JPS6336791A - Production of phospholipid by enzyme - Google Patents

Abstract

PURPOSE:To suppress formation of by-products, by bringing a raw material phospholipid and a hydroxyl group-containing receptor into contact with phospholipase D adsorbed on a carrier to carry out reaction and minimizing water content in the reaction system. CONSTITUTION:A raw material phospholipid such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, etc., and a hydroxyl group- containing receptor such as serine, methanol, ethanol, etc., are reacted with phospholipase D adsorbed on a carrier in the presence of an organic solvent. Or a carrier having adsorbed the raw material phospholipid is blended with the receptor and the carrier having adsorbed phospholipase D and reacted. The water content in the reaction system is <=1%. Active carbon, activated clay, silicic acid, silica gel, diatomaceous earth, zeolite, alumina, etc., may be used as the carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing phospholipids using an enzyme, and particularly to a method for producing phospholipids whose base structure has been converted.

(Prior Art) In recent years, phospholipids have attracted attention not only for their use as emulsifiers but also for their application as liposome base materials, drug carriers, artificial blood 1, artificial cells, etc. Phospholipids themselves have physiological activities and As substances with pharmacological effects, various uses in the medical, pharmaceutical, and engineering fields are being considered. In order to meet such diverse demands, it is of great industrial significance to develop a method for efficiently producing phospholipids having structures suitable for each use.

As a method for producing phospholipids using enzymes, a technique is known in which phospholipase D is applied to phospholipids in the presence of an arbitrary receptor, and a phospholipid having a target base is produced using a phosphatidyl group transfer reaction. (S, F, Yan
g, et al., J. Biol. Chem.

242, (3) 477-484 (1967))
: CR, M, C, Dawson.

Biochem, J., 102.205-210 (
1967) ).

When attempting to exchange the base moiety of a phospholipid using a phosphatidyl group transfer reaction by phospholipase D, a two-phase system of an aqueous phase and an organic solvent phase is generally used. namely, enzymes, receptors, pH buffers, which are primarily water soluble;
This is a reaction system in which an aqueous solution containing inorganic salts and the like and an organic solvent phase containing mainly lipophilic raw material phospholipids are stirred and mixed. Including the technique 4i mentioned above, subsequent research CK, Br
uzik andM, Tsai, Biochemis
try 23. (8) ], 656466H198
4) etc.] is also widely used.

(Problems to be Solved by the Invention) However, in such a conventionally used reaction system, the presence of a large amount of water as a solvent for water-soluble components essentially causes phospholipase D to have no hydrolytic activity. Because of this, hydrolysis occurs as a side reaction, producing phosphatidic acid (hereinafter abbreviated as PA).

The generation of PA through hydrolysis not only makes it difficult to separate and purify the target phospholipid after the reaction, but also because the raw material phospholipid is consumed by the hydrolysis reaction, it is difficult to separate and purify the target phospholipid after the reaction. When attempting to transfer a phosphatidyl group to 7 to a receptor, it was virtually impossible to obtain the desired product because the reaction rate was extremely low compared to the hydrolysis reaction rate.

Such problems are unavoidable as long as water is present, since phospholipase D itself is originally a hydrolytic enzyme.

In order to solve this problem by reducing the water content in the reaction system to the limit without deactivating the enzyme, the inventors of the present invention have conducted various studies and found that conventional methods of adding water to the reaction system in the form of an aqueous solution. The inventors have discovered a new reaction system in which components are adsorbed or supported on a carrier and added, leading to the present invention.

(Means for Solving the Problems) The present invention is characterized in that a raw material phospholipid and a receptor having a hydroxyl group are brought into contact with phospho-1jbase D adsorbed on a carrier to carry out a reaction.

The raw material phospholipid used in the present invention may be any phospholipid extracted from nature, purified after extraction, or synthesized, as long as it can serve as a substrate for phospholipase D. Moreover, commercially available products or products prepared by known methods may be used.

Examples include defatted soybean lecithin, egg yolk lecithin, phosphatidylcholine (hereinafter abbreviated as PC), phosphatidylethanolamine (hereinafter abbreviated as PE), phosphatidylserine (hereinafter abbreviated as PS), phosphatidylglycerol (hereinafter abbreviated as PG), etc., or mixtures thereof. can be given. In order to maximize the effects of the present invention, it is more convenient in terms of purification of the reaction product to use purified phospholipids or pure 9i of 1) as the raw material phospholipid.

In addition, from the viewpoint of raw material cost, ease of labor, and reactivity to enzymes, PC, PP, or PS are particularly preferred because they are highly effective industrially.

The reaction is preferably carried out in the presence of an organic solvent that dissolves or clouds the raw material phospholipid, and any solvent system that does not deactivate the enzyme can be used. Examples include petroleum ether, diethyl ether, methyl ethyl ether, chloroform, dichloromethane, carbon tetrachloride, dichloroethane, n-hexane, cyclohexane, n-octane, isooctane,
l1) Solvents such as ethyl acetate, dioxane, benzene, etc., or combinations thereof, or mixed solvent systems in which these are combined with polar solvents such as acetone and acetonitrile. However, since alcohols serve as substrates for the desired reaction, it is not very preferable to use them for purposes other than adding them as substrates.

As phospholipase D, any phospholipase D can be used, as long as it has phosphatidyl group transfer activity, regardless of whether it is commercially available or prepared by a known method. For example, Boehringer Manheim
nheim Gmb! cabbage-derived phospholipase D (PLDP) manufactured by Toyo Jozo Co., Ltd.; known methods [--for example, Kates and P, S.

``Methods in E
zymology” (J, M.

Lowenstein+ ed.), vol. I
4. pp197-203゜Academic Pre
For example, enzyme preparations extracted and purified or partially purified by the method (Ss., New York (1969)), or extracted crude enzymes can be mentioned.

Receptors include not only compounds conventionally known as receptors for phosphatidyl group transfer reactions such as choline, methanol, ethanol, ethanolamine, serine, glycerol, and glucose, but also ni-amino-2-propanol and 1-orthomethylglucoside. It is also possible to use compounds having a primary or secondary alcohol structure containing sugars, such as trehalose, which were conventionally considered not to be receptors for phosphatidyl group transfer reactions.

Examples of carrier materials that adsorb or support phospholipase D include activated carbon, activated clay, silicic acid, silica gel, diatomaceous earth, zeolite, alumina, porous glass, and ceramic "J'".
Examples include j=, Ln, resin, etc., and the shape is preferably granular or bead-like with a particle size of about 0.02 to 0.5 mm. These carriers can also adsorb or support raw material phospholipids and receptors.

Examples of methods for adsorbing or supporting phospholipase D and receptors on a carrier include contacting an aqueous solution of the components with the carrier, lyophilizing excess water after filtration, or deactivating the enzyme using diethyl ether or chloroform. There are methods for removing the components by repeatedly or continuously bringing them into contact with dry organic solvents that do not cause oxidation, and methods for further mixing by adding a small amount of water to a mixture of the powdered component and the carrier.

A method for bringing into contact a carrier on which enzymes, receptors, and other components have been adsorbed or supported with an R-containing solvent in which a raw material phospholipid is dissolved or suspended is to disperse and suspend the carrier in a solvent system in a container. Methods include stirring, or filling a column with the carrier and circulating the solvent system.

Examples of methods for adsorbing or supporting raw material phospholipids on a carrier include methods in which raw material phospholipids dissolved in a low polar organic solvent are brought into contact with the carrier and adsorbed, and then the solvent is removed by filtration, or methods in which raw material phospholipids are dissolved There is a method in which a carrier is impregnated with an organic solvent and then the solvent is distilled off.

By suppressing the water content of the entire reaction system to preferably 1% or less, the hydrolysis reaction can be suppressed compared to conventional reaction systems, but it is further preferably suppressed to 0.5% or less. In particular, by controlling the water content to 0.2% or less, receptors that could not react in conventional reaction systems can be reacted, and the effects of the present invention are maximized.

The reaction temperature may be any optimum temperature for the enzyme used, and is usually 3.
It ranges from 0 to 40'C. However, this does not apply when the solvent used has a low boiling point.

The reaction time is 0.5 to 36 hours, preferably 4 to 24 hours.

The target phospholipid having any base produced in this manner can be easily purified by appropriately using known means such as solvent fractionation, silicic acid or silica gel chromatography, alumina chromatography, DIEAE-cellulose chromatography, etc. Be able to do that.

(Effects of the Invention) The present invention uses an essentially non-aqueous reaction system in order to reduce the water content in the reaction system to the limit without deactivating the enzyme, so it is different from that seen in conventional reaction systems. The production of a large amount of PA was suppressed by trn, and the separation and purification of the target phospholipid after the reaction was facilitated, resulting in an improved yield.

Furthermore, it has become possible to produce many types of target phospholipids that could not be obtained using conventional reaction systems.

(Example) Hereinafter, the present invention will be specifically described based on Reference Examples, Examples, and Comparative Examples.

In addition, the compositional analysis and purity test of phospholipids were performed by thin layer chromatography (TLC). T t, c +Ii(
20-100 μg of lipid sample in Merck m5721)
Spotted to a diameter of 3 to 5 mm, chloroform-methanol-water (120 Nia 0:5) or chloroform-acetone-acetic acid-methanol-water (50:20:15:1)
0:5). For detection, Jittmer reagent, 50%
Sulfuric acid, ninhydrin reagent or Anthrone reagent were used depending on the purpose. For quantitative measurement, the color developed with the Jittmer reagent was measured using a high-speed thin layer chromatography scanner (Model C5-920, manufactured by Shimabara Seisakusho).

In addition, unless otherwise specified, the reaction products were identified by comparing Rf values with standard products on TLC and by color reaction with various reagents.

Reference Example 1 Spinach phospholipase D was prepared by an example of the above-mentioned known method, Kates and P.
.

Extracted according to the method of S., 5astry).

Fresh spinach (Barreto species) obtained from a nearby farmer was washed with water and shredded, and 20 g of water (1 ml) was added to 100 g.
Homogenization was performed for 5 minutes under water cooling. The filtrate filtered through 5 layers of gauze was centrifuged at 2,000×g for 15 minutes at 4° C. to obtain 210 ml of supernatant. Transfer this supernatant to 1 liter of water at 4°C.
Dialyzed 3 times against Il, 15,000 x g at 4°C.
195 ml of the supernatant obtained by centrifugation for 1 minute was used as a spinach crude enzyme solution. It was confirmed that this crude enzyme solution did not contain a detectable amount of phospholipid.

Reference example 2 Soybean PC and PE were prepared by Paldan (Von H.

Pardun's method (Fette 5etfen A)
nstrichmitte average, (2) 55-62
(1984)).

Commercially available defatted soybean lecithin powder (PC24%, PE21%,
Phosphatidylinositol 14%, PA 8%) 20g
isopropanol-methanol-water (50:45:5
) It was dispersed in 100mff1 and dissolved by heating and stirring at 40°C. The mixture was cooled to 20°C with stirring and kept at 20°C for 1 hour. The insoluble matter was centrifuged or filtered under reduced pressure using a glass filter while maintaining the temperature at 20°C. The collected supernatant was dried under reduced pressure, and PC and PF were concentrated (PC68%, P
(E17%, PA7%, PS not included) 9.7g was obtained.

Reference example 3 PC was purified from egg yolk lecithin by a conventional method.

Commercially available egg yolk lecithin (PC67%, PE19%, PA8%
, Lyso PC 3%) was transferred to a silica gel column (diameter 3.8
cm x 60 cm). Chloroform-methanol (5:1) as elution solvent 1.57! ,
Pour 3p of chloroform-methanol (3:1) and
Both parts containing a large amount of C were collected and dried under reduced pressure to form PC (PC97).
%) 4.3 g was obtained.

Reference example 4 Purified egg yolk PC was supported on porous glass.

500mg of purified egg yolk PC obtained in Reference Example 3 was added to 100mf
was dissolved in n-hexane and impregnated into 5 g of porous glass crushed to a diameter of 0.5 mm or less, and then the n-hexane was dried under reduced pressure to be supported.

Reference example 5 M, Lees method (“Metho”) from cow brain
ds in Enzymology” (S, P, Colo
wick and N, 0. Kaplan ed,)+
vo1.3+ pp328. Academic
Press, Neh York (1957)) and purified by DEAE-cellulose column chromatography.

300 g of fresh bovine brain membranes and blood vessels removed obtained from a local slaughterhouse were homogenized in 1.2β acetone and extracted. 1.2β of the filtration residue again
Extract with acetone. The filter residue is 1.2 I! Extract with ethanol. The filtration residue was similarly extracted twice with 1.21 petroleum ether, the extracts were collected and dried under reduced pressure,
3.9 g of crude cephalin fraction was obtained.

D was dissolved in chloroform and prepared in acetic acid form.
EAE-cellulose column (Whatman DE32,
2.5 cm x 20 cm). After washing the column with 16 chloroform-methanol (1:4), 750 m of acetic acid! Both eluted fractions were collected. An equal volume of chloroform was added to the @ acid elution fraction, and the mixture was washed four times with 2 volumes of water. The chloroform layer was dried under reduced pressure, and PS (PS98%
) 0.8 g was obtained.

Reference example 6 Commercially available diatomaceous earth was purified by washing with water and solvent.

1) 100 g of h503 Celite (manufactured by Johns Manville Sails, trade name) was suspended in 21 water, washed, and at the same time fine particles were removed by decantation. In the same manner, it was further washed twice with water, once with methanol, and once with chloroform, and finally, the suspension in 500 ml of acetone was filtered under reduced pressure. After air drying, dry at 120℃ for 5 hours,
74 g of purified Celite was obtained.

Example I L-serine 50 g 1 cabbage phospholipase D (manufactured by Boehringer Mannheim) 0.5 g, k7jx
Calcium chloride dihydrate 0.25g to 500ml 5
The mixture was dissolved in mM acetate buffer pH 5.6, 40 g of special grade activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred at room temperature for 30 minutes.

After filtration, the mixture was freeze-dried for 8 hours to obtain 53.7 g of activated carbon adsorbed with enzymes, receptors, and other substances.

500 g of soybean pc and PE concentrate obtained in Reference Example 2
53.5 g of activated carbon, which had been dissolved in ml of diethyl ether and adsorbed with enzymes, receptors, etc., was added and dispersed. The reaction mixture was kept at 37° C. in a closed container and stirred at 500 rpm for 17 hours.

The reaction solvent was collected by filtration, and the activated carbon was collected at 200 mff1.
Washed three times with chloroform. The reaction solvent and washing liquid were combined and dried under reduced pressure to form a phospholipid mixture (PC8%, PE8%,
6.9 g of PA9%, P372%) was obtained.

Chloroform this stuff? A DEAE-cellulose column (Whatman DE32, diameter 3.8 x 2
7 cm). After washing the column with chloroform-methanol (1:4N, !M), PS (PS
99%) was recovered.

Example 2 A mixed solution of 25 ml of IM ethanolamine aqueous solution adjusted to pi (5.0 with hydrochloric acid) and 25 ml of the spinach crude enzyme solution prepared in Reference Example 1 was added with purified Celite obtained in Reference Example 6.
5 g was added and stirred at room temperature for 30 minutes.・After filtration, add yL to a small amount of diisopropyl ether! , 'IiJ' column (diameter I x 3.5 cm). After absorbing and removing excess water by passing 2.51 g of dry diisopropyl ether, commercially available egg yolk lecithin (67% PC, 19% PE) was added.
, PA 8%, Lyso PC 3%) was dissolved in 20 ml of diisopropyl ether at a flow rate of 0.3 using a metering pump.
It was circulated and reacted at ~0.5 d/min at 30° C. for 6 hours.

The reaction solvent was collected and the column was washed three times with 10 ml of chloroform. The reaction solvent and washing liquid were combined and dried under reduced pressure to obtain 2.7 g of a phospholipid mixture (PC1%, PE85%, PAIO%, LysoPC2%).

Dissolve this in chloroform and use a silica gel column (
2.5 x 40 cm). Chloroform-
Methanol-water (65:25:2) 21? Discharge,
Fractions containing PE were collected and dried under reduced pressure to obtain PE (PE97%
) 1.6g was obtained.

Example 3 333mM 1-amino-2 adjusted to pH 5.5 with hydrochloric acid
- Cabbage phospholipase D in propanol aqueous solution
(manufactured by Boehringer Mannheim) and 0.5 mg of calcium chloride dihydrate were dissolved, 200 mg of purified Celite obtained in Reference Example 6 was added, and the mixture was shaken well for 15 minutes. The precipitate obtained by centrifugation at 12,000 x g for 15 minutes was freeze-dried for 6 hours to obtain 278 mg of Celite adsorbed with enzymes, receptors, and the like.

Porous glass 60 with purified egg yolk PC obtained in Reference Example 4 supported on 10 ml of n-hexane-acetone (95:5)
0 mg, and 276 mg of Serai to which enzymes, receptors, etc. were adsorbed were suspended and dispersed. The mixture was stirred at 500 rpm for 24 hours at 37°C to react.

The reaction solvent was collected by filtration and the carrier was washed three times with 5 ml of chloroform. The reaction solvent and washing liquid were combined and dried under reduced pressure to obtain 42 mg of a phospholipid mixture.

This substance contains 4% PA, but no PC was detected, and the remaining 94% was positive for both Shiso I-Mar reagent and ninhydrin reagent, and the Rf value on TLC was that of standard P.
Since it was very similar to E, it was determined that the target phospholipid was 1-amino-2-propanol with a phosphatidyl group.

Example 4 100 mg of freeze-dried powder of the spinach crude enzyme solution obtained in Reference Example 1 and 300 mg of activated carbon were thoroughly mixed, and 60 mg of glycerol (water content 3%) was added and mixed well.

This material was dispersed in 15 ml of chloroform in which 50 mg of bovine brain PS obtained in Reference Example 5 was dissolved, and the mixture was heated at 35°C for 5 hours.
The mixture was stirred at 00 rpm to react.

The reaction solvent was collected by filtration and the activated carbon was washed three times with 5 ml of chloroform. The reaction solvent and washing liquid were combined and dried under reduced pressure to obtain a phospholipid mixture (PS28%, PA3%,
PC67%) 44mg obtained.

Example 5 600 mg of 1-orthomethyl glucoside, 1 g of phospholipase D (Toyo Jozo Co., Ltd. (Horizona PLDP) 12/, 5 m
Dissolved in 1 liter of 5mM acetate buffer pH 5,6 and 1 liter of activated carbon.
g in the same manner as in Example 3, and freeze-dried.

Dipalmitoyl PC45mg? Disperse 950 mg of activated carbon adsorbed with enzymes, receptors, etc. in 15 mf of chloroform-isooctane (1:1), and heat at 38°C for 1
500 rpm T: Stirred for 2 hours to react.

48 mg of a phospholipid mixture was obtained in the same manner as in Example 4.

This was mixed with chloroform-methanol-water (12
0nia 0:5) was used as a developing solvent to obtain 29 mg of unidentified phospholipid.

This phospholipid was analyzed using a JMS-DX303 mass spectrometer (manufactured by JEOL ES) under the following conditions.
The parent peak on the cation side was m/e 847, and the parent peak on the anion side was m/e 823.

Measurement conditions Ionization method CFAB method Impact gas: Xe Partial ion acceleration voltage: 5kV Filament current: 20mA Detector: Conversion dynode extrusion voltage: 15 to V Matrix Nitriethanolamine (sodium chloride added for cations) Data processing: JM 8-DA5000 These values are based on the assumption that a phosphatidyl group has been introduced into 1-orthomethylglucoside, respectively, and the sodium salt (molecular weight 824 + 23) and anion (molecular weight
fi 824-1), it was identified as the target phospholipid.

Example 6 1 g of levalose I and 10 t!g of phospholipase D (Toyo Jozo Co., Ltd. (Kikai PLDP)) were dissolved in 5 ml of water, dried commercially available beaded styrene-divinylbenzene resin was added, and the mixture was stirred at 4°C for 5 hours. and impregnated.

Dipalmitoyl PC was lyophilized for 18 hours after filtration.
It was dispersed in 2Qml of dry dichloromethane in which 45mg was dissolved, and reacted in a rotary trowel at 4Orpm at 37°C for 24 hours.

The reaction solvent was collected by filtration and the resin was washed three times with 2Qml of dichloromethane. The reaction solvent and washing liquid were combined and dried under reduced pressure to obtain 43 mg of a phospholipid mixture.

This one contained 38% PC and 5% PA, but 54%
Since the phospholipid occupying No. 94 was positive to Jittmer's reagent and Anthrone's reagent, it was determined that it was the target phospholipid in which a phosphatidyl group was introduced into trehalose.

Furthermore, in the same manner as in Example 5, a portion was fractionated by TLC and subjected to mass spectrometry, and a cation side-view peak m/e 9
95, anion side viewing peak m/e 971, computational molecule! ! Each sodium salt of (972) (molecular weight 972+
23), it was identified as the target phospholipid because it matched with the anion (molecular weight 972-1).

Comparative Example 1 A reaction was carried out using a conventional reaction system under conditions as close as possible to those of Example 1.

L-saison 200mM, calcium chloride 40mM
, Phospholipase D (manufactured by Boehringer Mannheim)
50mM acetate buffer pH 5,6 containing 2.5mg
rrl.

and 40 m of the soybean pc and PE concentrate obtained in Reference Example 2.
5 ml of diethyl ether dissolved in
Stirred at 500 rpm.

Five identical samples were reacted in parallel for 1, 2, 4, 8, and 16 hours each. After the reaction, add 5 ml of diethyl ethyl
The lipids were extracted and the phospholipid N composition was analyzed. The results are shown in Table 1.

Table 1 Composition (%) Comparative Example 2 A reaction was carried out using a conventional reaction system under conditions as close as possible to those of Example 2.

1M ethanolamine aqueous solution adjusted to pH 5.0 with hydrochloric acid 0.35mf and spinach crude enzyme solution obtained in Reference Example 1 0.
．． 375mA! , 4.215 ml of water, 30% of diisopropyl ether 5- dissolved in 45 mg of commercially available egg yolk lecithin.
The mixture was stirred at 500 rpm.

Four identical samples were reacted in parallel for 1, 2, and 4.6 hours each, and analyzed in the same manner as in Comparative Example 1.

The results are shown in Table 2.

Table 2 Composition (%) Comparative Example 3 A reaction was carried out using a conventional reaction system under conditions as close as possible to those of Example 5.

1-ortho-methylglucoside 600mg, phosphor,
Lipase D (Toyo Jozo (4 Kiku!!! PLDP) 12μ
5 mR of 50mM acetic acid aM fJi solution at pH 5,6 in which 100 g of chloroform-isooctane (1:1) in which 45 mg of dipalmitoyl PC was dissolved was mixed at 38°C.
, and stirred at 500 rpm to react.

Analysis was carried out over time in the same manner as in Comparative Examples 1 and 2, but the substrate 7), which corresponds to the target phospholipid observed in Example 5, was not observed on TLC, and the substrate was completely removed after 8 hours of reaction. Analysis was discontinued due to decomposition.

Claims (6)

[Claims] (1) In producing a phospholipid with a converted base structure, an enzymatic phospholipid is used, which is characterized in that the reaction is carried out by bringing the raw material phospholipid and a receptor having a hydroxyl group into contact with phospholipase D adsorbed on a carrier. Method for producing lipids. (2) The manufacturing method according to claim 1, wherein the reaction is carried out in the presence of an organic solvent. (3) The production method according to claim 1, wherein a carrier adsorbed with a raw material phospholipid and a carrier adsorbed with a receptor and phospholipase D are mixed and reacted. (4) The carrier is activated carbon, activated clay, silicic acid, silica gel,
The manufacturing method according to claim 1, wherein the material is diatomaceous earth, zeolite, alumina, porous glass, earthenware, porcelain, or resin. (5) Claim 1, wherein the receptor is any one of serine, ethanolamine, 1-amino-2-propanol, 1-orthomethyl-glucoside, and trehalose;
The manufacturing method according to item 2, 3 or 4. (6) The manufacturing method according to any one of claims 1 to 5, wherein the reaction is carried out in a state where the water content in the reaction system is 1% or less.