JP2007110951A - Base exchange method of phospholipid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for increasing yield of produced phospholipid and a method for simply separating the produced phospholipid, in base exchange reaction of phospholipid and in a two phase system. <P>SOLUTION: The base exchange method of the phospholipid, which method comprises a step for reacting the phospholipid with a hydroxy-containing compound, in the presence of phospholipase D and in the two phase system comprising a nonpolar solvent, a polar solvent and water, is provided, wherein the content of the polar solvent is more than 20% but less tan 80% on a volume basis per the total amount of the nonpolar solvent and the polar solvent and the molar ratio of the hydroxy-containing compound to the phosphatide is 4 or more but 30 or less. The base exchange method of phospholipid, which method comprises reacting the phosphatide with the hydroxy-containing compound, in the presence of an edible oil and the phospholipase D, in the two phase system comprising a nonpolar solvent, a polar solvent and water, is provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a phospholipid base exchange method using phospholipase D. More specifically, in the phospholipid base exchange reaction using phospholipase D, the reaction is performed by limiting the mixing ratio of the nonpolar solvent and the polar solvent and the mixing ratio of the phospholipid and the hydroxyl group-containing compound, or The present invention relates to a method for performing a reaction using cooking oil.

Phospholipase D (hereinafter abbreviated as PLD) is known to catalyze the base exchange reaction of phospholipids. PLD catalyzes a base exchange reaction between a phospholipid such as phosphatidylcholine (hereinafter abbreviated as PC) or phosphatidylethanolamine (hereinafter abbreviated as PE) and an alcohol as a receptor. For example, when glycerol is used as the alcohol, phosphatidylglycerol (hereinafter abbreviated as PG) is produced. When serine is used as the alcohol, phosphatidylserine (hereinafter abbreviated as PS) is produced. On the other hand, when water is used instead of alcohol as a receptor, phospholipid hydrolysis proceeds to produce phosphatidic acid (hereinafter abbreviated as PA).

In general, the base exchange reaction of phospholipids using PLD is carried out in an aqueous system or a two-phase system composed of an aqueous phase and an organic solvent phase. A hydrolysis reaction also proceeds as a reaction. In particular, in the case of a two-phase system, when the content of water in the reaction system increases, the hydrolysis reaction is promoted more than the base exchange reaction, and the yield of the base exchange reaction is deteriorated. In particular, when heptane, acetone and water are used, if the amount of water increases, there is a problem that it becomes difficult to separate the products in addition to a decrease in yield.

Therefore, various methods have been reported to increase the yield of the base exchange reaction.
In the case of an aqueous system, a method of adding a salt (calcium salt or the like) or a surfactant to suppress a hydrolysis reaction or a method of promoting a base exchange reaction is known.
In the two-phase system, a method of suppressing the hydrolysis reaction by minimizing the amount of water (10% or less) has been devised. In the case of a two-phase system, the aqueous phase mainly contains receptors such as PLD and alcohol. For example, a method in which an aqueous phase containing these components is enclosed in reverse micelles and added to the reaction system (see Patent Document 1). There is a method in which a phospholipid and a receptor are brought into contact with a PLD adsorbed on a carrier and the phospholipid and the receptor are reacted in a state where the water content in the reaction system is 1% by weight or less (Patent Document 2). reference).
Although the production of PA was suppressed by these methods, the yield of the target phospholipid was not sufficient, and it is desired to further increase the yield.

In order to further increase the yield in the base exchange reaction, it is necessary to increase the amount of the acceptor such as alcohol. However, since the solubility of the acceptor in water is limited, the amount of the acceptor added is increased. Inevitably, the amount of water must be increased.

On the other hand, for separation of the product after the base exchange reaction, dilution oil is added to the PS-containing solution obtained by the base exchange reaction before the concentration step or the solvent removal step in the PS production method by the base exchange reaction. Thus, a method for efficiently obtaining a PS composition having good fluidity has been reported (see Patent Document 3).
However, a method with good yield in the base exchange reaction and easy separation of the product is not yet known.
Japanese Patent Publication No. 7-16426 Japanese Patent Publication No. 3-67676 JP 2003-304814 A

In view of the above situation, the present invention provides a method for increasing the yield of phospholipid produced and a method for simplifying separation in a base exchange reaction of phospholipid in a two-phase system.

As a result of intensive studies to solve the above-mentioned problems, the present inventors limited the blending ratio of the nonpolar solvent and the polar solvent in the reaction system and the blending ratio of the phospholipid and the hydroxyl group-containing compound. It has been found that the yield of the product is improved. Moreover, when performing base exchange reaction of the phospholipid by a two-phase system, it discovered that the isolation | separation of a product became easy, without reducing a yield by adding an edible oil in a reaction system.
That is, the first present invention includes a step of reacting a phospholipid and a hydroxyl group-containing compound in a two-phase system composed of a nonpolar solvent, a polar solvent and water in the presence of phospholipase D. However, the amount is more than 20% and 80% or less on a volume basis with respect to the total amount of the nonpolar solvent and the polar solvent, and the molar ratio of the hydroxyl group-containing compound to the phospholipid is 4 or more and 30 or less. This is a base exchange method for phospholipids.
The second present invention includes a step of reacting a phospholipid and a hydroxyl group-containing compound in a two-phase system comprising a nonpolar solvent, a polar solvent and water in the presence of edible oil and phospholipase D. This is a lipid base exchange method.

The present invention is described in detail below.
In the phospholipid base exchange method according to the first aspect of the present invention, in the presence of phospholipase D, in a two-phase system comprising a nonpolar solvent, a polar solvent and water, the content of the polar solvent is nonpolar solvent and polar solvent. The phospholipid and the hydroxyl group-containing compound are reacted with each other in an amount of more than 20% and 80% or less based on the volume of the total amount.
The non-polar solvent is not particularly limited, and examples thereof include aliphatic hydrocarbons such as heptane, hexane, and petroleum ether; cyclic aliphatic hydrocarbons such as cyclopentane and cyclohexane; ethers such as diethyl ether and tetrahydrofuran; methyl acetate, Examples include esters such as ethyl acetate; halogenated hydrocarbons such as carbon tetrachloride and chloroform. As the nonpolar solvent, heptane and hexane are preferable.
The polar solvent is not particularly limited, and examples thereof include ketones such as acetone, methyl ethyl ketone, and diethyl ketone. As the polar solvent, acetone is preferable.

In the base exchange method of the present invention, when the content of the polar solvent is 20% or less or exceeds 80%, the yield of the target phospholipid is lowered. The content of the polar solvent is preferably 25 to 50%.

Examples of water used in the base exchange method of the present invention include ion exchange water, purified water, distilled water, and tap water. Furthermore, it is good also as a buffer solution for pH adjustment by containing acetic acid etc. in these. Preferably, ion exchange water, purified water or distilled water is used.

In the base exchange method of the present invention, the content of water is preferably 30 to 250%, more preferably 30 to 200%, based on the volume with respect to the total of the nonpolar solvent and the polar solvent. More preferably, it is 50 to 100%.

In addition, the base exchange method of the present invention is performed under a condition where the molar ratio of the hydroxyl group-containing compound to the phospholipid is 4 or more and 30 or less. When the molar ratio of the hydroxyl group-containing compound is less than 4, the yield of the phospholipid to be produced is low. The molar ratio is preferably 6 or more, and more preferably 8 or more. Moreover, when the said molar ratio exceeds 30, cost will become high and it is unrealistic.

The phospholipid used in the base exchange method of the present invention may be of any origin, and natural products containing phospholipids, extracts from natural products, purified products of the extracts, synthetic phospholipids, etc. are preferably used. It is done. Examples of such phospholipids include phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), and mixtures thereof. The above phospholipids may be used singly or in combination of two or more. Generally, soybean lecithin, egg yolk lecithin, rapeseed lecithin and the like are used.
The purity of the phospholipid is preferably 20 to 99.5% by mass based on the total mass of the compound. Moreover, it is preferable to use 0.1-60 mass% of said phospholipids in this reaction system. The ratio of the phospholipid to the organic solvent composed of the nonpolar solvent and the polar solvent is not particularly limited, but it is preferable that the phospholipid is dissolved in the organic solvent phase during the base exchange reaction. The organic solvent is 1 to 10 ml.

Examples of the hydroxyl group-containing compound include alcohols, nitrogen-containing alcohols, saccharides, polyols, and hydroxy cyclic compounds.
Examples of alcohols include methanol, ethanol, propanol, ascorbic acid and the like.
Examples of nitrogen-containing alcohols include amino acids such as serine; 1-amino-2-propanol.
Examples of the saccharide include nucleosides such as adenosine, guanosine, inosine, xanthosine, deoxyadenosine, deoxyguanosine; glucose, trehalose, N-acetyl-D-glucosamine and the like.
Examples of polyols include glycerol, ethylene glycol, propylene glycol and the like.
Examples of the hydroxy cyclic compound include succinic acid and arbutin.
In the base exchange method of the present invention, the ratio of the hydroxyl group-containing compound and water is not particularly limited, but it is preferable that the hydroxyl group-containing compound is dissolved in water during the base exchange reaction. When using serine, it is preferable to mix | blend 3-10 ml of water with respect to 1g of serine.

The base exchange method of the present invention is performed in the presence of phospholipase D (PLD). The PLD is not particularly limited as long as it can hydrolyze and / or base-exchange the phospholipid base moiety, and examples thereof include plant-derived PLD and microorganism-derived PLD. Examples of plant-derived PLD include cabbage and soybean-derived PLD, and examples of microorganism-derived PLD include actinomycetes, filamentous fungus-derived PLD, and the like.
The concentration of PLD in this reaction system is preferably 5 to 200 U, more preferably 20 to 100 U, with respect to 1 g of phospholipid. In addition, 1U uses 95% soybean phosphatidylcholine as a substrate, 37 M 0.2M acetate buffer (pH 4.0, 10 mM CaCl ₂ , 1.3% Triton X-100) 37%. It is the amount of enzyme that liberates 1 μmol of choline per minute when reacted at ° C.

The base exchange reaction is preferably performed under conditions of pH 3.5 to 10, and more preferably pH 4 to 9. 10-40 degreeC is preferable and, as for reaction temperature, 20-30 degreeC is more preferable.
Moreover, since the reaction system is a two-phase system, it is preferable to stir the reaction system during the base exchange reaction.

The base exchange method of the present invention also preferably includes edible oil in the two-phase system. By performing the base exchange reaction in the presence of edible oil, the aqueous phase and the organic solvent phase are separated, and the phospholipid produced is easily separated. About the addition of edible oil, it can carry out similarly to the base exchange method of the 2nd this invention mentioned later.

By the base exchange reaction, for example, PC and PE in phospholipid react with a hydroxyl group-containing compound such as serine to obtain PS and the like.
After performing the base exchange reaction, for example, the PLD is deactivated by a treatment such as heating, and the organic solvent layer and the aqueous layer are separated by a centrifugal method or the like to obtain the organic solvent layer. Concentrate by removing. Next, crystallization is performed with acetone or ethanol, a solid is obtained by solid-liquid separation, and dried, whereby the phospholipid produced by the base exchange reaction can be isolated.

Next, the base exchange method for phospholipid according to the second invention will be described.
The base exchange method of the second aspect of the present invention includes a step of reacting a phospholipid and a hydroxyl group-containing compound in a two-phase system composed of a nonpolar solvent, a polar solvent and water in the presence of edible oil and phospholipase D. .
Examples of the edible oil include those derived from plants, those derived from animals such as fish oil, and those derived from microorganisms, such as coconut oil, coconut oil, palm oil, grapefruit oil, and medium chain fatty acid triglycerides (MCT). Oils rich in saturated fatty acids; or soybean oil, sesame oil, perilla oil, peanut oil, rice bran oil (rice germ oil), wheat germ oil, corn oil, rapeseed oil, cottonseed oil, pumpkin seed oil, olive oil, guava seed oil, date palm Any of oils rich in unsaturated fatty acids such as seed oil, grape seed oil, camellia seed oil, sunflower oil, evening primrose seed oil, safflower oil, etc., or a mixed oil thereof can be used. The edible oil may be natural or processed. The edible oil may be liquid, or may be solid as long as it becomes liquid during the reaction.

The amount of edible oil added is preferably 5 parts by weight or more with respect to 100 parts by weight of phospholipid. If the amount is less than 5 parts by weight, the aqueous phase and the organic solvent phase may not be sufficiently separated, making it difficult to separate and purify the phospholipid. More preferably, it is 5 to 80 parts by weight.

The edible oil may be added before starting the base exchange reaction, or may be added after starting the reaction, but in the present invention, it is preferably added before starting the reaction. When added before the start of the base exchange reaction, the aqueous phase and the organic solvent phase are well separated, and separation and purification of the phospholipid after the reaction is easy.

The phospholipid, hydroxyl group-containing compound, phospholipase D, nonpolar solvent, polar solvent and water type and amount used in the present invention, pH and reaction temperature during the reaction, and isolation of the phospholipid after the base exchange reaction are as described above. This is the same as the first aspect of the present invention.

Until now, in the base exchange reaction in a two-phase system, separation of the phospholipid after the reaction was difficult because the aqueous phase and the organic solvent phase were emulsified, but in the method of the present invention, edible oil was added to the reaction system. By adding, without reducing the yield of phospholipid, the aqueous phase and the organic solvent phase are well separated after the reaction, and purification of the phospholipid becomes easy.

In the base exchange method according to the first aspect of the present invention, by limiting the blending ratio of the nonpolar solvent and the polar solvent and the blending ratio of the phospholipid and the hydroxyl group-containing compound, the efficiency can be improved even if the reaction system contains a lot of water. Since the base exchange reaction proceeds well, a large amount of the hydroxyl group-containing compound can be blended, and the yield of the target phospholipid can be increased.
In the base exchange method of the second aspect of the present invention, by adding edible oil to the reaction system, the aqueous phase and the organic solvent phase are well separated after the reaction without reducing the yield of phospholipid. Separation and purification of lipid becomes simple, and high-purity phospholipid is obtained.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
Example 1
SLP-PC55 (manufactured by Sakai Oil Co., Ltd., PC 54.4% by weight, PE 12.2% by weight) as lecithin was dissolved in a mixed solution of heptane and acetone so as to be 20% (weight / volume). The mixing ratio of heptane and acetone was heptane: acetone = 8.0: 2.0, 7.5: 2.5, 7.0: 3.0, 6.5: 3.5 and 6.0: 4. 0 (capacity: capacity). Hereinafter, this solution is referred to as a lecithin solution.
Next, L-serine (manufactured by Kyowa Hakko) was dissolved in 0.6 M sodium acetate buffer (pH 4.0) so as to be 25% (weight / volume). Hereinafter, this solution is referred to as a serine solution.
As described above, the total amount of the prepared lecithin solution and serine solution was such that the molar ratio of lecithin to serine was lecithin: serine = 1: 2, 1: 4, 1: 6, 1: 8 and 1:10. Was dispensed into a 2 ml Eppendorf tube so that 1 ml was 1 ml. Furthermore, phospholipase D (manufactured by Nagase ChemteX) was added so as to be 90 U per 1 g of lecithin, and the reaction was carried out by stirring at 30 ° C. for 16 hours using BioShaker M-BR-022UP manufactured by TITEC. . Hereinafter, the mixed solution of the lecithin solution and the serine solution is referred to as a reaction solution.
The phospholipid composition was prepared by dissolving 50 μl of the reaction solution in a mixed solution of chloroform: methanol = 1: 1 (volume: volume), removing insoluble matters by centrifugation, and then performing high performance liquid chromatography (HPLC) under the following conditions. Analyzed in
Column used: Unisil Q NH ₂ (inner diameter 4.6 mm × 25 cm) manufactured by GL Sciences
Mobile phase: acetonitrile: methanol: 10 mM ammonium dihydrogen phosphate = 619: 291: 90 (v / v / v)
Flow rate: 1.3 ml / min
Column temperature: 37 ° C
Detection: UV 205nm
Further, the PS production rate represents the amount of PS produced relative to the amount of PC and PE before the reaction.
PS production rate% = (generated PS amount / PC + PE amount before reaction) × 100
The production rate of PS is shown in Table 1 and FIG. In addition, the separation state of the lecithin solution and the serine solution after the reaction became worse as the amount of the serine solution increased.

Example 2
Squid-derived phospholipids (manufactured by Nippon Kagaku Feed Co., Ltd., PC 49.1% by weight, PE 8.7% by weight) as lecithin were dissolved in a mixture of heptane and acetone so as to be 20% (weight / volume). The mixing ratio of heptane and acetone is heptane: acetone = 7.0: 3.0, 6.5: 3.5, 6.0: 4.0, 5.0: 5.0 and 4.0: 6. 0 (capacity: capacity). Hereinafter, this solution is referred to as a lecithin solution.
A serine solution was prepared in the same manner as in Example 1.
As described above, the prepared lecithin solution and serine solution were prepared in a molar ratio of lecithin to serine: lecithin: serine = 1: 8, 1:10, 1:12, 1:14, 1:16, 1:18, and It was dispensed into 2 ml Eppendorf tubes so that the total volume was 1 ml at a ratio of 1:20. Furthermore, phospholipase D (manufactured by Nagase ChemteX Corporation) was added so as to be 90 U per 1 g of lecithin, and the mixture was stirred at 30 ° C. for 5 hours using BioShaker M-BR-022UP manufactured by TITEC.
Analysis of the phospholipid composition was carried out by the method described in Example 1. The production rate of PS is shown in Table 2 and FIG. In addition, the separation state of the lecithin and the serine solution after the reaction was bad.

Example 3
SLP-PC55 (manufactured by Sakai Oil Co., Ltd., PC 54.4% by weight, PE 12.2% by weight) as lecithin was dissolved in a mixed solution of heptane and acetone so as to be 20% (weight / volume). The mixing ratio of heptane and acetone was heptane: acetone = 7: 3 (volume: volume). Furthermore, 0-100 parts by weight of rapeseed oil (manufactured by Nacalai Tesque) was added to 100 parts by weight of lecithin as shown in Table 3 to obtain a lecithin solution.
A serine solution was prepared in the same manner as in Example 1.
As described above, the prepared lecithin solution and serine solution were dispensed into 500 ml stoppered bottles so that the molar ratio of lecithin and serine was lecithin: serine = 1: 10, and the total amount was 100 ml, The reaction was performed by stirring with a magnetic stirrer in a 30 ° C. water bath for 5 hours.
After the reaction, the mixture was allowed to stand for 2 hours, and the separation state of the lecithin solution and the serine solution, the turbidity of the lecithin solution, and the like were visually confirmed. The PS production rate was measured by the method described in Example 1. The results are shown in Table 3.

Example 4
Squid-derived phospholipids (manufactured by Nippon Kagaku Feed Co., Ltd., PC 49.1% by weight, PE 8.7% by weight) as lecithin were dissolved in a mixture of heptane and acetone so as to be 20% (weight / volume). The mixing ratio of heptane and acetone was heptane: acetone = 6: 4 (volume: volume). Furthermore, 0 to 50 parts by weight of rapeseed oil (manufactured by Nacalai Tesque) was added to 100 parts by weight of lecithin as shown in Table 4 to obtain a lecithin solution.
A serine solution was prepared in the same manner as in Example 1.
As described above, the prepared lecithin solution and serine solution were mixed at a ratio of lecithin: serine molar ratio of lecithin: serine = 1: 8, 1:12, 1:16, 1:18, and 1:20. The total amount was 1 ml, and dispensed into 2 ml Eppendorf tubes. Furthermore, phospholipase D (manufactured by Nagase ChemteX) was added so as to be 90 U per 1 g of lecithin, and the mixture was stirred at 30 ° C. for 5 hours using BioShaker M-BR-022UP manufactured by TITEC.
Analysis of the phospholipid composition was carried out by the method described in Example 1. After the reaction, the mixture was allowed to stand for 2 hours, and the separation of the lecithin solution and the serine solution was visually confirmed. The results are shown in Table 4.

In the base exchange method according to the first aspect of the present invention, by limiting the blending ratio of the nonpolar solvent and the polar solvent and the blending ratio of the phospholipid and the hydroxyl group-containing compound, the efficiency can be improved even if the reaction system contains a lot of water. Since the base exchange reaction proceeds well, a large amount of the hydroxyl group-containing compound can be blended, and the yield of the target phospholipid can be increased.
In the base exchange method of the second aspect of the present invention, by adding edible oil to the reaction system, the aqueous phase and the organic solvent phase are well separated after the reaction without reducing the yield of phospholipid. Separation and purification of lipid becomes simple, and high-purity phospholipid is obtained.

3 is a graph showing a PS generation rate in Example 1. 6 is a graph showing a PS generation rate in Example 2.

Claims (5)

Reacting a phospholipid with a hydroxyl group-containing compound in the presence of phospholipase D in a two-phase system consisting of a nonpolar solvent, a polar solvent and water,
The content of the polar solvent is an amount exceeding 20% and 80% or less on a volume basis with respect to the total amount of the nonpolar solvent and the polar solvent,
A method for base exchange of a phospholipid, wherein the molar ratio of the hydroxyl group-containing compound to the phospholipid is 4 or more and 30 or less. The method according to claim 1, wherein the water content is 30 to 200% on a volume basis with respect to the sum of the nonpolar solvent and the polar solvent. The method according to claim 1 or 2, further comprising edible oil in the two-phase system. A method for base exchange of phospholipid, comprising a step of reacting a phospholipid and a hydroxyl group-containing compound in a two-phase system comprising a nonpolar solvent, a polar solvent and water in the presence of edible oil and phospholipase D. The process according to claim 4, wherein the edible oil is added before the base exchange reaction is started.

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