JPH0716426B2 - Method for producing phospholipid by enzyme - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a phospholipid by an enzyme, and particularly to a method for producing a phospholipid having a converted base structure.

(Prior art) Phospholipids are not only used as emulsifiers, but in recent years, their application to drug carriers, artificial blood, artificial cells, etc. as a base material for liposomes has been attracting attention, and their own physiological activity
Various applications in the fields of medicine, pharmacy, and engineering are considered as those having a pharmacological action. In order to meet such various demands, it is industrially significant to develop a method for efficiently producing a phospholipid having a structure suitable for each application.

As a method for producing a phospholipid by an enzyme, a technique is known in which phospholipase D is allowed to act on a phospholipid in the presence of an arbitrary receptor and a phospholipid having a desired base is produced by utilizing a phosphatidyl group transfer reaction. (SF Yang, et a
L., J. Biol. Chem., 242, (3) 477-484 (1967)]:
[RMC Dawson, Biochem. J., 102, 205-210 (1967)].

When the phosphatidyl group transfer reaction by phospholipase D is used to exchange the base portion of phospholipid, a two-phase system of an aqueous phase and an organic solvent phase is generally used. That is, it is a reaction system in which an aqueous solution mainly containing a water-soluble enzyme, a receptor, a pH buffer solution, an inorganic salt and the like and an organic solvent phase mainly containing a lipophilic raw material phospholipid are stirred and mixed. Starting with the above-mentioned technology, subsequent research [K. Bruzik and
M. Tsai, Biochemistry 23 , (8) 1656-1661 (1984)].

(Problems to be solved by the invention) However, such a reaction system that has been conventionally used is caused by the presence of a large amount of water as a solvent of a water-soluble component, and phospholipase D has essentially no hydrolytic activity. Therefore, it has a drawback that hydrolysis occurs as a side reaction to form phosphatidic acid (hereinafter abbreviated as PA).

The production of PA by hydrolysis not only makes it difficult to separate and purify the target phospholipid after the reaction, but also consumes the raw material phospholipid by the hydrolysis reaction, so that the reactivity of sugars and secondary alcohols is low. When trying to transfer the phosphatidyl group to the acceptor, it was virtually impossible to obtain the desired product because the reaction rate was extremely low relative to the hydrolysis reaction rate.

Such a problem is unavoidable in the presence of water as long as phospholipase D itself is a hydrolase.

Therefore, the inventors of the present invention have conducted various investigations in order to solve this problem by reducing the water content in the reaction system to the limit within the range where the enzyme is not inactivated. The present invention was accomplished by devising a new reaction system in which the components added to the above were encapsulated in reverse micelles (w / o type microemulsion) and added.

In the present specification, the reverse micelle refers to a state in which an amphipathic molecule is associated in a low-polarity solvent with a lipophilic group facing outward and a hydrophilic group facing inward with a very small amount of an aqueous phase as a center.

(Means for Solving Problems) In the present invention, the reaction is performed by adding an aqueous phase containing a receptor having a hydroxyl group and phospholipase D in a reverse micelle in an organic solvent in which a raw material phospholipid is dissolved. It is characterized by performing.

As the raw material phospholipid used in the present invention, any material that can be a substrate for phospholipase D can be used regardless of whether it is extracted from nature, purified after extraction, or synthesized. In addition, a commercially available product or a product prepared by a known method 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 PE).
Examples thereof include PS) and phosphatidylglycerol (hereinafter abbreviated as PG), etc., or a mixture thereof. In order to maximize the effect of the present invention, it is more convenient in terms of purification of the reaction product to use purified phospholipid as a raw material or one having a simple composition. Further, PC, PE or PS is particularly preferable because it is industrially effective from the viewpoints of raw material cost, availability, and reactivity with enzymes.

The reverse micelle is preferably formed by a surfactant.

When most of the raw material phospholipid is PC, PE, PG or PS having two acyl groups having a carbon chain length of 12 or more, or a mixture thereof, the raw material phospholipid itself is a surfactant for forming reverse micelles. Acts as. However, the raw phospholipid has PC, PE, P having two acyl groups with carbon chain length of 10 or less.
When it is difficult to form reverse micelles with the raw phospholipid itself as in the case of S, PG or lyso PC, lyso PE, lyso PS, lyso PG, a surfactant that satisfies the following conditions may be added separately. preferable.

(A) It can be dissolved in the reaction solvent at a concentration equal to or higher than the critical micelle concentration.

(B) Reverse micelles can be formed.

(C) It does not give a decisive loss in enzyme activity.

(D) It is desirable to have no alcohol residue.

Examples include sodium lauryl sulfate, sodium di (2-ethylhexyl) sulfosuccinate, sodium di-n-octylsulfosuccinate, polyoxyethylene nonylphenyl ether, and the like. The amount of these surfactants added is 0.5 to 5 mol per mol of the raw material phospholipid,
It is preferably 1 to 3 mol.

Any reaction solvent can be used as long as it satisfies the following conditions.

(A) The raw material phospholipid can be dissolved at a concentration equal to or higher than the critical micelle concentration.

(B) Almost no water is mixed or dissolved.

(C) It does not give a decisive loss in enzyme activity.

That is, it may have a double bond or an ether bond in the molecule, may have a functional group such as an alkyl group, and has a linear or branched chain or cyclic group having 5 to 10 carbon atoms. Hydrocarbon compounds having 1 to 2 carbon atoms or a mixture thereof, and examples thereof include n-hexane, isooctane, diethyl ether, diisopropyl ether, cyclohexane, benzene, xylene, chloroform, carbon tetrachloride and dichloroethane. And mixed systems of two or more of these. A compound having an alcohol structure serves as an acceptor for a phosphatidyl group transfer reaction, and therefore it is not preferable to add it except for using it as a substrate.

As the phospholipase D, any commercially available one or one prepared by a known method can be used as long as it has a phosphatidyl group transfer activity. As an example, cabbage-derived phospholipase D manufactured by Boehringer Mannheim GmbH, microbial-derived phospholipase D (PLDP) manufactured by Toyo Brewing Co., Ltd., a known method [as an example, Kates and Sustrii PSSa
stry), "Methods in Enzymology" (JMLowens
tein, ed.), vol.14, pp197−203, Achademic Press, New Y
ork (1969)] and purified or partially purified enzyme preparation, or extracted crude enzyme.

As the receptor, not only compounds such as choline, methanol, ethanol, ethanolamine, serine, glycerol and glucose which are conventionally known as receptors for phosphatidyl group transfer reaction, but also 1-amino-2-propanol and 1-ortho Compounds having a primary or secondary alcohol structure, including saccharides such as methyl glucoside and trehalose, which are conventionally not considered as an acceptor of the phosphatidyl group transfer reaction, can also be used.

To enclose enzymes, receptors, etc. in reverse micelles, a solution of enzymes, receptors, etc. is dropped into an organic solvent in which the raw material phospholipid is dissolved at a critical micelle concentration or higher, and immediately shaken or sonicated. A known method such as, for example, can be used.

At the same time as forming reverse micelles, in order to suppress the by-product of PA at the time of phosphatidyl group transfer reaction, the water content in the reaction system is 6 to 6 mol with respect to 1 mol of the raw material phospholipid or the surfactant.
It should be 15 moles, preferably 8-12 moles for best results.

The reaction temperature may be the optimum temperature of the enzyme to be used, usually 30
It is in the range of ~ 40 ° C. Preferably, the stability of the reverse micelle is improved by keeping it constant from the formation of the reverse micelle to the end of the enzymatic reaction. 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 thus produced is easily purified by appropriately using known means such as solvent fractionation, silicic acid or silica gel chromatography, alumina chromatography, and DEAE-cellulose chromatography. be able to.

Further, since the production of PA is suppressed, the unreacted substrate can be easily recovered.

(Effect of the Invention) By using the reaction system of the present invention, the production of PA as seen in the conventional reaction system is suppressed, and separation and purification of the target phospholipid after the reaction is facilitated.

Further, it has become possible to produce a target phospholipid that could hardly or hardly be obtained by the conventional reaction system.

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

The composition analysis and purity test of phospholipids were performed by thin layer chromatography (TLC). TLC plate (Merck No.572
A lipid sample (20 to 100 μg) was spotted on 1 to 3 to 5 mm in diameter, and chloroform-methanol-water (120: 70: 5) or chloroform-acetone-acetic acid-methanol-water (50: 2) was added.
It was deployed at 0: 15: 10: 5). Detecter Reagent, 50 for detection
% Sulfuric acid, ninhydrin reagent or anthrone reagent was used depending on the purpose. For quantitative measurement, the color developed with the Zitmer reagent was measured with a high-speed thin-layer chromatography scanner (CS-920 manufactured by Shimadzu Corporation).

Reference Example 1 Soybean PC and PE were paldan (Von H. Pardun) method [Fe
tte Seifen Anstrichmitte 86 , (2) 55-62 (1984)]
Were separated and fractionated by.

20 g of commercially available defatted soybean lecithin powder (PC24%, PE21%, phosphatidylinositol 14%, PA8%) was dispersed in 100 ml of isopropanol-methanol-water (50: 45: 5) and dissolved by heating with stirring at 40 ° C. Cool to 20 ℃ with stirring and
Hold at ℃ for 1 hour. The insoluble matter was centrifuged or vacuum-filtered with a glass filter while keeping the insoluble matter at 20 ° C. The collected supernatant was dried under reduced pressure to obtain PC and PE concentrates (PC68%, PE17%,
PA7%, PS not included) 9.7 g was obtained.

Reference Example 2 Beef brain to M. Lees ["Methods in Enzym
ology "(SPColowick and NOKaplan ed.), vol.3, pp
328, Achademic Press, New York (1957)], and crude cephalin was extracted and purified by DEAE-cellulose column chromatography.

300 g of fresh bovine brain debris and blood vessels from a local slaughterhouse were homogenized in 1.2 acetone and extracted. The filter residue is extracted once again with 1.2 acetone. The filter residue is extracted with 1.2 ethanol. The filtration residue was extracted twice with 1.2 petroleum ether in the same manner, and the extracts were collected and dried under reduced pressure to give a crude sephaline fraction.
3.9 g was obtained.

This product was dissolved in chloroform to prepare DE
AE-Cellulose column (Whatman DE32, diameter 2.5 cm
X 20 cm) was used for fractionation. After washing the column with chloroform-methanol (1: 4) 1, fractions eluted with 750 ml of acetic acid were collected. An equal volume of chloroform was added to the fraction eluted with acetic acid, and the fraction was washed 4 times with 2 volumes of water. The chloroform layer was dried under reduced pressure to obtain 0.8 g of PS (PS98%).

Reference Example 3 Commercially available sodium dioctylsulfosuccinate was purified.

50% methanol solution of sodium dioctyl sulfosuccinate (Nippon Yushi Co., Ltd., trade name Lapizole B-90)
100 ml was left standing at 4 ° C. for 3 hours and then centrifuged at 4 ° C. at 2,000 × g for 15 minutes to remove the precipitate.

5 g of special grade activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the supernatant and gently stirred for 18 hours. Activated carbon was filtered off and methanol was distilled off.

The dried product was dried in a vacuum desiccator containing phosphorus pentoxide for one day or more and used as purified sodium dioctylsulfosuccinate.

Reference Example 4 M.Kates and PSSastry
From cabbage soluble fraction according to the method of
Was partially purified.

The inner leaf of fresh cabbage obtained from a nearby farm was washed with water, then shredded, 200 ml of water was added to 100 g, and the mixture was homogenized for 5 minutes under ice cooling. The filtrate obtained by filtering with 5 layers of gauze is 4
Centrifugation at 12,000 xg for 30 minutes at 190C gave 190 ml of supernatant. This supernatant was heat-treated at 55 ° C. for 5 minutes and immediately cooled with ice. Centrifuge at 12,000 xg for 30 minutes at 4 ℃, and supernatant 175ml
Got

Acetone (350 ml) cooled to −15 ° C. was added little by little with stirring, and the mixture was allowed to stand for 10 minutes and then centrifuged at 4 ° C. at 12,000 × g for 30 minutes to collect a precipitate.

The precipitate was dissolved in 15 ml of 50 mM acetate buffer pH 5.6 and dialyzed 3 times against 500 ml of the same buffer at 4 ° C.

The insoluble matter was centrifuged at 10,000 xg for 15 minutes at 4 ° C and used as partially purified cabbage phospholipase D.

It was confirmed that this enzyme solution did not contain a detectable amount of phospholipid.

Example 1 L-serine 1.5M, cabbage phospholipase D (Boehringer Mannheim) 7 mg / ml, and 10 mM calcium chloride dihydrate were dissolved in 0.5 ml of 5 mM acetate buffer pH 5.6 to obtain an aqueous phase. 1.5 g of the soybean PC and PE concentrate obtained in Reference Example 1 was dissolved in 40 ml of dried diisopropyl ether to obtain an organic phase.

Keep the organic phase at 35 ℃, NS100-2U type ultrasonic cleaner (output 1
00W, manufactured by Nippon Seiki Seisakusho, Ltd.), water phase 0.3 while sonicating
5 ml was added dropwise and sonication was further performed for 1 minute to obtain an almost transparent reverse micelle solution.

This was reacted by shaking back and forth at 35 ° C. for 12 hours at 15 rpm.

When a part of the reaction mixture was taken and directly analyzed by TLC, PC4
8%, PE14%, PA11%, the standard PS and Rf match, and it is positive for the gitmer reagent and ninhydrin reagent.
It contained 24% of the product identified as PS.

The reaction mixture was directly fractionated using a DEAE-cellulose column (DE32 manufactured by Whatman, diameter 2.5 × 20 cm) prepared in the form of acetic acid.

The column was eluted with 500 ml of chloroform-methanol (1: 4) and dried under reduced pressure to recover unreacted substrate (PC73%, PE27%).

Further, the column was eluted with 400 ml of acetic acid. The acetic acid elution fractions were collected, an equal volume of chloroform was added, and the mixture was washed 4 times with 2 volumes of water. Chloroform layer was dried under reduced pressure, PS (PS98%) 294m
g was recovered.

Example 2 1-amino-2-propanol 1.5 adjusted to pH 5.7 with hydrochloric acid in the same manner as in Example 1 at 38 ° C. using 1 ml of isooctane in which 40 mg of commercially available hydrogenated egg yolk lecithin (PS99%) was dissolved as an organic phase. M, cabbage phospholipase D (Boehringer Mannheim) 7 mg / ml, calcium chloride dihydrate
A reverse micelle was formed by dropwise addition of 11 μ of an aqueous phase consisting of 10 mM.

This product was reacted by shaking back and forth at 38 ° C. for 8 hours at 15 rpm.

A part of the reaction mixture was taken and directly analyzed by TLC.
At 2% and 5% PA, the standard PE and Rf were close to each other and showed positive for the Jitmer reagent and ninhydrin reagent, so 1-amino-
The product contained 24% of the product judged to be a target phospholipid having a phosphatidyl group introduced into 2-propanol.

Example 3 Using 1 ml of cyclohexane in which 40 mg of bovine brain PS (PS98%) obtained in Reference Example 2 was dissolved as an organic phase, and at 38 ° C., glycerin 1.5M and cabbage phospholipase D (Boehringer Mannheim) 9 mg of aqueous phase consisting of 7 mg / ml, calcium chloride / dihydrate 10 mM, 5 mM acetate buffer pH 5.6
Was added dropwise to form reverse micelles.

This was reacted by shaking back and forth at 38 ° C. for 12 hours at 15 rpm.

A part of the reaction mixture was taken and directly analyzed by TLC.
The product contained 25% of the product identified as PG because the Rf was in agreement with the standard PG at 0% and PA3% and was positive for the Zitmer reagent.

Example 4 Using 2.5 ml of benzene in which 100 mg of commercially available dipalmitoyl PC was dissolved as an organic phase, 1-orthomethyl glucoside 1.5M and phospholipase D (PLDP manufactured by Toyo Brewery Co., Ltd.) 1 mg / ml at 35 ° C. in the same manner as in Example 1. Then, 20 μm of an aqueous phase consisting of 5 mM acetate buffer (pH 5.6) was added dropwise to form reverse micelles.

This was reacted by shaking back and forth at 35 ° C. for 12 hours at 15 rpm.

A part of the reaction mixture was taken and directly analyzed by TLC.
It contained 4% and 4% PA, and 19% unidentified phospholipids.

The reaction mixture was directly used for No.5745 preparative TLC plate (manufactured by Merck) and chloroform-methanol-water (120: 70:
Fractionation and fractionation using 5) as developing solvent, unidentified phospholipid 16m
got g.

When this phospholipid was analyzed under the following conditions using a JMS-DX303 mass spectrometer (manufactured by JEOL Ltd.), the parent peak on the cation side was m / e847 and the parent peak on the anion side was m / e847.
It was e823.

Measurement conditions Ionization method: FAB method Impact gas: Xe Temporary ion acceleration voltage: 6kV Filament current: 20mA Detector: Onversion dynode Extrusion voltage: 15kV Matrix: Triethanolamine (sodium chloride added for cation) Data processing: JMA-DA5000 These values are sodium salt (molecular weight 824 + 23) and anion (molecular weight 8), which are assumed to have a phosphatidyl group introduced into 1-orthomethylglucoside.
Since it coincided with 24-1), it was identified as a target phospholipid.

Example 5 Chloroform in which 100 mg of commercially available dipalmitoyl PC was dissolved
2.5 ml of isooctane (35:65) was used as an organic phase, and trehalose 1.5M, phospholipase D (PLDP manufactured by Toyo Brewery Co., Ltd.) 1 mg / ml, 5 mM acetate buffer p at 35 ° C in the same manner as in Example 1.
A reverse micelle was formed by dropping 17 μ of an aqueous phase containing H5.6.

This was reacted by shaking back and forth at 35 ° C. for 12 hours at 15 rpm.

A part of the reaction mixture was taken and directly analyzed by TLC.
Since 6% and 5% PA simultaneously showed positive results for the gitmer reagent and the anthrone reagent, 17% of the phospholipids judged to be the target phospholipid having a phosphatidyl group introduced into trehalose were included.

16 mg of the phospholipid obtained by fractionating the reaction mixture in the same manner as in Example 4 was subjected to mass spectrometry in the same manner as in Example 4, and the cation side parent peak m / e995 and the anion side parent peak were obtained. m /
At e971, it was identified as the target phospholipid because it coincided with the calculated sodium (972) sodium salt (molecular weight 972 + 23) and anion (molecular weight 972-1).

Example 6 3 mg of n-hexane in which 100 mg of commercially available dioctyl PC and 90 mg of purified dioctyl sulfosuccinate sodium sodium obtained in Reference Example 3 were dissolved as an organic phase, and the pH was adjusted to 6.0 with hydrochloric acid at 37 ° C. in the same manner as in Example 1. 1M in 1.5M ethanolamine
A solution of phospholipase D (/ ml PLDP manufactured by Toyo Shuzo Co., Ltd.) in an amount of 36 μm was added dropwise as an aqueous phase to form reverse micelles.

This was shaken back and forth at 37 ° C. for 10 hours at 15 rpm to react.

A part of the reaction mixture was taken and directly analyzed by TLC.
It contained 22% of phospholipids judged to be the target phospholipids, since they had Rf values close to those of standard PE at 1% and PA 7%, and showed positive positivity for jidmer reagent and ninhydrin reagent at the same time.

Example 7 5 ml of dichloromethane-isooctane (3: 7) in which 200 mg of commercially available 1-stearoyl lyso PC and 100 mg of sodium lauryl sulfate (manufactured by Wako Pure Chemical Industries, Ltd., for biochemistry) were dissolved were used as an organic phase, and Example 1 was used at 35 ° C. In the same manner as above, 65 μ of a solution of 1.5 M glycerin and 10 mM of calcium chloride / dihydrate in the cabbage partially purified enzyme solution obtained in Reference Example 4 was dropped as an aqueous phase to form reverse micelles.

This was reacted by shaking back and forth at 35 ° C. for 15 hours at 15 rpm.

The reaction mixture was directly fractionated using a DEAE-cellulose column (DE52 manufactured by Whatman, diameter 1.8 × 5 cm) prepared in the form of acetic acid. After washing the column with 100 ml of chloroform-methanol (1: 4), chloroform containing 50 mM ammonium acetate-
Fractions eluted with 100 ml of methanol (1: 4) were collected. An equal volume of chloroform was added to the fraction eluted with acetic acid, and the fraction was washed 4 times with 2 volumes of water.

26.7 mg of phospholipid obtained by drying the chloroform layer under reduced pressure
Was subjected to mass spectrometry in the same manner as in Example 4 to find that the cation side parent peak m / e534 and the anion side parent peak m / e511
Calculated molecular weight (512) each sodium salt (molecular weight 512 + 2
3), since it coincided with the anion (molecular weight 512-1),
It was identified as the desired 1-stearoyl lyso PG.

Comparative Example 1 The reaction was carried out in the conventional reaction system under the conditions as close as possible to Example 1.

PH containing L-serine 200 mM, calcium chloride 40 mM, and phospholipase D (Boehringer Mannheim) 2.5 mg
5 ml of diisopropyl ether in which 2.5 ml of 50 mM acetate buffer of 5.6 and 40 mg of the soybean PC and PE concentrate obtained in Reference Example 1 were dissolved
l was stirred at 37 ° C. and 500 rpm.

Five identical samples were reacted in parallel for 1, 2, 4, and 12 hours, respectively. After the reaction, the lipid was extracted three times with 5 ml of diethyl ether, and the phospholipid composition was analyzed. The results are shown in the table below.

Comparative Example 2 The reaction was carried out in the conventional reaction system under the conditions as close as possible to Example 4.

1-Orthomethyl glucoside 1.5M, phospholipase D
(PLDP made by Toyo Brewery Co., Ltd.) 50 μm with 20 μg dissolved in pH 5.6
2.5 ml of M acetate buffer and 2.5 ml of benzene in which 100 mg of dipalmitoyl PC was dissolved were stirred at 35 ° C. and 500 rpm to cause a reaction.

When analyzed in the same manner as in Comparative Example 1 over time, no spot corresponding to the target phospholipid found in Example 4 was found on TLC, and the substrate was completely decomposed at the time of reaction for 16 hours. The analysis was terminated.

Comparative Example 3 The reaction was carried out in the conventional reaction system under the conditions as close as possible to Example 5.

Trehalose 1.5M, phospholipase D (Toyo Brewing Co., Ltd.)
2.5 ml of 50 mM acetate buffer of pH 5.6 containing 20 μg of PLDP
And chloroform containing 100 mg of dipalmitoyl PC dissolved in
2.5 ml of isooctane (35:65) was stirred at 35 ° C and 500 rpm,
It was made to react.

When analyzed over time in the same manner as in Comparative Example 1, no spot corresponding to the target phospholipid found in Example 5 was found on TLC, and the substrate was completely decomposed at the time of reaction for 16 hours, The analysis was terminated.

Claims (3)

[Claims] 1. A method for producing a phospholipid having a converted base structure, wherein an aqueous phase containing a receptor having a hydroxyl group and phospholipase D is encapsulated in a reverse micelle in an organic solvent in which a raw material phospholipid is dissolved. A method for producing a phospholipid by an enzyme, which comprises adding and performing a reaction. 2. The production method according to claim 1, wherein the reverse micelles are formed from a surfactant, and the raw material phospholipid itself is used as the surfactant. 3. Receptor is serine, ethanolamine, 1-
The production method according to claim 1, which is any one of amino-2-propanol, 1-orthomethyl-glucoside, glycerol and trehalose.