JPH0279990A - Production of phosphatidylserine - Google Patents

Abstract

PURPOSE:To obtain high-purity phosphatidylserine through a safe, simple process by reaction between phosphatidylcholine and serine using phospholipase D stemmed from microorganisms classified as Streptomyces. CONSTITUTION:For example, using a reactor equipped with an agitator and temperature controller, each specified amount of (A) calcium chloride, serine and phospholipase D stemmed from microorganisms classified as Streptomyces is added to (B) an aqueous acetic acid- or phosphoric acid buffer solution to effect dissolution. Thence, phosphatidylcholine is dissolved in e.g., ethyl acetate or ether, and a specified amount of the resultant solution is added to the above- mentioned aqueous solution followed by keeping the resultant system at 25-35 deg.C which is agitated and made to react, thus obtaining the objective high-purity phosphatidylserine.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a new method for producing phosphatidylserine.

(Conventional technology) Phospholipids exist as components of biological membranes of cells, and in biological membranes, they form a double layer membrane with a bimolecular structure, and are used for the permeation of substances, selective transport, etc. along with proteins, cholesterol, etc. fulfills functions essential to life phenomena. Phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin exist in living organisms, and each plays an important role. A common feature of these phospholipids is that they have a hydrophilic group and a hydrophobic group within their molecules, which allows them to create microvesicles called liposomes, which are characteristic of phospholipids, and drugs can be contained therein. Development of this technology into drug delivery systems is being planned. On the other hand, medical developments such as those typical of PAF are also being developed by utilizing its physiologically active effects.

Although phosphatidylserine is widely distributed in the living world, its quantity is not very large.However, when phosphatidylserine is added to extracellular fluid, it has physiologically active effects such as enhancing the release of histamine, and has been used in the field of genetic engineering. It is known for its uses such as being used for cell fusion.

Conventionally, phosphatidylserine has been produced by extracting it from plant or animal tissues and then purifying it.
r, J, Biochem, +42+483(1
974)) or synthesis by cabbage phospholipase D (Yang et al., J. Biol. Chew.
242.477 (1967)) are known. Chemical synthesis methods are also known (Baer
et al., J. Biol.

Chell, 212 39 (1955), Dee
nen et al., Rec, Trav.

Chim, 83.99 (1964), Turne
r et al., J. Lipid Res.

Engineering, 616 (1964), etc.).

(Problems to be Solved by the Invention) In the method of extracting phosphatidylserine from animals and plants, as mentioned above, the quantitative ratio of phosphatidylserine is small and there are many types of phospholipids, so purification and separation is extremely difficult. It is difficult to In addition, there is a problem in that the type of bound fatty acid cannot be freely selected, and only phosphatidylserine that is a mixture of many fatty acid types can be obtained.

Furthermore, enzymatic base exchange and synthesis of phospholipids using phospholipase D from large intestine and cabbage is an excellent method, but the problem is that conventionally known enzymes have low base exchange ability. There is.

The chemical synthesis method has the advantage of being able to produce large quantities of phosphatidylserine with the necessary fatty acids attached, but the reaction process is very long and serine is reactive, making it difficult to remove the protective group. The problem is that this process is extremely difficult industrially.

This invention is intended to solve the above-mentioned problems, and is to obtain phosphatidylserine by reacting phosphatidylcholine and serine using phospholipase D derived from a specific microorganism. Another object of the present invention is to provide a method for producing phosphatidylserine using simple steps and equipment.

(Means for Solving the Problems) The present invention is a method for producing phosphatidylserine, which is characterized by reacting phosphatidylcholine and serine using phospholipase D originating from a microorganism of the genus Streptomyces.

That is, it is characterized in that the choline group of phosphatidylcholine is exchanged with a serine group by phospholipase D originating from a microorganism of the genus Streptomyces.

The phosphatidylcholine used in the present invention is a natural mixed fatty acid type or a synthetic 1.2-position defined fatty acid type, and since it is an enzymatic reaction, phosphatidylcholine containing highly unsaturated fatty acids can also be easily used.

Phosphatidylserine can be obtained according to the structure of phosphatidylcholine.

The other raw material, serine, can be used in either the L-form or the 0-form, and the phospholipase D derived from a microorganism of the genus Streptomyces used in the present invention can also synthesize stereoisomers of phosphatidylserine. Phospholipase D has been extracted from spinach, cabbage, carrots, etc. and has been studied, but it is generally believed that Ca' is required to express its activity. , promoted by esters,
It is also promoted by dodecyl sulfate, phosphatidic acid, and phosphatidylinositol. On the contrary, cationic surfactants, choline, ethanolamine, etc. act in an inhibitory manner.

Although phospholipase D is used in the base exchange reaction in the present invention, it also has hydrolytic activity as shown in the following formula.

Therefore, it is also important to have a technique to reduce the hydrolysis effect and advance the reaction toward the synthesis reaction side. That is, it is important that the phospholipase D used in the reaction has low hydrolyzability.

O- (phosphatidylserine) (phosphatidic acid) Phospholipase D used in the present invention is characterized by low hydrolysis, and depending on the amount of water used during the reaction,
Its hydrolyzability is hardly affected, and the base exchange reaction proceeds preferentially, making it particularly suitable for the synthesis of phosphatidylserine, which is specifically produced by Streptomyces, especially Streptomyces. Ru. Streptomyces genus bacteria include, for example, Streptomyces chromophoracus, Streptomyces prunicola, Streptomyces xansporia, Streptomyces chromodienia, Streptomyces hachijoensis, and Streptomyces verticidumcinano. There are things like nium.

Commercially available phospholipase D is available for use in the present invention.

Specifically, the phosphatidylserine synthesis reaction in the present invention is carried out as follows.

That is, in a reaction apparatus equipped with a stirring device and a temperature control device, an acetate buffer or phosphate buffer aqueous solution (approximately 0.2 M, pH varies depending on the enzyme type, but the p
Calcium chloride (H5-7 is suitable) and calcium chloride (0.01-7
0.2 M), phospholipase D (the required amount varies depending on the enzyme activity, but the reaction is possible if it is 0.1 unit or more. The number of units is the number of micromoles of phosphatidic acid produced in 1 minute at 30°C) ) and serine (higher concentration is preferable, basically saturation concentration (approximately 5M)
gives the maximum rate of phosphatidylserine production) and dissolve.

Next, dissolve phosphatidylcholine in ethyl acetate, ether, etc., add a predetermined amount (the larger the serine/phosphatidylcholine ratio, the higher the reactivity), and add 25
Keep at ~35°C and stir for 1-5 hours to react. The reaction product is extracted into a chloroform layer using the Folch method, and purified and separated using a silica gel column using a chloroform/methanol-based eluent.

By performing the above steps, highly pure phosphatidylserine having desired fatty acid species can be produced.

(Effects of the Invention) According to the present invention, phosphatidylcholine and serine can be subjected to a base exchange reaction using phospholipase D originating from a microorganism belonging to the genus Streptomyces without causing almost any hydrolysis reaction. Phosphatidylserine can be produced in high yield.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Calcium chloride 6 in 0.2M phosphate buffer 10
10 mg (0.1 unit) of phospholipase D derived from the genus Streptomyces (manufactured by Yakult) and 4.18 grams of monoserine were dissolved and kept at 30°C.

Next, while maintaining the temperature at 30 DEG C., 600 µm of phosphatidylcholine purified from egg yolk phospholipid dissolved in 20-degree ethyl acetate was added, and the mixture was reacted at 30 DEG C. with stirring at pH 7 for 2 hours.

Transfer the reaction solution to a separatory funnel, add 2:1 of chloroform, 500 g of methanol and 180 g of water, concentrate the chloroform layer, and remove phosphorus! 530 mg was obtained. Add this to developing agent: chloroform/methanol/acetic acid 15% NaHSOz
=40/15/6/2, and was analyzed by Iatroscan and found to contain 95.2% phosphatidylserine. This phosphatidylserine was transferred to a silica gel column (chloroform/methanol/water = 65/25/4).
420 IIw of pure phosphatidylserine was obtained.

Example 2 Calcium chloride 60 to 0.2M acetate buffer 10
20 mg (0.2 units) of ff1r and Streptomyces-derived phospholipase D (manufactured by Toyo Brewery) and 2.05 g of shiichiserine were dissolved and kept at 30°C.

Then, also kept at 30°C, 600 μl of dipalmitoylphosphatidylcholine dissolved in 20% ethyl acetate was added.
was added, and the mixture was reacted at 30° C. with stirring at pH 5, 6 for 4 hours.

The same procedure as in Example 1 was carried out to obtain 510 ml of dipalmitoyl phosphatidylserine having a purity of 93.3%. Purification was carried out in the same manner as in Example 1 to obtain 400 mr of pure dipalmitoylphosphatidylserine.

Example 3 Calcium chloride 6 in 0.2M phosphate buffer 10
0■ and Streptomyces-derived phospholipase D (manufactured by Yakult) 10■ (0.1 units) and 5.
3 g of D-serine was dissolved and reacted and treated in the same manner as in Example 1. As a result, 520 µm of phosphatidylserine with a purity of 92.9% was obtained, and 430 µm of pure serine-type phosphatidylserine was obtained after silicage Jl/$# production.

Example 4 Calcium chloride 6 in 0.2M phosphate buffer 10
0■ and Streptomyces-derived phospholipase D (manufactured by Yakult) 20■ (0.2 units) and 4.
No. 18 Serine was dissolved and kept at 30°C.

Next, 1.2 g of silyl phosphatidylcholine dissolved in 20 d of ethyl acetate was added while keeping the temperature at 30° C., and the mixture was reacted at 30° C. under an Nt atmosphere at pH 1 for 4 hours.

It was treated in the same manner as in Example 1 to obtain 1.03 g of silyl phosphatidylserine with a purity of 82.2%.

Purification was carried out in the same manner as in Example 1 to obtain 740 ml of pure silylylphosphatidylserine.

Example 5 Using 0.2M phosphate buffer 50-1, the reaction and purification were carried out in the same manner as in Example 1, to obtain 510 ml of phospholipid containing 93.8% phosphatidylserine. Purification was carried out in the same manner as in Example 1 to obtain 410 ml of pure phosphatidylserine.

Comparative Example 1 About 3 kg of cabbage was processed with a juicer, and after centrifugation, the top layer was treated at 50° C. for 5 minutes, then rapidly cooled to 10° C., and centrifuged again. Twice the volume of acetone cooled to -20°C was added to the supernatant to precipitate.

The precipitate was collected and lyophilized to obtain crude phospholipase D.

Calcium chloride 6 in 10ml 0.2M acetate buffer
0■ and the crude phospholipase D50 derived from the above cabbage
(0.8 units) and 4.1 g of L-serine were dissolved, and 1.2 g of dipalmitoylphosphatidylcholine was added.
were reacted and treated in the same manner as in Example 2. As a result, 22
．． 430 ml of crude dipalmitoylphosphatidylserine with a purity of 3% was obtained. The phospholipids other than phosphatidylserine were 74.9% dipalmitoylphosphatidic acid and 2.8% unreacted phosphatidylcholine.

Comparative Example 2 D-serine was reacted with cabbage phospholipase D obtained in Comparative Example 1 in the same manner as in Comparative Example 1, but no phosphatidylserine was produced.

Claims (1)

[Claims] (1) A method for producing phosphatidylserine, which comprises reacting phosphatidylcholine and serine using phospholipase D originating from a microorganism of the genus Streptomyces.