sn2 position can be obtained.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Haruo Akazawa 1-12-1 Yurakucho, Chiyoda-ku, Tokyo Inventor Nichiro Central Research Institute

Claims (1)

[Claims] 1. When obtaining a phospholipid having a highly unsaturated fatty acid at the Sn2 position by enzymatic reaction of the enzyme phospholipase A ₂ from a lyso-type phospholipid having an acyl group at the Sn1 position and a highly unsaturated fatty acid or an ester thereof. And a method for producing a highly unsaturated fatty acid-containing phospholipid, which comprises adding glycerol to the reaction system.