JPH0367675B2 - - Google Patents

Description

Detailed Description of the Invention (a) Industrial Application Field The present invention relates to a method for rearranging phosphatidyl groups, which is characterized by reacting phospholipids and alcohols in the presence of phospholipase D and a specific amount of water. By utilizing this rearrangement reaction, mixed phospholipids can be concentrated and novel phospholipid derivatives can be produced. (b) Conventional technology Phospholipids can be isolated and concentrated from various living organisms, and can also be manufactured by chemical synthesis methods, and products manufactured by both methods are used industrially. ing. However, although it is possible to purify to a fairly high concentration with the former method (concentration from living organisms), if you want to obtain a single component, you must use a solvent method, column chromatography, etc. In the latter method (chemical synthesis method), although it is possible to produce a single component, the combined fatty acids and phosphate esters deteriorate due to the thermal history during synthesis. At present, it is necessary to adopt a complicated and economically disadvantageous process for coloring and refining it. Attempts to enzymatically convert phospholipids have been studied for some time. For example, it has been reported that when lecithin and ethanol are reacted in the presence of phospholipase D, the ester bond in the phosphoric acid-alcohol moiety of the phospholipid is hydrolyzed and, at the same time, phosphatidyl ethanol is generated through a phosphatidyl group transfer reaction. (RMDawson,
Biochem.J., 102 , 205 (1967), SFYang, J.
Biol.Chem., 242 , 477 (1967), etc.). In addition, British Patent No. 1581810 discloses that cabbage-derived phospholipase D It is stated that when the alcohol transfer reaction is carried out by the action of C5, the reaction occurs only with primary alcohols having less than _C5 , and in the case of alcohols having more than _C5 , the main product is the corresponding phosphatidic acid. Furthermore, in addition to these, phospholipase D derived from microorganisms is also known to
-187786), it has been shown that the reaction of sphingophospholipids with primary alcohols, secondary alcohols, and phenol glycosides (JP-A-59-187787) causes a phosphatidyl group transfer reaction. (c) Problems to be Solved by the Invention However, in such conventional reaction systems, for example, 1% phospholipid emulsion (0.1 ml), 0.4 M acetate buffer (0.1 ml), 0.1 M calcium chloride aqueous solution (0.05 ml),
Distilled water (0.1ml), 10% alcohol solution (0.1ml)
and phospholipase D aqueous solution (0.1 ml), and the amount of water coexisting in the reaction system is approximately 80% or more, which is approximately 40 times the weight of the substrate (phospholipid and alcohol). water coexists. This is not necessarily satisfactory from the viewpoint of reaction efficiency. On the other hand, phospholipase D has been produced from cabbage for a long time.
It is known to be contained in higher plants such as spinach, carrots, and radish, fungal cells (Streptomyces chromofuscus), and rat brain microsomes, and these are also known to cause phosphatidyl group rearrangement reactions (for example, Hanahan D .
J., J.Biol.Chem., 172 , 191 (1948), Imamura,
S., J.Biochem., 85 , 79 (1979), etc.). Similar reactions have also been inferred for asparagus, carrots, spurge, cottonseed, wheat, barley, etc. (VEVaskovsky, J. Chromatogr.,
261, 324 (1983), etc.). However, phospholipase D in rice bran is not known to cause a phosphatidyl group transfer reaction, and the existence of phospholipase D derived from soybean, rapeseed, sunflower, and sesame is unknown.
Or, even if known, the transfer reaction has not been clarified. An object of the present invention is to provide a method for efficiently proceeding with a phosphatidyl group rearrangement reaction of phospholipids, and to carry out the above reaction using phospholipase D, which has not been known to cause such a rearrangement reaction. There is a particular thing. By using the reaction method of the present invention, phospholipids such as phosphatidylcholine and phosphatidylethanolamine, which were conventionally purified from living organisms through complicated extraction and concentration steps, can be purified at around room temperature. It is possible to efficiently produce high purity and high quality under mild reaction conditions near neutral pressure and neutrality, and it is also possible to obtain new alcohol derivatives of phospholipids. It can be produced more easily and economically than chemical synthesis methods. (d) Means for Solving the Problems As a result of intensive research, the present inventors have discovered that the above object can be achieved when the above reaction is carried out in the presence of a specific water content. The present invention has been completed based on this knowledge, and consists of combining phospholipids and alcohols having a primary hydroxyl group in the presence of phospholipase D originating from rice and soybeans, and by adjusting the total weight of phospholipids and alcohols. This is a phosphatidyl group rearrangement method characterized in that the reaction is carried out in the presence of an amount or less of water. The phospholipid used in the present invention is, for example, one represented by the following formula () and/or (). [Formula] [Formula] However, in the formulas () and (),
R ₁ and R ₂ may be the same or different and are H or a saturated or unsaturated fatty acid residue having 8 to 24 carbon atoms, and A is -H or -(CH ₂ ) ₂₊ N (CH ₃ ) ₃ , −(CH ₂ ) ₊ NH ₃ , −CH ₂ CH(NH ₂ )COOH, −CH ₂ CH ₂ N(CH ₃ ) ₂ , −CH ₂ CH ₂ NH(CH ₃ ), -
Either _CH2 -CH(OH) _-CH2 (OH) or a mixed group. Examples of the phospholipids include lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidyl-N-methylethanolamine, phosphatidyl-N,N-dimethylethanolamine, phosphatidylserine, phosphatidic acid, and phosphatidylserol. These fatty acids include caproic acid, caprylic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α- and γ-linolenic acid, and behenic acid. , erucic acid, eicosapentaenoic acid, docosahexanoic acid, arachidonic acid, and tetracosatetraenoic acid, and may be in the form of a free hydroxyl group that is not substituted with these fatty acids. Among these fatty acids, fatty acids having physiological activities such as so-called thrombolysis and platelet aggregation, such as eicosapentaenoic acid, γ-linolenic acid, and arachidonic acid, are particularly important. Furthermore, these base moieties (A in formulas () and ()) and fatty acid moieties may each be used singly or in combination. These phospholipids may be extracted and concentrated from natural products, or may be synthetic products. Next, the alcohols used in the present invention have a primary hydroxyl group, such as one or two of a hydroxyl group, an amino group, a mono-, di- or trialkylamino group, a carboxyl group, an acetyl group, or a halogen group. An alcohol having at least one linear or side-chain primary hydroxyl group, substituted or unsubstituted with one or more substituents, which may be used alone or in combination in the present invention. I can't hold back in any way. Examples of the alcohols include methanol, ethanol, 1-propanol, 1-butanol, 1-octanol, 1-nonanol, 1-decanol,
1-dodecanol, 1-hexadecanol, 2-
Ethylhexanol, 1,2-propanediol, 1,3-propanediol, 2-ethyl-
1,3-hexanediol, ethylene glycol, diethylene glycol, diethylene glycol monobutyl ether, propylene glycol,
Dipropylene glycol, glycerin, diglycerin, monostearin, phosphatidylglycerol, 1-amine-2-propanol and hydrochloride, choline and choline chloride, ethanolamine and ethanolamine hydrochloride, serine, serine ethyl ester, N-methyl ethanolamine,
Examples include N,N-dimethylethanolamine, but the present invention is not limited thereto. Further, these alcohols can be used either as natural products or as synthetic products. In the present invention, the above phospholipid and alcohol are reacted in the presence of phospholipase D.
The present inventors isolated phospholipase D from plants in which the existence of phospholipase D was not known at all, or in which the phosphatidyl group transfer effect was not clear even though the existence of phospholipase D was known. The effect was confirmed, and such phospholipase D can be used in the present invention. Examples of such plants include rice and soybeans, and their roots, stems, leaves, and other plant organisms, seeds, and residues obtained by physically treating them or treating them with chemicals such as organic solvents (for example, by treating the seeds with chemicals such as organic solvents) (residue from pulverization and degreasing with hexane)
The phospholipase D can be collected from, for example. To collect the phospholipase D, plants or their residues can be extracted with water, and the aqueous solution can be subjected to conventional enzyme isolation and purification methods.
For example, salting out with salt, ammonium sulfate, etc., ethanol,
Treatment methods such as precipitation with an organic solvent such as acetone, dialysis, ion exchange or adsorption chromatography, gel filtration, and adsorption can be used. The enzyme thus obtained can be made into a liquid or solid form by adding stabilizers such as carbohydrates, proteins, and various salts as necessary, and by vacuum concentration, drying, freeze-drying, and other treatments. Furthermore, in the present invention, the phospholipase D can be used in the form of an immobilized product in a reaction system using the phospholipase D. In general, enzymes are sometimes immobilized for the purpose of improving pH and stability, maintaining activity, reusing, etc. Immobilization carriers include cellulose, dextran, polystyrene, polyacrylamide, polyvinyl alcohol, ion exchange resin, magnetic silica, activated carbon, alumina, photocrosslinkable resins, alginates, various gelling agents, etc. are used. In the present invention, the phospholipase D is adsorbed onto these immobilization carriers, ionic bonding,
It can be used by covalently bonding or entrapping it to form immobilized phospholipase D in the form of particles, films, or sheets. In the present invention, when phospholipids and alcohols are reacted using the above-mentioned phoslipase D, the amount of water is set to be equal to or less than the weight of the reaction substrate, that is, the phospholipid and alcohol, preferably, It is characterized by limiting the amount of water to an amount equal to or less than the weight of the lipid, which makes it possible to efficiently proceed with the phosphatidyl group transfer reaction. If a water content exceeding the above-mentioned content coexists in the reaction system, the reaction product will contain a hydrolyzate or the hydrolyzate will be the main component, and the highly pure phospholipid or its derivative targeted by the present invention will not be produced. I can't get it. According to the method of the present invention, phospholipids and alcohols are reacted as follows. That is,
A composition of one or more types of phospholipids is placed in a suitable glass or stainless steel container, and alcohols or alcohols and a non-aqueous solvent in a molar equivalent to about 100 times the molar amount of the phospholipid used are added. Dissolve in a mixed solvent of As a non-aqueous solvent,
n-heptane, n, which is a so-called inert organic solvent
Examples include hexane, benzene, xylene, acetone, dimethyl ether, diethyl ether, and ethyl acetate. Next, additives known as activators of phospholipase D, such as sodium dodecyl sulfate, cholic acid, deoxycholic acid or salts thereof, calcium chloride, anionic surfactants, etc., and buffers for maintaining the stability of the enzyme are added. For example, acetic acid, phosphoric acid, citric acid,
Addition of hydrochloric acid, etc. as an aqueous solution, and further anti-phospholipid
Add 0.1-10% by weight of enzyme in powdered form, granules or as an aqueous solution. Water required for the reaction system is usually supplied as water in an aqueous solution of the above-mentioned activator, buffer, and the like.
Therefore, these water contents are the amounts specified in the present invention,
That is, it is necessary to calculate in advance so that the amount is equal to or less than the total weight of phospholipid and alcohol as reaction substrates. Next, the contents are uniformly emulsified by shaking or using a suitable stirrer such as a homomixer, and the pH is adjusted to 4.
~8 Preferably PH5~7.5, 20~50℃ Preferably
The reaction is carried out at 35-45°C for about 1 hour to 24 hours. In addition, when this reaction is carried out using a column method filled with immobilized phospholipase D, the immobilized lipase is packed in a glass or stainless steel cylindrical tube, and the reaction liquid emulsified with the above-mentioned composition is pumped through a liquid circulation pump or the like. continuously from one side of the column.
Alternatively, the reaction may be carried out by flowing it under slight pressure. The reaction process in which the desired high-purity phospholipid or phospholipid derivative is produced by the phosphatidyl group transfer reaction is, for example, TLC (thin layer chromatography),
Analytical methods such as HPLC (high-performance liquid chromatography) can be used to monitor the progress of the reaction, which also allows the reaction time to be controlled. After the reaction is complete, heat the enzyme to 70 to 80°C for a short time or add an enzyme activity inhibitor, such as an aqueous EDAT solution, to lose the enzyme activity. The desired product can be obtained by performing purification treatments such as adsorption column chromatography, solvent fractionation, and precipitation. (e) Examples Example 1 Four-neck flask with stirrer and cooling tube (500
ml), add 10 g of dimyristoylphosphatidylcholine (manufactured by Sigma, USA), and add 50 g of diethyl ether.
Dissolved in ml. Furthermore, 5 g of N-methylethanolamine hydrochloride was added, followed by 5 ml of 0.5 M phosphate buffer (PH7.0) containing 5 mM calcium chloride. On the other hand, 5 times the volume of purified water was added to the rice germ, homogenized at 4°C, and centrifuged at 10,000 x g for 20 minutes. To the supernatant, 1.5 times the volume of acetone was added while cooling at 4°C, and phospholipase was added as a precipitate. I got a D.
1 g of this was made into 2 ml of an aqueous solution, and the above mixture was added dropwise with stirring to form an emulsified state. The reaction was allowed to proceed for 10 hours with stirring at 40°C, and a portion of the reaction product was extracted using chloroform/methanol/acetic acid as a developing solvent.
TCL (thin layer chromatography) analysis revealed that the spots of dimyristoylphosphatidylcholine had disappeared and that dimyristoylphosphatidyl-N-methylethanolamine had been produced, and that the corresponding phosphatidyl acid had disappeared. I confirmed that it was not generated. Note that the diethyl ether layer of this reaction product was collected, washed with water, and then the solvent was distilled off. When TLC analysis was performed under the same conditions as above, substantially pure dimyristoylhofsuphatidyl-N-metalethanolamine with a purity of 98% or more was obtained. For comparison, the reaction was carried out in the same manner as above except that 40ml of 0.5M phosphate buffer (PH7.0) containing 5mM calcium chloride was used, and the reaction product was analyzed, and the main component was dimyristoyl, a hydrolyzate. phosphatidic acid (85%), the raw material dimyristoylphosphatidylcholine (8%), and the transition product (target product) dimyristoylphosphatidyl-N-methylethanolamine.
It was only %. Example 2 Concentrated phosphatidylcholine PC-70 (manufactured by Nisshin Oil Co., Ltd.; phospholipid composition: 70% phosphatidylcholine, 10% phosphatidylethanolamine, lysophosphate) in a glass Erlenmeyer flask (1). Acidylcholine 2%; fatty acid composition: palmitic acid 16%, stearic acid 5%, oleic acid 9%, linoleic acid 65%
%, linolenic acid 5%) was dissolved in 300 ml of n-hexane/diethyl ether (=1:1),
100 ml of 0.3M Tris-HCl buffer (PH7.5) containing 60g of ethanolamine hydrochloride, 5mM calcium chloride and 4mM sodium dodecyl sulfate was added. On the other hand, phospholipase was collected and purified from raw rice bran according to the method described in the literature (Takahashi et al., Tokyo University of Agriculture Agricultural Bulletin, 28 (No. 3), 262 (1984)).
3.5 g of D was added as a powder to the above mixture while stirring to obtain an emulsified state, and the mixture was reacted at 37° C. for 5 hours with gentle shaking. The solvent layer of the reaction product was collected, washed several times with water, and then the solvent was distilled off. When TLC analysis was performed in the same manner as in Example 1, phosphatidylcholine and phosphatidic acid were not detected, and the main component was phosphatidylcholine. It was atidylethanolamine. As a result, it was confirmed that mixed phospholipids could be converted into high-purity phospholipids with a single composition. Example 3 Egg yolk lecithin (phospholipid composition: 70% phosphatidylcholine, 5% lysophosphatidylcholine, 15% phosphatidylethanolamine, 3% lysophosphatidylethanolamine) was prepared under the same conditions as in Example 2. , phosphatidylinositol 1%, sphingomyelin 2%; fatty acid composition: palmitic acid 20%, stearic acid 5%, oleic acid 14%, linoleic acid 55%, linolenic acid 6%) and choline were reacted and analyzed. As a result, no ethanolamine-bound phospholipids were detected in the raw material phospholipids. Example 4 The filamentous fungus Cunninghamallaelegans (NRRL 1378) was cultured, and phospholipids containing γ-linolenic acid (C ₁₈ : ₃ ω6) as a main component were obtained by solvent extraction from the cells.
The phospholipid composition is phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, and sphingomyelin, and the constituent fatty acid composition is the above-mentioned γ-linolenic acid. The main ingredients were linoleic acid, oleic acid, stearic acid, and palmitic acid. To 10 g of such phospholipids, 5 g of choline chloride, 50 ml of diethyl ether, 5 mM calcium chloride and 1%
5 ml of 0.5 M Tris-HCl buffer (PH7.0) containing Triton X-100, 10 g of granular activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.)
5 g of the immobilized product in which 4 g of rice bran-derived phospholipase D obtained in Example 2 was adsorbed in an aqueous solution was added,
The mixture was emulsified using a homomixer while cooling. This mixture was placed in an Erlenmeyer flask, continued to be gently shaken, and reacted at 40°C for 15 hours. According to the TLC analysis results of the reactant, no ethanolamine-bound phospholipid was detected in the raw material. Example 5 50 g of 1,2-dilinoleoylphosphatidylcholine (synthetic product, purity 98%), 20 g of diethylene glycol monobutyl ether (reagent, manufactured by Wako Pure Chemical Industries, Ltd.)
g, 0.4M acetate buffer containing 10mM calcium chloride and 20mM sodium deoxycholate (PH6.0)
In the same manner as in Example 2, using 50 g of ammonium sulfate fractionated from soybean seeds and 2 g of dialyzed phospholipase D.
The reaction was carried out for 10 hours. When the composition of the reactant was checked by TLC analysis, free choline, unreacted raw materials, and a new esterified product resulting from the transfer reaction between the phospholipid and alcohol were detected. For comparison, the above contains 10mM calcium chloride and 20mM sodium deoxycholate.
The reaction was carried out in the same manner except that 500 g of 0.4M acetate buffer (PH6.0) was used, and the reaction product was analyzed and found that the main components were 1,2-dilinoleoylphosphatidic acid and unreacted raw materials. No object (object) was detected. (f) Effects of the Invention The present invention combines natural or synthetic phospholipids and alcohols having a primary hydroxyl group in the presence of phospholipase D derived from rice or soybean under specific reaction conditions, that is, under the condition of moisture in the reaction system. This is a method of efficiently producing a phosphatidyl group transfer reaction by regulating the amount of the base moiety.This method allows the base moiety to easily make a specific component of a mixture of phospholipids highly pure. The economic benefits of this are significant, as the amount of material that used to go through complicated processes such as concentration has been significantly reduced. In addition, compared to chemical synthesis methods, the thermal history is small and the reaction is simple, so side reactions can be almost completely suppressed, making it possible to stably and economically obtain the desired product with excellent quality. can.
Further, according to the method of the present invention, novel phospholipid derivatives can also be produced with the same advantages as described above. Furthermore, it has been shown that by using the method of the present invention, it is possible to perform a phosphatidyl group transfer reaction on alcohols with a long carbon number, which was not possible with conventional phospholipase D such as cabbage. Furthermore, in the present invention, by using phospholipase D as an immobilized product, the target product can be continuously produced in a bioreactor using a column method, a membrane method, or other methods. The products, high-purity phospholipids and new phospholipids, are used in many industrial fields including food, pharmaceuticals, cosmetics, and agricultural chemicals, as well as in the pharmaceutical field, as well as surfactants, liposome bases, therapeutic agents, emulsifiers, and insecticides. It is expected to have useful uses, such as for the purpose of carrying or protecting physiologically active substances, and for easily converting physiologically active polyunsaturated fatty acids into forms that are easy to test for pharmacological activity.

Claims (1)

[Claims] 1. Phospholipids and alcohols having a primary hydroxyl group are mixed in the presence of phospholipase D originating from rice or soybeans in an amount equal to or less than the total weight of the phospholipids and the alcohols. A phosphatidyl group rearrangement method characterized by carrying out the reaction in the presence of water. 2. The method according to claim 1, wherein the phospholipid is represented by the following general formula () and/or (). [Formula] [Formula] (However, R ₁ and R ₂ are the same or different and are H or a saturated or unsaturated fatty acid residue having 8 to 24 carbon atoms, and A is -H or -( CH ₂ ) ₂₊ N (CH ₃ ) ₃ , −(CH ₂ ) ₂ N ₊ H ₃ , −CH ₂ CH(NH ₂ )COOH, −CH ₂ CH ₂ N(CH ₃ ) ₂ , −CH ₂ CH ₂ NH( _CH3 ), _-CH2
-CH(OH) _-CH2 (OH) or a mixed group. 3. Claim 2, wherein either or both of R ₁ and R ₂ in the general formulas () and () is a fatty acid residue of eicosapentaenoic acid, γ-linolenic acid, or arachidonic acid. Method. 4 Alcohols have hydroxyl groups, amino groups, mono-
A linear or side chain primary hydroxyl group that is substituted or unsubstituted with one or more substituents of a di- or trialkylamino group, a carboxyl group, an acetyl group, or a halogen group. The method according to claim 1, wherein the alcohol has at least one alcohol. 5. The method according to claim 1, wherein phospholipase D is an immobilized product. 6. The method according to claim 5, which uses a column packed with immobilized phospholipase D.