JPH027633B2 - - Google Patents

Description

Detailed Description of the Invention The present invention relates to a method for producing phospholipid primary alcohol derivatives by an enzymatic method, and relates to a method for producing phospholipid primary alcohol derivatives using an enzymatic method. This invention relates to a method for producing phospholipid primary alcohol derivatives using an enzymatic method that enables the production of alcohol derivatives. In more detail, phospholipase D derived from cabbage (optimum temperature 40℃ or less, optimum pH 5.4 ~
Unlike 5.6), the optimum temperature is 60 to 70℃, and the optimum pH is
This invention relates to a method for producing a phospholipid primary alcohol derivative, which involves reacting a phospholipid with a primary alcohol in the presence of phospholipase DM. In the present invention, a phospholipid primary alcohol derivative refers to an ester bond between the phosphoric acid structure of a phospholipid as a starting material and the (base or) alcohol structure of the phospholipid,
It refers to a new phospholipid that is different from the starting material and is derived by being hydrolyzed by the action of DM and simultaneously transferred to the primary alcohol used in the above reaction. In particular, the present invention provides the following formula () However, in the formula, A represents the following (i) or (ii) [Formula] [Formula], where both R ₁ and R ₂ are -O-COR ₁₁
or both are -O-R ₁₂ , or in formula (i), R ₁ and R ₂ together represent [formula] [where n represents a number from 11 to 19] In the above, R ₁₁ and R ₁₂ may be the same or different and each represents a C ₇ to C ₂₁ saturated or unsaturated aliphatic hydrocarbon group, and B is -(CH ₂ ) ₂ N( _CH3 ) ₃ ,− _{( CH2} ) _2NH2 ,−
CH ₂ CH (NH ₂ )COOH, −CH ₂ CH ₂ NH (CH ₃ ), −
CH ₂ CH ₂ N(CH ₃ ) ₂ , -CH ₂ CHOHCH ₂ OH or -(CH ₂ ) _n H [Here, m represents a number from 1 to 5]
A phospholipid represented by the following formula () R-OH ... () where R represents a member selected from the group consisting of the following (1) to (3), (1) C ₆ (excluding hexanol) ~ _C26 saturated or unsaturated aliphatic or aromatic hydrocarbon residue, where the hydrocarbon residue is substituted with a substituent selected from halogen, amino, acetyl, and hydroxyl group or the above-mentioned hydrocarbon residue having a bond selected from the group consisting of ether, ester, and amide bonds in the molecule, (2) pregnane steroid compound residue, (3) galactono-γ-lactone , N(2-hydroxyethyl)phthalimide, 2-(3-indole)ethanol, 2-(2-hydroxyethyl)pyridine, pyridoxine, N(2-hydroxyethyl)morpholine, 5-hydroxymethylcytosine, cytidine, uridine, A heterocyclic compound residue selected from the group consisting of arabinocytidine, thiamine, 2-(2-hydroxyethyl)piperazine, adenosine, guanosine and cyclocytidine is reacted with a primary alcohol represented by: in the presence of phospholipase DM. The following formula () is characterized by However, in the formula, A and R have the same meanings as above,
This invention relates to a method for producing a phospholipid primary alcohol derivative represented by: It has been known that phospholipase D hydrolyzes the choline base-phosphate ester of phospholipids, such as phosphatidylcholine, and catalyzes the reaction that produces the free base and phosphatidic acid [M.Kates Can.J. Biochem.Physiol. 32 571
(1954)]. Furthermore, when a phospholipid, such as lecithin, is reacted with ethyl alcohol in the presence of phospholipase D, the ester bond between the phosphoric acid structure of the phospholipid and the alcohol structure of the phospholipid is hydrolyzed, and at the same time, phosphotidyl group transfer occurs. It has been reported that phosphatidylethanol is produced by the action [RM.Dawson;Biochem.J.,
102, 205 (1967)]: [SFYang; J.Biol.Chem.,
242, 477 (1967)]. Since the above-described phosphatidyl group transfer effect of phospholipase D was known, research in this field has progressed, and the proposal of British Patent No. 1581810 (corresponding to West German Publication No. 2717547) is known. According to this proposal, a phospholipid represented by the general formula of this proposal and a primary compound having a straight or branched alkyl group up to C ₅ which may be substituted with a hydroxyl group, halogen, amino or other substituent A primary alcohol transfer reaction using the enzymatic action of the cabbage-derived phospholipase D with alcohol is disclosed. and the reaction occurs only with primary alcohols containing no more than 5 carbon atoms,
If the alcohol contains more than 5 carbon atoms, the main product of the reaction is stated to be the corresponding phosphatidic acid. Furthermore, the proposal also states that there are no particular restrictions on the selection of the alcohol component as long as it is a primary alcohol that satisfies the above requirements. In addition, the inventors of the above proposal, S. Kovatchev and H. Eibl, et al., Adv. Exp. Med. Biol., Vol. 101,
221 (1978), regarding the primary alcohol transfer reaction using the enzymatic action of phospholipase D.
They reported that no rearrangement reaction was observed with _C7 - _C10 alkanols, but a 20% rearrangement reaction occurred with _C6 hexanol. on the other hand,
Rokhimov, MM, Uzb.Biol.Zh., Vol.3, 6
-9 (1979), alcohols with _C5 or higher, e.g.
They reported that no rearrangement reaction occurred with _C6 hexanol. The present inventors have discovered the existence of a microorganism that has a phospholipase D production ability that differs from the conventionally known Cavell-derived phospholipase D in terms of its optimum temperature, optimum pH, etc., and has already filed the patent application No. 56-161076. , Tokugansho
No. 56-163475 was proposed. Further research revealed that the enzyme produced by the phospholipase D-producing microorganism contains primary alcohols of _C6 or higher (excluding hexanol), and is conventionally thought to be incapable of forming phospholipid primary alcohol derivatives. and the surprising fact that it exhibits an enzymatic catalytic action that enables a transfer reaction with a primary alcohol selected from the group consisting of (1), (2), and (3) above, which has never been mentioned before. discovered. According to the research conducted by the present inventors, the present invention has newly developed phospholipase DM, which catalyzes the formation of phospholipid primary alcohol derivatives between phospholipids and, for example, geraniol, which is a _C10 primary alcohol.
There is an enzyme called phospholipase.
By reacting the phospholipid represented by the above formula () with the primary alcohol of the above formula () in the presence of DM, new derivatives including phospholipid primary alcohol derivatives, which were previously said to be impossible to produce, can be created. It was discovered that it could be manufactured. Thus, new phospholipid primary alcohol derivatives can be successfully obtained by enzymatic methods without the need for complicated and disadvantageous chemical synthesis methods, under mild and easy conditions, and without the risk of side reactions. It was found that it can be manufactured at a high rate. Therefore, an object of the present invention is to provide a new enzymatic method for producing phospholipid primary alcohol derivatives. The above objects and many other objects and advantages of the present invention will become more apparent from the following description. The raw material phospholipid used in the method of the present invention is represented by the following formula (). However, in the formula, A represents the following (i) or (ii) [Formula] [Formula], where both R ₁ and R ₂ are -O-COR ₁₁
or both are -O-R ₁₂ , or in formula (i), R ₁ and R ₂ together represent [formula] [where n represents a number from 11 to 19] In the above, R ₁₁ and R ₁₂ may be the same or different and each represents a C ₇ to C ₂₁ saturated or unsaturated aliphatic hydrocarbon group, and B is -(CH ₂ ) ₂ N( _CH3 ) ₃ ,− _{( CH2} ) _2NH2 ,−
CH ₂ CH (NH ₂ )COOH, −CH ₂ CH ₂ NH (CH ₃ ), −
CH ₂ CH ₂ N(CH ₃ ) ₂ , -CH ₂ CHOHCH ₂ OH or -(CH ₂ ) _n H [Here, m represents a number from 1 to 5]
shows. The raw material phospholipid of the formula () is a known compound and is available on the market, and can be extracted from natural products or synthesized by methods known per se. For example, lecithin, kephalin, phosphatidylserine, phosphatidyl-N-methylethanolamine, phosphatidyl-N,N-dimethylethanolamine, phosphatidylglycerol, phosphatidic acid alkyl ester obtained by extraction from animal and plant tissues by known means. They can be used alone or as a mixture, as they are, or after being purified. β-type phospholipids and alkyl ether-type phospholipids can also be used by chemically synthesizing part or all of their structures by methods known per se. can be used. In the method of the present invention, the primary alcohol reacted with the raw material phospholipid of the above formula () in the presence of phospholipase DM is represented by the following formula (). R-OH...() However, in the formula, R represents a member selected from the group consisting of the following (1) to (3), (1) C ₆ (excluding hexanol) to C ₂₆ saturated or an unsaturated aliphatic or aromatic hydrocarbon residue, where the hydrocarbon residue may be substituted with a substituent selected from halogen, amino, acetyl, and hydroxyl, or ether, ester, and the above hydrocarbon residue having a bond selected from the group consisting of an amide bond, (2) a pregnane steroid compound residue, (3) galactono-γ-lactone, N(2-hydroxyethyl)phthalimide, 2-(3) -indole) ethanol, 2-(2-hydroxyethyl)pyridine, pyridoxine, N(2-hydroxyethyl)morpholine, 5-hydroxymethylcytosine, cytidine, uridine, arabinocytidine, thiamine, 2-(2-hydroxyethyl) A heterocyclic compound residue selected from the group consisting of piperazine, adenosine, guanosine and cyclocytidine. Specific examples of such primary alcohols of formula () include the following primary alcohols. Examples of primary alcohols belonging to the group (1) above include aliphatic alcohols with no substituents such as 1-heptanol (C ₇ ), 1-octanol (C ₈ ), and 2-ethyl-1 -Hexanol ( _C8 ), 1-nonanol ( _C9 ), 1-decanol ( _C10 ), 1-undecanol (C11), 1-dodecanol ( _{C12 )} , 1-tetradecanol ( _C14 ), 1
-hexadecanol ( _C16 ), 1-octadecanol ( _C18 ), 1-docosanol ( _C20 ), 1-eicosanol ( _C22 ), 1-hexacosanol ( _C26 ),
Geraniol (C ₁₀ ), citronellol (C ₁₀ ), falnesol (C ₁₅ ), phytol (C ₂₀ ), etc.;
Polyhydric alcohols include 1,6-hexanediol (C ₆ ), 1,2,6-hexanetriol (C ₆ ), sorbitol (C ₆ ), mannitol (C ₆ ),
2-n-butyl-2-ethyl-1,3-propanediol (C ₉ ), 2-ethyl-1,3-hexanediol (C ₈ ), 1,1,1-tris(hydroxymethyl)propane (C ₆ ), 3,3-bis(hydroxymethyl)heptane ( _C9 ), 1,10-decanediol ( _C10 ), 1,12-dodecanediol ( _C12 ), trimethylolpropane, dipentaerythritol, galactitol etc.; Those with amino groups include 6-amino-1-hexanol (C ₆ ), triethanolamine, N-butyldiethanolamine, serine ethyl ester, dihydroxyethylglycine, sphingosine (C ₁₈ ),
N-methylpentanolamine, N-methylhexanolamine, N-ethylbutanolamine,
N-ethylpentanolamine, N-ethylhexanolamine, N-propylpropanolamine, N-propylbutanolamine, N-propylpentanolamine, N-propylhexanolamine, N-butylethanolamine, N-butylpropanolamine, N-butylbutanolamine, N-butylpentanolamine, N-butylhexanolamine, N-pentylethanolamine, N-hexylethanolamine, N,
N-dimethylbutanolamine, N,N-dimethylpentanolamine, N,N-dimethylhexanolamine, N,N-diethylethanolamine, N,N-diethylpropanolamine, N,
N-diethylbutanolamine, N,N-diethylpentanolamine, N,N-diethylhexanolamine, N,N-dipropylethanolamine, N,N-dipropylpropanolamine,
N,N-dipropylbutanolamine, N,N-
Dipropylpentanolamine, N,N-dipropylhexanolamine, N,N-dibutylpropanolamine, N,N-dibutylbutanolamine, N,N-dibutylpentanolamine,
N,N-dibutylhexanolamine, N,N-
Dipentylethanolamine, N,N-dihexylethanolamine, N,N,N-trimethylpropanolamine, N,N,N-trimethylbutanolamine, N,N,N-trimethylpentanolamine, N,N,N-trimethyl Hexanolamine, N,N,N-triethylethanolamine, N,N,N-triethylpropanolamine, N,N,N-triethylbutanolamine,
N,N,N-triethylpentanolamine,
N,N,N-triethylhexanolamine,
N,N,N-tripropylethanolamine,
N,N,N-tripropylpropanolamine,
N,N,N-tripropylbutanolamine,
N,N,N-tripropylpentanolamine,
N,N,N-tripropylhexanolamine,
N,N,N-tributylethanolamine, N,
N,N-tributylpropanolamine, N,
N,N-tributylbutanolamine, N,N,
N-tributylpentanolamine, N,N,N
-tributylhexanolamine, N,N,N-
Tripentylethanolamine, N,N,N-trihexylethanolamine, N,N-dibutylethanolamine, N-(3-aminopropyl)
Diethanolamine, etc.; Those with a carboxyl group include 16-hydroxyhexadecanoic acid (C ₁₆ ), gluconic acid, galactonic acid, etc.; Those with a halogen group include 6-chloro-1-hexanol (C ₆ ), etc., and ester bonds. Monolaurin (C ₁₅ ), monoolein (C ₂₁ ), 1,2-dilaurin (C ₂₇ ), 1,2-distearin (C ₃₉ ), 2-hydroxyethyl methacrylate (C ₆ ), ethylene glycol Monolaurate, diethylene glycol monolaurate, ethylene glycol monostearate, diethylene glycol monostearate, ethylene glycol monooleate, diethylene glycol monooleate, ethylene glycol monopalmitate, diethylene glycol monopalmitate, etc.; Pantothenyl alcohol as having an amide bond ( _C9 ), pantetheine ( _C11 ), tri-L-serine,
L-seryl-L-leucine, L-seryl-L-methionine, L-seryl-L-arginine, L-seryl-L-lysine, L-seryl-L-glutamine, N-capryloylethanolamine, N-cap loylethanolamine, N-lauroylethanolamine, N-myristoylethanolamine, N-palmitoylethanolamine, N-stearoylethanolamine, N-oleoylethanolamine, N-palmitooleoylethanolamine, N-linoloylethanolamine,
N-linoleylethanolamine, N-arachidonoylethanolamine, N-eicosanoylethanolamine, N-docosanoylethanolamine, lauric acid diethanolamide, stearic acid diethanolamide, oleic acid diethanolamide, palmitic acid diethanolamide, Oxyethylene lauric acid amide, tetraoxyethylene lauryl ether triethanolamine sulfate, triethanolamine lauryl sulfate, etc.; as those having an ether bond, triethylene glycol (C ₆ ), diethylene glycol monobutyl ether (C ₈ ), ethylene glycol monolauryl Ether, diethylene glycol monolauryl ether, ethylene glycol monocetyl ether, diethylene glycol monocetyl ether, ethylene glycol monostearyl ether, diethylene glycol monostearyl ether, ethylene glycol monooleyl ether, diethylene glycol monooleyl ether, ethylene glycol monobutyl ether, ethylene glycol mono(2) -diethylaminoethyl) ether, ethylene glycol monohexyl ether, ethylene glycol monotolyl ether, diethylene glycol monoethyl ether,
Diethylene glycol monohexyl ether, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, octaethylene glycol, decaethylene glycol, dodecaethylene glycol, diglycerol, triglycerol, tetraglycerol, pentaglycerol, hesysaglycerol, heptaglycerol, octaglycerol Examples include aromatic alcohols such as benzyl alcohol (C ₇ ),
β-phenethyl alcohol (C ₈ ), 3-phenyl-1-propanol (C ₉ ), cinnamic alcohol (C ₉ ) L-seryl-L-tyrosine, L-seryl-
L-phenylalanine, L-serine benzyl ester, L-serine β-naphthylamide, L-dinitrophenyl-L-serine, N-(1-dimethylaminonaphthalene-5-sulfonyl)-L-serine, ethylene glycol monobenzyl Ether, ethylene glycol monooctyl phenol ether, diethylene glycol monooctyl phenol ether, ethylene glycol monononyl phenol ether, diethylene glycol monononyl phenol ether, etc.; Those with substituents include p-chlorobenzyl alcohol (C ₇ ), p-aminophenol ether, etc. enethyl alcohol ( _C8 ),
N-ethyl-N-(2-hydroxyethyl)-m-
Toluidine (C ₁₁ ), β-hydroxyethylaniline (C ₈ ), N-(2-cyanoethyl)-N-(2-
hydroxyethyl)-aniline (C ₁₁ ), N-phenyldiethanolamine (C ₁₀ ), anis alcohol (C ₈ ), 1,4-di(2-hydroxyethoxy)benzene (C ₁₀ ), ethylene glycol monophenyl ether ( C ₈ ), Mehuenesin (C ₁₀ ), 2
-Hydroxyethylsalicylic acid (C ₉ ) ethylene glycol monochlorophenyl ether, etc.; polycyclic compounds such as O,O-bis(2-hydroxyethyl)tetrabromobisphenol (C ₁₉ ), 2-naphthalene ethanol (C ₁₂ ), etc. For example, among alicyclic alcohols, retinol (C ₂₀ ),
Examples include 1,4-dihydroxymethylcyclohexane ( _C8 ). Examples of primary alcohols belonging to the group (2) above include aldosterone, corticosterone, cortisone, dehydrocorticosterone, deoxycorticosterone, hydrocortisone, prednisolone, prednisone, tetrahydrocortisol, tetrahydrocortisone, triamcinolone, etc. can be exemplified. Furthermore, examples of primary alcohols belonging to the group (3) above include galactono-γ-lactone, N-(2
-hydroxyethyl)phthalimide, 2-(3-
indole) ethanol, 2-(2-hydroxyethyl)pyridine, pyridoxine, pyridoxal, pyridoxamine, N-(2-hydroxyethyl)morpholine, 5-hydroxymethylcytosine, cytidine, uridine, arabinocytidine, adenosine, guanosine, cyclocytidine , adenine deoxyriboside, cytosine deoxyriboside, guanine deoxyriboside, 5-hydroxymethyluracil, thymine deoxyriboside, uracil deoxyriboside, inosine, orotidine,
Examples include 2-(2-hydroxyethyl)piperazine, thiamine, and toxopyrimidine. The primary alcohol of formula () as exemplified above can be used as either a natural product or a synthetic product, but it is preferable to use it after it has been purified in advance using an appropriate known means so that it does not contain any alcohol other than the intended primary alcohol. . Examples of such purification means include distillation, recrystallization, column chromatography using alumina, silica gel, activated carbon, ion exchange resin, etc., thin layer chromatography, and appropriate combinations of these purification means. can. According to the method of the present invention, the formula () as exemplified above
A phospholipid and a primary alcohol of formula () as exemplified above are reacted in the presence of phospholipase DM. The phospholipase DM used at this time is:
It is distinguished from known phospholipase D by having an optimum temperature of 60 to 70°C and an optimum pH of around 7, compared to the conventional phospholipase D extracted from cabbage which has an optimum temperature of 40°C or less and an optimum pH of 5.4 to 5.6. An example of this is phospholipase DM produced by a phospholipase DM producing bacterium. The phospholipase DM can also be distinguished from the known phospholipase D in that it catalyzes the formation of a phospholipid primary alcohol derivative between geraniol, a C ₁₀ primary alcohol, and a phospholipid of formula (). As an example of such phospholipase DM producing bacteria, there is
Phospholipase DM belonging to the genus Nocardiopsis disclosed in No. 161076
For example, phospholipase DM producing bacteria belonging to the genus Actinomadura disclosed in Japanese Patent Application No. 163475/1983 filed by the same applicant, such as Norcadeiopsis strain NO779 [FERM-P No. 6133]. For example, Actinomadeura spp.
Examples include the NO362 strain [FERM-P No.6132]. Table 1 below shows the differences in enzymatic properties between the phospholipase DM used in the method of the present invention and the known phospholipase D, as well as the differences in optimum temperature and optimum pH, as well as some other differences. [Table] [Table] The reason why a phospholipid primary alcohol derivative that cannot be obtained using the known phospholipase D can be formed by the method of the present invention is that the known phospholipase D involved in this enzymatic catalytic reaction and the method of the present invention can be formed. It is presumed that the difference in enzymatic properties as described above from the phospholipase DM used is involved. Of course, the method of the present invention is not subject to any restrictions based on the assumption of such effects. The phospholipase DM producing bacterium Nocardiopsis sp. NO779 strain [FERM-P No. 6133] which produces phospholipase DM that can be used in the method of the present invention
and Actinomadeura sp. NO362 strain [FERM-
P No. 6132], the titer measurement method and physicochemical properties of phospholipase DM produced by them are described below. Nocardiopsis strain NO779 [FERM-P
No. 6133] mycological properties: - (a) Morphology Grows well on glucose asparagine agar, glycerin asparagine agar, yeast malt agar, etc., and grows moderately on starch inorganic salt medium, forming a colony of aerial mycelia. live. The color of the bacterial flora on which the spores have settled varies slightly depending on the type of culture medium and the time of observation, but it is generally white or grayish-white to light gray. On sucrose nitrate agar, nutrient agar, and oatmeal agar media, aerial mycelia do not grow,
Only poorly established. When this fungal strain grown on an agar medium is observed under a microscope, the aerial hyphae are 0.5 to 0.8 μ in size, linear, loosely waved or curved, and extend long with branches, and the total aerial hyphae number from several tens to 100. It is formed by a chain of all spores of more than 1,000 spores. The size of the spores is 0.5 to 0.8 x 0.7 x 1.0μ, almost short cylindrical and slightly irregular in size. The basal hyphae are 0.5 to 0.8μ wide and elongate with branches, and although they do not always divide on agar media, they are finely divided in most cases when cultured in liquid. However, zoospores, sporangia, sclerotia, etc. are not formed. (b) Properties on various media The experimental methods described below are mainly based on E.B. Shearing (Int. J. Syst. Bacteriol.
313-340, 1966). The hue was determined using the "Color Standard" (Japan Color Research Institute, 1964), and the hue symbol was written in parentheses in the order of hue name, saturation number, and brightness number. Cultivation is carried out at 25℃, and 2 to 3 are the most vigorously growing.
Table 2 shows the observation results on each medium during the week. However, in Table 2, the color of the surface of the basal hyphae listed in the growth items shows the observation results during the first week of culture before spores have settled, and it is difficult to judge the color of the surface of the basal hyphae in a medium where spores are attached quickly. is not stated. [Table] Soluble Pigment that produces melanin-like brown pigment Zuru [Table] Soluble Pigment that does not produce
(c) Physiological properties 1 Growth temperature: Grows around 5℃~30℃,
Grows best at 30℃. 2 Liquefaction of gelatin: No liquefaction (cultivated on glucose-peptone-gelatin medium at 25°C for 3 weeks). 3 Starch hydrolysis: Decompose (cultivate on starch agar medium at 25°C for 3 weeks). 4. Coagulation and peptonization of skim milk: Neither coagulation nor peptonization was performed (cultured at 30°C for 3 to 4 weeks). 5 Production of melanin-like pigment: Produced on peptone yeast iron agar, tyrosine agar (25℃, 2
~4 days). (d) Assimilation of carbon sources (culture at 30°C for 10-16 days) L-arabinose-sucrose-D-xylose-inositol-D-glucose + L-rhamnose-D-fructose-raffinose- (e) Chemical analysis of cells Diaminopimelic acid of this strain is meso type and does not contain hydroxydiaminopimelic acid. The sugar composition of the cell wall does not contain arabinose, xylose, madeulose, rhamnose, etc.
Contains galactose, mannose, etc. Moreover, this strain does not have nocardomicolic acid. Regarding the above analysis results, Bergey's Manual of
the Dterminative Bacteriology 8th edition, 657
pp. 658 (1974), and L'Essier Vallier (Inter.J.
System.Bacteriol.Volume 20, pp. 435-443, 1970
Mayer (Int.J.Syst.Bacteriol.26, 487)
According to the classification method of p.-493 (1976), this bacterium is of the cell wall type.
Type, cell wall sugar pattern C
Becomes a mold. As described above, this bacterium has a cell wall type and a sugar composition type of C.
Therefore, according to the classification method of Reschievarie, it belongs to the genus Actinomadeura, Thermoactinomyces, Actinobiifidus, or Geodermatophyllus, all of which are dasonbiley types. However, in the morphology of this fungus, all of the aerial hyphae consist of long chains of spores, dividing the basal hyphae into small pieces, but since no endospores, zoospores, or sporangia were found, it was found that the hyphae of this fungus are of the Dasonbirei type. Genus Actinomadura
It is appropriate for classification to identify it as (dassonvillei type). In addition, in recent years, the genus Actinomadeula of the dasonbelay type has been integrated into the new genus Nocardiopsis proposed by Mayer, and is generally treated under the name of the genus Nocaldeiopsis. Therefore, this bacterium is Nocardiopsis spp. NO779.
(Genus Nocardiopsis sp NO779). This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is FERM-P No. 6133. This bacterium was discovered on March 21, 1980, at No. 512,
(FERM BP-512) was transferred to the international deposit under the Budapest Treaty. The bacteria used to produce the phospholipase DM used in the present invention include not only the genus Nocardiopsis NO779 and a mutant strain obtained by mutating this strain, but also the genus Nocardiopsis (formerly known as Actinomadeula dason). All bacteria that belong to the genus Belay type and produce phospholipase DM can be used. Actinomadeura sp. NO362 strain [FERM-P
No. 6132] mycological properties: - (a) Morphology It grows well on starch inorganic salt agar, tyrosine agar, yeast/malt agar, oatmeal agar, etc., and grows moderately on glycerin-asparagine agar, with no formation of aerial mycelia. Epiphytes the settlement. The color of the bacterial flora that has attached spores varies slightly depending on the type of culture medium and the time of observation, but it is generally grayish-white to gray with a slight purplish tinge. On seuucrose nitrate agar, nutrient agar, and glucose asparagine agar, aerial mycelia do not attach or attach only poorly. When this strain grown on an agar medium is observed under a microscope, the aerial hyphae are branched with a width of 0.8 to 1.2 μm, with some loops or spirals, and elongate mainly in a straight line with some bends. The tip is often loosely wound into a loop. The spores settle in chains of several tens to more than 100, forming almost the entire aerial hyphae. The size of the spores is 0.8-1.2 x 1.2-1.7μ, short cylindrical or oval, and somewhat irregular in size and shape. The basal hyphae are 0.6 to 1.0 μm in width and elongate with irregular branches and bends, and zoospores, sporangia, sclerotia, etc. are not formed. In addition, septa and hyphal division are not usually observed, but hyphal division may be observed in liquid culture. (b) Properties of various media The experimental methods described below are mainly based on E.
Bee Shearing (Int.J.Syst.Bacteriol.16
Vol. 313-340, 1966). The hue was determined using the "Color Standard" (Japan Color Research Institute, 1964), and along with the hue name, the hue symbol was written in parentheses in the order of hue name, saturation number, and brightness number. Cultivation is carried out at 25℃, and 2 to 3 are the most vigorously growing.
Table 3 shows the observation results on each medium during the week. However, in Table 3, the color of the surface of the basal hyphae listed in the growth items shows the observation results during the first week of culture before spores attach, and it is difficult to judge the color of the surface of the basal hyphae in a medium where spores attach quickly. There is no mention of. [Table] Soluble Not produced pigments
[Table] Soluble Not produced pigments
(c) Physiological properties 1 Growth temperature: Grows around 10℃~37℃, 20~37℃
Grows best at 30℃. 2 Liquefaction of gelatin: No liquefaction (cultivated on glucose-peptone-gelatin medium at 25°C for 3 weeks). 3 Starch hydrolysis: Decompose (cultivate on starch agar medium at 25°C for 3 weeks). 4 Coagulation and peptonization of skim milk: No coagulation,
Peptonize (cultivate at 30°C for 3 to 4 weeks). 5 Production of melanin-like pigment: Produced on peptone yeast iron agar, tyrosine agar (25℃, 2
~4 days). (d) Assimilation of carbon sources (culture at 30°C for 10 to 16 days) L-arabinose + sucrose - D-xylose + inositol ± D-glucose + L-rhamnose - D-fructose - raffinose - (e) Chemical analysis of cells The diaminopimelic acid of this strain is meso type, and the sugar composition of the cell wall does not contain arabinose, xylose, rhamnose, etc., and has only madeulose,
Contains galactose, mannose, etc. Regarding the above analysis results, Bergey's Manual of
The Determinative Bacteriology, 8th edition, pp. 657-658 (1974), and L'Essier Varier (Inter.J.
System. Bacteriol. vol. 20, pp. 435-443, 1970)
When judged according to the classification method of , etc., this bacterium is of cell wall type and cell wall sugar pattern type B. Below, this bacterium has a cell wall type and a sugar composition type of B.
Therefore, it belongs to the genus Micropispora, Streptosporangium, Spirillospora, Branomonospora, Dermatophilus, and Actinomadeula. However, this fungus forms a spore chain consisting of many spores, and since no sclerotia, sporangia, or zoospores were found, it was found that the genus Actinomadeura (Genus
Taxonomically, it is most appropriate to identify it as ``Acinomadura''. Therefore, this bacterium is Actinomadera genus NO362.
(Actinomadura sp NO362). This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is FERM P-6132. This bacterium was transferred to the International Deposit under the Budapest Treaty on March 21, 1981 as FERM BP-511. The bacteria used to produce the phospholipase DM used in the present invention include not only the above-mentioned Actinomadeula genus NO362 and a mutant strain obtained by mutating this strain, but also strains belonging to the Actinomadeula genus that produce phospholipase DM. Any bacteria can be used. The phospholipase DM used in the method of the present invention is
In order to use the phospholipase DM producing bacteria as exemplified above, phospholipase DM as exemplified above is used.
The producing bacteria may be cultured in a medium, and phospholipase DM may be collected from the culture. Although both liquid culture and solid culture can be used as the culture form, it is advantageous industrially to perform deep aeration agitation culture. The culture sources used include carbon sources, nitrogen sources, inorganic salts, and other micronutrients commonly used for microbial culture, as well as phospholipases belonging to the genus Nocardiopsis and Actinomadeula.
Any nutrient source available to the DM-producing microorganism can be used. Examples of carbon sources for the culture medium include glucose, fructose, sucrose, lactose, starch, glycerin, dextrin, molasses, sorbitol, etc., as well as fatty acids, fats and oils, crude lecithin, alcohol, and organic acids. Or they can be used in combination. As the nitrogen source, either an inorganic nitrogen source or an organic nitrogen source can be used, and examples of the inorganic nitrogen source include ammonium nitrate, ammonium sulfate, urea, sodium nitrate, monoammonium phosphate, diammonium phosphate, ammonium chloride, etc. Examples of organic nitrogen sources include flour, bran, and defatted lees of soybeans, rice, corn, cottonseed, rapeseed, and wheat, as well as corn steep liquor, peptone, yeast extract, meat extract, casein, and amino acids. Examples of inorganic salts and micronutrients include salts such as phosphoric acid, magnesium, potassium, iron, aluminum, calcium, manganese, and zinc;
Suitable substances that promote bacterial growth and phospholipase DM production, such as vitamins, nonionic surfactants, and antifoaming agents, can be used as appropriate. Cultivation is carried out under aerobic conditions. The culture temperature can be changed and selected as appropriate within the temperature range in which the bacteria grow and produce phospholipase DM, but particularly preferred is approximately
20 to about 35°C. Although the culture time varies depending on the conditions, it is sufficient to culture until the maximum production amount of phospholipase DM is reached. In the case of liquid culture, the period is, for example, about 1 to 3 days. Phospholipase DM produced in the culture is mainly dissolved in the culture medium in submerged culture, so
Phospholipase DM can be collected from the culture solution obtained by separating the solid matter from the culture solution. To collect phospholipase DM from the culture solution, any method commonly used for enzyme purification can be used. For example, methods such as salting out with ammonium sulfate, common salt, etc., precipitation with organic solvents such as acetone, ethanol, methanol, etc., dialysis, ion exchange chromatography, adsorption chromatography, gel filtration, adsorbent, and isoelectric precipitation can be used. . Furthermore, by appropriately combining these methods,
If the purification effect of phospholipase DM increases,
It can be done in combination. Phospholipase DM, which can be obtained as described above and can be used in the method of the present invention, can be prepared by concentration under reduced pressure, with or without addition of various salts, carbohydrates, proteins, lipids, surfactants, etc. as stabilizers, for example. It can be made into a liquid or solid form of phospholipase DM by methods such as vacuum drying and freeze drying. The enzymatic activity of phospholipase DM used in the method of the present invention is determined by acting on the substrate glycerophospholipid to decompose the ester bond between phosphoric acid and a nitrogen-containing base and measuring the amount of base produced. Phospholipase DM activity was measured by the choline oxidase method described below unless otherwise specified. Titer determination method: 0.1 ml of 1% egg yolk purified lecithin emulsion (0.1 g lecithin, 1 ml ethyl ether, 10 ml distilled water ultrasonic emulsion), 0.1 ml of 0.2 MPH7.2 Tris-HCl buffer, 0.1 M CaCl ₂ Aqueous solution 0.05ml, distilled water 0.15
ml, add 0.1ml of enzyme solution to this, and incubate at 37℃.
After 20 minutes of reaction, 0.2 ml of 1M Tris-HCl buffer (PH8.0) containing 50 mM EDTA-2Na is added and immediately boiled for 5 minutes to completely stop the reaction. Next, add 4 ml of a solution of the choline coloring agent contained in the kit of cholinesterase measurement reagent [manufactured by Nippon Shoji Co., Ltd.] dissolved in the coloring solution, react at 37°C for 20 minutes, and then measure the absorbance at 500 nm. Measure. As a control, the absorbance of a similar reaction using an enzyme solution that has been heat-inactivated in advance is measured. The enzyme activity that releases 1 μmol of choline per minute is defined as 1 unit. Using the aforementioned Nocardiopsis genus NO779 strain and Actinomadeula genus NO362 strain,
The physicochemical properties of phospholipase DM that can be used in the method of the present invention using an enzyme preparation purified by the method described in 9. Purification method below will be described below. 1. Action Decomposes the ester bond between phosphoric acid of glycerophospholipid and nitrogen-containing base to liberate phosphatidic acid and base. 2 Substrate specificity Using 0.1 ml of emulsion containing 0.5 μmol of one of lecithin, lysolecithin, and sphingomyelin as a substrate, and using 1% instead of distilled water.
Except for using an aqueous solution containing Triton X-100,
The amount of choline released by the reaction was measured in the same manner as the titer measurement method described above, and the phospholipase DM activity against each substrate was measured. As a result, the relative activities when the activity against lecithin was set as 100 were: Nocardiopsis phospholipase DM: - lysolecithin 4.9; sphingomyelin 0.3; Actinomadeula phospholipase DM: - lysolecithin 3.6; sphingomyelin 0.3; It was hot. 3 Optimum PH Instead of buffer solution used in titer measurement method, PH
For 3.0 to 4.0, use formic acid/sodium formate buffer, PH4.0~
Acetic acid/sodium acetate buffer for pH 5.5, Tris/maleic acid/caustic soda buffer for pH 5.5 to 8.5, pH
For 7.0 to 9.0, use Tris/HCl buffer, PH9.0 to 10.0
Then, the activity of phospholipase DM was measured using a glycine/caustic soda buffer, and the optimal pH was determined. Also, instead of 0.15 ml of distilled water used in the same measurement method, 0.15 ml of 1% Triton X-100 (Wako Pure Chemical Industries) aqueous solution was used.
The optimal pH when using ml was also determined. As a result, when distilled water was used, Nocardiopsis phospholipase DM: - around the optimum pH 7 (6.5 to 7.0), Actinomadeula phospholipase DM: - around the optimum PH 7, and 1% Triton X - When a 100% aqueous solution was used, Nocardiopsis phospholipase DM: - optimum pH around 5, Mactinomadeula phospholipase DM: - optimum pH around 5.5. 4 Optimum temperature In the titer measurement method, the reaction temperature condition is 10,
20, 25, 37, 40, 50, 55, 60, 70, 80 and 90
Enzyme activity was measured at °C. As a result, Nocardiopsis phospholipase DM: - Optimal temperature 60°C to 80°C, especially 60°C to 70°C, Actinomadeula phospholipase DM: - Optimal temperature 55°C to 80°C, especially 60°C 〜70℃, was observed. 5 PH stability 0.1 ml of enzyme solution, 0.2 ml for Nocardiopsis phospholipase DM, 0.9 for Actinomadeula phospholipase DM
ml of 0.1M various buffers, i.e. glycine-hydrochloric acid buffer for pH 3.0-3.5, acetic acid-sodium acetate buffer for pH 3.5-7.0, Tris-maleic acid-caustic soda buffer for pH 5.0-8.0, PH7.0～
Tris/hydrochloric acid buffer was added for pH 9.0, and glycine/caustic soda buffer was added for pH 9.0 to 9.5, and the mixture was kept at 25°C for 2 hours. Then, in these enzyme buffer solutions
0.5M Tris-HCl buffer (PH7.2) for Nocardiopsis phospholipase DM.
1.2ml, Mactinomadeula phospholipase
In the case of DM, 9.0 ml was added to adjust the pH to 7.0 to 7.3. Using 0.1 ml of this solution, the titer was measured according to the titer measurement method, and the stable PH range was investigated. As a result, Nocardiopsis phospholipase DM: - Particularly stable PH4.0 to 7.0, Actinomadeula sp. Phospholipase DM: - Recognized as particularly stable PH4.0-8.0. In addition, when using 0.15 ml of 1% Triton Not much had changed. 6 Thermostability Add 0.1M Tris-HCl buffer (PH7.2) to 0.1ml of the enzyme solution, 4ml for Nocardiopsis phospholipase DM, 9.9ml for Actinomadeula phospholipase DM,
After standing at 20, 30, 37, 40, 50, 60 and 65°C for 30 minutes, the remaining enzyme activity was measured. As a result, Nocardiopsis phospholipase DM: It was hardly inactivated by heat treatment at -30℃ for 30 minutes, and 50
Actinomadeula phospholipase DM: 80% activity remains after heat treatment at -30℃ for 30 minutes; 80% activity remains after heat treatment at -30℃ for 30 minutes;
The results showed that 60% of the activity remained after heat treatment at ℃ for 30 minutes. 7. Effects of various substances In the titer measurement method, 0.05 ml of aqueous solutions of various substances were added in place of the CaCl ₂ aqueous solution, and the activity was measured at a concentration of 1 mM in the enzyme reaction system.
The results are based on the activity when water is added as 100, and the relative activities that have an activation effect are Nocardiopsis phospholipase DM: - For example, AlCl ₃ , CuSO ₄ , ZnSO ₄ , CoCl ₂ ,
_CaCl2 , _FeCl3 , _FeSO4 , _MgCl2 , _SnCl2 , sodium deoxycholate, ethanol, isopropanol, t-butanol, Triton X-100, Actinomadeula phospholipase DM: - For example, _AlCl3 , _CaCl2 , FeSO ₄ , _FeCl3 ,
MgCl ₂ , SnCl ₂ , sodium deoxycholate, ethanol, isopropanol, t-butanol, while those that had an inhibitory effect on Nocardiopsis phospholipase DM: - For example, sodium dodecyl sulfate, cetylpyridinium chloride, Actinomadeula phospholipase DM: - For example, cetylpyridinium chloride. 8. Method for measuring titer As described above. 9 Purification method Soybean flour 3.0g, corn steep liquor 1.0%,
Peptone 0.5g, powdered yeast extract 0.1%, glucose 1.0g, NH ₄ NO ₃ 0.25%, K ₂ HPO ₄ 0.4%,
MgSO ₄ 7H ₂ O0.01%, Tween -
Medium consisting of 85 0.1% (PH6.0) approx. 15 to 30
Place in a jar fermenter, sterilize at 120℃ for 15 minutes, inoculate with 1.5 seed culture solution, and incubate at 27℃.
Culture was performed for 40 hours. The above seed culture solution contains 1% starch ( _NH4 ), 0.25% _H2PO4 , and peptone _.
0.25%, _K2HPO4 0.2 _% , _MgSO4・_7H2O0.01 %
Pour 100 ml of an aqueous medium (PH6.8) containing the following into a 500 ml Sakaguchi flask, and after steam sterilization, use Nocardiopsis sp. NO779 strain [FERM-P No.6133] or Actinomadeula sp. NO362 strain [FERM-P
No. 6132] was inoculated with a loopful of spores, and the culture temperature was
It was prepared by culturing with shaking at 30°C for 2 days at 120 revolutions/min. After culturing, the bacterial solid matter was removed by centrifugation, and the centrifugal supernatant 13 (Nocardiopsis spp.
When using FERM-P No.6133 strain, 0.54u/
ml; Actinomadeula FERM-P No.
When strain 6132 was used, it was 1.7u/ml. ) was obtained. After cooling this centrifuged supernatant to 5°C, -20
C. acetone was added, and a precipitate containing phospholipase DM corresponding to an acetone concentration fraction of 30 to 70% was collected by centrifugation. When using the FERM-P No. 6133 strain of the genus Nocardiopsis, the precipitate was PH6.0;
PH6.5 when using FERM-P No.6132 strain
Trimer dissolved in maleate buffer, 0.02M
After dialyzing against the same buffer, the cellulose was passed through a column of DEAE-cellulose equilibrated with the same buffer, and the passed-through fraction was collected. Next, the method of Horiuchi et al. [J.Biochem.
81, 1639 (1977)] was packed into a column, thoroughly washed with water, and then
The DEAE-cellulose permeate was injected to adsorb the activity. This was mixed with 0.05M Tris-HCl buffer (PH
After washing with 7.2), the same buffer containing 0.2% Triton X-100 was added to elute the activity. After collecting the active fraction and concentrating it using an ultrafiltration membrane (Type G-10T) manufactured by Bio Engineering, it was injected into a packed column of Toyo Pearl HW-55F (manufactured by Toyo Soda Co., Ltd.) as a gel carrier, and distilled. The active fraction was collected and lyophilized using water. This dry powder was dissolved in 0.025M imidazole-hydrochloric acid (PH7.4) for Nocardiopsis phospholipase DM, and 0.025M for Actinomadeula phospholipase DM.
After dissolving in Tris-acetic acid (PH8.3), it was passed through a column packed with a polybuffer exchanger PBE ^TN 94 (20ml) manufactured by Pharmacia Fine Chemicals to adsorb the activity, and then a polybuffer exchanger (PH8.3) manufactured by the same company was used for elution.
5.0) using a PH gradient. The eluted active fraction of phospholipase DM was collected and concentrated using an ultrafiltration membrane, passed through a Sephadex G-75 packed column, and the active fraction of phospholipase DM was collected and freeze-dried. Thus, in the case of Nocardiopsis phospholipase DM, the activity recovery rate is about 40%, with a specific activity of 178.3 u/mg protein, and in the case of Actinomadeula phospholipase DM, the activity recovery rate is about 43%. Phospholipase DM was recovered as a protein with a specific activity of 218.3 u/mg. Isoelectric point Nocardiopsis phospholipase DM: -4.85±0.1 (measured by ampholine electrophoresis) Actinomadeula phospholipase DM: -6.4±0.1 (measured by ampholine electrophoresis) Transfer action Conventionally known phospholipase D is , as mentioned above, generates phosphatidic acid from lecithin,
It is known that this is transferred to a linear primary alcohol having 1 to 6 carbon atoms to form an ester. As a result of similarly examining the transfer effect of phospholipase DM, we found that this enzyme transfers to a wider range of alcohols to form esters, including primary alcohols that are known to not undergo transfer with known phospholipase D. There was found. The phospholipase DM used in the method of the present invention is
The reaction is carried out according to the experimental method for transfer reaction described below (method for confirming the formation of transfer products by TLC), and the reaction is catalyzed to form a phospholipid primary alcohol derivative between a C ₁₀ primary alcohol, such as geraniol, and a phospholipid, such as lecithin. , forming the primary alcohol derivative of the phospholipid. Known phospholipase D does not form the above derivatives. According to the method of the present invention, the formula () as exemplified above
A phospholipid and a primary alcohol of formula () as exemplified above are added to the phospholipase DM described in detail above.
By reacting in the presence of the following formula () However, in the formula, A and R have the same meanings as described above. A phospholipid primary alcohol derivative represented by the following can be produced. At this time, phospholipase
DM does not need to be used as a refined product, and may be a crude product. Furthermore, it can also be used by being supported and immobilized on a suitable immobilization carrier, such as a polypropylene membrane, celite grains, glass beads, etc., or on a granular or film-like material made of various polymer resins or inorganic materials. The reaction can be carried out by bringing a phospholipid of formula () into contact with a primary alcohol of formula () in the presence of phospholipase DM, preferably in the presence of a solvent. Examples of the solvent to be used include aqueous solvents and mixed solvents of aqueous solvents and organic solvents. The primary alcohol of formula () itself can also serve as a solvent. Also available are solvents containing any other additives that do not inhibit the enzymatic catalytic action of phospholipase DM, such as solvents containing suitable additives that promote this action or serve to stabilize the enzyme. be able to. For example, it can be an aqueous solvent containing a buffer such as acetic acid, citric acid, or phosphoric acid, or a neutral salt such as calcium chloride. Furthermore, examples of organic solvents include primary alcohols of the formula () themselves, aliphatic hydrocarbons such as n-heptane, n-hexane, etc.; alicyclics such as cyclopentane, cyclohexane, cyclobutane, etc. Continuing hydrocarbons;
Aromatic hydrocarbons such as benzene, toluene, xylene etc.; Ketones such as acetone, methyl isopropyl ketone etc.; Ethers such as dimethyl ether, diethyl ether, diisopropyl ether etc.; Esters such as methyl acetate, ethyl acetate etc.; Examples include halogenated hydrocarbons such as carbon chloride, chloroform and methylene chloride; amide solvents such as dimethylformamide; and sulfoxide solvents such as dimethylsulfoxide. When using an aqueous solvent in the form of a mixed solvent with an organic solvent, the mixing ratio of both can be selected appropriately, but for example, the ratio of aqueous solvent: organic solvent (v/v ratio) is 50:
A mixing ratio of 1 to 1:10 can be exemplified. The reaction molar ratio, the amount of phospholipase DM used, the amount of solvent used, etc. can be selected as appropriate, but for example, the ratio of primary alcohol of formula () to 1 mole of phospholipid of formula () from about 1:1 to about 1:1000, Preferably, a reaction molar ratio of about 1:10 to about 1:100 mol can be exemplified. The amount of phospholipase DM to be used is, for example, about 10 to about 100,000 units, preferably about 100 to about 1,000 units per gram of phospholipid of formula (). Further, the amount of the solvent to be used may be, for example, about 10 times to about 500 times (by volume) the amount of the phospholipid of formula (). Since the reaction proceeds at room temperature, there is no particular need for cooling or heating, but cooling or heating conditions can be adopted as appropriate, if desired. for example,
Examples of reaction temperatures include about 0°C to about 90°C, preferably about 20°C to about 60°C. The reaction time can also be selected as appropriate, for example from about 1 minute to about 10 minutes.
Examples of reaction times include days, preferably about 0.1 hour to about 72 hours, more preferably about 1 hour to about 72 hours, particularly preferably about 1 hour to about 24 hours. If desired, the reaction time can be changed as appropriate by tracking the reaction progress using a technique such as TLC (thin layer chromatography) and confirming the formation of the desired target product. The mode of contacting the phospholipid of formula () with the primary alcohol of formula () in the presence of phospholipase DM can be selected as appropriate, but it is usually carried out under stirring or shaking conditions. In addition, when phospholipase DM is used in the form of an immobilized enzyme supported and immobilized on a suitable granular or film-like carrier as described above, for example, it can be transferred via an immobilized enzyme membrane or an immobilized enzyme particle layer. The reaction can be carried out in such a manner that the reaction composition liquid is passed through using a circulation pump. After carrying out the reaction as described above, the phospholipid primary alcohol derivative of formula () formed can be separated and utilized as it is or in the form of a salt by precipitation. Further, separation and purification can be carried out using appropriate known methods such as silicic acid column chromatography, alumina column chromatography, high performance liquid chromatography, countercurrent distribution, gel filtration, and adsorption chromatography. According to the method of the present invention, as described above, a phospholipid of formula () and a primary alcohol of formula () are combined into
A phospholipid primary alcohol derivative of formula () can be produced by reaction in the presence of phospholipase DM. The resulting phospholipid primary alcohol derivative of formula () has excellent surfactant action and has a large effect on cell membrane permeability. In this sense, the derivative of formula () can be used as a liposome-forming base material, and
It is useful as an emulsifier that is useful for skin physiology when incorporated into cosmetics such as creams and emulsions, and is also useful in a wide range of emulsifier applications, including as an emulsifier for fatty drugs, and as an emulsifier for agricultural chemicals such as insecticides and herbicides. Furthermore, in many cases, it is known that each phospholipid has a unique physiological activity, and since many of the formula () derivatives obtained by the method of the present invention have similar structures, they have various physiological activities. You can expect it to be active. In addition, by transferring a pharmacologically active compound having a primary alcohol hydroxyl group or into which a primary alcohol hydroxyl group has been introduced to a phospholipid, the pharmacological side effects of the compound can be weakened or the pharmacological effect can be increased and its dosage can be reduced. It can also be expected to reduce the Furthermore, as a carrier for the pharmacologically active compound to transfer the pharmacologically active compound to phospholipids and concentrate the compound accurately in the affected area,
Furthermore, it can be expected to play a useful role as a protecting group for pharmacologically active compounds. Furthermore, it is useful as an intermediate for chemical synthesis including various pharmaceuticals, and for example, derivatives obtained by transferring alcohols having highly reactive halogen or amine substituents can be used. Furthermore, tritium
Labeled phospholipid derivatives can be obtained by transferring ¹⁴ C-labeled primary alcohols, which can also be used to elucidate metabolic pathways of phospholipids. Hereinafter, several embodiments of carrying out the method of the present invention will be illustrated in more detail with reference to Examples. Reference Example 1 Preparation of phospholipase DM. According to the purification method section, Nocardiopsis strain NO779 [FERM-P No. 6133] and Actinomadeula strain NO362 [FERM-P No.
6132] to obtain phospholipase DM with the activity recovery and specific activity as described in that section. Example 1 (Run No. 1 to No. 91) Phospholipid substrate of the following formula () shown in Table 4 below: L-α-lecithin, β, γ-dimyristoyl (Sigma) (1,2- ditetradecanoyl-sn-glycerol-3-phosphorylcholine) Substrate: L-α-lecithin, β, γ-dihexadecyl (Calbiochem-Behring) (1,2-dihexadecyl-sn-glycerol-3-phosphorylcholine) Substrate: L -α-lecithin, β,γ-hexadecylidine (same as above) (1,2-cyclohexadecylidene-sn-glycerol-3-phosphorylcholine) Substrate: β-lecithin-α,γ-dipalmitoyl (same as above) (1 , 3-dihexadecanoyl-glycerol-2-phosphorylcholine) and a number of primary alcohols of the formula () shown in Table 4 below, and confirmed the formation of a transfer product (phospholipid primary alcohol) by TLC below. According to the method, reacting in the presence of phospholipase DM,
Formation of transfer products was confirmed. The Rf values are shown in Table 4 below. Method for confirming the formation of transfer products by TLC: - 1% phospholipid emulsion with the following composition 0.1ml 0.4M acetate buffer (PH5.7) 0.1ml 0.1M calcium chloride aqueous solution 0.05ml Distilled water 0.1ml 10% primary alcohol solution 0.1 ml of reaction solution, add 0.1 ml of phospholipase DM aqueous solution.
(0.2 to 1.0 u/0.1 ml) was added and allowed to stand at 37°C for 1 to 5 hours. The above 1% phospholipid emulsion contains 100 phospholipids.
1 ml of diethyl ether and 10 ml of distilled water are added to 1 ml of diethyl ether and subjected to ultrasonication for 5 minutes at 600 W and 20 KHz under ice-cooling conditions. When forming the reaction solution, if necessary, water or an organic solvent such as diethyl ether or acetone was added to prepare the 10% primary alcohol solution. After the above-mentioned standing, 0.2 ml of 50 mM EDTA (ethylenediaminetetraacetic acid) aqueous solution was added, and further 5 ml of chloroform-methanol mixture (2:1 volume ratio) was added and stirred vigorously to extract lipids (products). This suspension was centrifuged at 2000 x g for 10 minutes, the lower chloroform layer was collected, dried under reduced pressure at 30°C, and then dissolved in 75μ of a chloroform-methanol mixture (1:1 volume ratio). This was used as a sample for TLC. Of this, 10μ was applied to a thin layer of silica gel (Funagel 60Å, 20μ
cm x 20cm (Funakoshi Yakuhin) and developed using diisobutylketone-acetic acid-water (40:25:5) as a developing solvent. The following reagents were used to detect spots. undegraded substrate at the detected spot,
When a spot of phospholipid other than phospholipid and its hydrolyzate (phosphatidic acid and its analogs) was detected, this was recognized as a transfer product. Color development of detection reagent phosphoric acid: Zinzade reagent (Beiss.UJ
Chromatog. 13 104, 1964) Color development of primary amines: Ninhydrin reagent (0.25% acetone solution of ninhydrin) Color development of secondary amines: Hypochlorous acid-benzidine reagent (Bischel MC et al. Biochim, Biophys, Acta
70 598 1963) Color development of purines and pyrimidines: fluorescein-ammonia reagent (Wieland T. et al. Angew
chem. 63 511 1951) Coloration of glycol bonds: periodic acid-Schiff's reagent (Shaw N.Biochim.Biophys.Acta. 164 435
1968) Color development of 17-ketosteroids: m-dinitrobenzene reagent (Jisboa BPJChromatog. 16 136
1964) Comparative Example 1 In Example 1, in place of phospholipase DM, known phospholipase D (P-
The same procedure as in Example 1 was carried out except that L Biochemicals Inc.) was used. As a result, no transfer product was observed for any of the primary alcohols shown in Table 4 below. [Table] [Table] [Table] Colors were developed using reagents.
Example 2 (Run No. 1 to No. 23) L-α-lecithin, β, γ-dimyristoyl (manufactured by Sigma Chemical Company, purity 98%)
Put 400mg of diethyl ether, 1ml of diethyl ether, and 10ml of distilled water into an ultrasonic cell, and heat at 600W and 20K while cooling on ice.
Ultrasonication was performed at Hz for 5 minutes to obtain a milky white emulsion. 2ml of this lecithin emulsion (lecithin 80mg),
Put 2ml of 0.4M acetate buffer (PH5.7), 1ml of 0.1M calcium chloride aqueous solution, and 2ml of 10% β-hydroxyethylaniline diethyl ether solution into a test tube with a stopper, and add phospholipase DM aqueous solution (2.5u/
After adding 2 ml of ml) and mixing well, it was left standing at 37°C for 3 hours. After terminating the reaction by adding 0.5 ml of 0.5N hydrochloric acid to the reaction solution, 15 ml of a chloroform-methanol (2:1) mixture was added and vigorously mixed to extract phospholipids. This mixture was centrifuged at 2000×g for 10 minutes, and the lower chloroform layer was separated. Add 10 ml of chloroform to the upper aqueous layer again, perform the same extraction procedure, combine the extracts,
It was then washed with 10 ml of 0.02N hydrochloric acid. The chloroform layer was separated again from this mixture by centrifugation, dried under reduced pressure, and then dissolved in 1 ml of a mixture of n-hexane-2-propanol-water (60:80:7). When 20μ of this sample was spotted on a thin layer of silica gel (Funagel Funakoshi Yakuhin) and developed with a solvent system of diisobutylketone-acetic acid-water (40:25:5), three types of phospholipids were detected, two of which were One had an Rf value that matched that of phosphatidic acid and lecithin. This sample was purified by high performance liquid chromatography. The column was a radial pack cartridge made of silica 8 mm x 10 cm (manufactured by Waters), and the eluent was the above-mentioned n-hexane-2-propanol.
Water = 60:80:7, flow rate 2 ml/min, peak detection using 441 ultraviolet detector (Waters)
Absorption at 214 nm and R401 differential refractometer (same model) were used. The sample was divided into four injections of 0.25 ml each. First, the three components of β-hydroxyethylaniline, phosphatidic acid, and β-hydroxyethylaniline ester of phosphatidic acid mixed in this eluent are separated, and then the eluent is extracted with n-hexane-2-propanol-water. = 60:80:14, and undecomposed lecithin adsorbed on the column was eluted. The three types of phospholipids obtained were each purified once again using high performance liquid chromatography in the same manner. The three types of phospholipids are approximately 10% phosphatidic acid;
The rearrangement product was about 80% undegraded lecithin and about 10% (molar ratio), and about 40 mg of purified rearrangement product β-hydroxyethylaniline ester of phosphatidic acid was obtained. The IR spectrum of this compound was measured using a JASCO A202 infrared spectrophotometer.
Measured by liquid film method. The results are shown in Table 5 (Run No. 14). The same procedure as above was carried out using other primary alcohols of formula () shown in Table 5. The results are shown in Table 5 (Run Nos. 1-13, 15-24 and 26-33).
However, when adding the primary alcohol to the reaction solution,
Depending on the solubility of the alcohol, it was added as a solution in water, diethyl ether, or acetone, as appropriate. [Table] [Table] [Table] [Table] Example 3 (Run No. 1 to No. 24) L-α-lecithin β, γ-dihexadecyl (manufactured by Calbiochem-Behring) 400 mg, diethyl ether 1 ml, and distilled water Example 2: 10ml of the mixture
The mixture was emulsified in the same manner as in Example 2, and 2 ml of this emulsion was used to carry out a reaction in the same manner as in Example 2. However, a 10% aqueous solution of thiamine hydrochloride was used as the primary alcohol. This reaction solution was treated in the same manner as in Example 2 to obtain 30 mg of a transfer product. The IR spectrum of this compound is shown in Table 6 (Run No. 23). The same procedure as above was carried out using other primary alcohols of formula () shown in Table 6. The results are shown in Table 6 (Run Nos. 1-22 and 24). [Table] [Table] Example 4 (Run No. 1 to No. 24) 400 mg of L-α-lecithin β, γ-hexadecylidine (manufactured by Calbiochem-Behring) was emulsified in the same manner as in Example 2. Using an emulsion equivalent to 80 mg, geraniol was added as a receptor for the transfer reaction, and the reaction, extraction, and purification were carried out in the same manner as in Example 2. As a result, 50 mg of the transfer product was obtained.
The IR spectrum of this compound is shown in Table 7 (Run No. 4). The same procedure as above was carried out using other primary alcohols of formula () shown in Table 7. The results are shown in Table 7 (Run Nos. 1-3 and 5-24). [Table] [Table] [Table] Example 5 (Run No. 1 to No. 24) 400 mg of β-lecithin and α, γ-dihexadecanoyl (manufactured by Calbiochem-Behring) were emulsified in the same manner as in Example 2. Then, using an emulsion equivalent to 80 mg and adding pantothenyl alcohol as an acceptor for the transfer reaction, reaction, extraction, and purification were carried out in the same manner as in Example 2. As a result, 25 mg of transfer product
I got it. The IR spectrum of this compound is shown in Table 8 (Run No. 9). The same procedure as above was carried out using other primary alcohols of formula () shown in Table 8. The results are shown in Table 8 (Run Nos. 1-8 and 10-24). [Table] [Table] [Table] Example 6 (Run No. 1 to No. 5) L-α-phosphatidylethanolamine,
β,γ-dimyristoyl (), L-α-phosphatidyl-N-methylethanolamine, β,γ
- Dimyristoyl (), L-α-phosphatidyl-DL-glycerol β, γ-dimistoyl () (both Calbiochem-Behring), L-α-phosphatidylserine () (Sigma), and SFYang (J.Biol.Chem 242 ,
477, 1967), L-α-phosphatidiethanol, β,γ-dimyristoyl ()
was emulsified in the same manner as in Example 2. Put 1.5 ml of the emulsion containing 50 mg of each phospholipid into separate stoppered test tubes, and add geraniol as alcohol.
Add 10 ml of 10% diethyl ether solution, 1 ml of 0.4 M acetate buffer, 1 ml of distilled water, and 0.5 ml of 0.01 M calcium chloride aqueous solution, and then heat the reaction solution at 600 W.
Ultrasonication was performed at 20KHz for 1 minute. Next, 1 ml of phospholipase DM aqueous solution (8 u/ml) was added to each reaction solution, and the mixture was allowed to stand at 37°C for 6 hours. Hereinafter, extraction and purification of phospholipids were performed in the same manner as in Example 2 to obtain phosphatidic acid-geraniol ester, which is a common transfer product. Yield: 12mg, 20mg, 10mg, 24mg,
The amount was 22 mg. Its IR spectrum is shown in Table 9. 【table】

Claims (1)

[Claims] 1. The following formula () However, in the formula, A represents the following (i) or (ii) [Formula] [Formula], where both R ₁ and R ₂ are -O-COR ₁₁
or both are -O-R ₁₂ , or in formula (i), R ₁ and R ₂ together represent [formula] [where n represents a number from 11 to 19] In the above, R ₁₁ and R ₁₂ may be the same or different and each represents a C ₇ to C ₂₁ saturated or unsaturated aliphatic hydrocarbon group, and B is -(CH ₂ ) ₂ +N( _CH3 ) ₃ ,- _{( CH2} ) _2NH2 ,-
CH ₂ CH (NH ₂ )COOH, −CH ₂ CH ₂ NH (CH ₃ ), −
CH ₂ CH ₂ N(CH ₃ ) ₂ , -CH ₂ CHOHCH ₂ OH or -(CH ₂ ) _n H [Here, m represents a number from 1 to 5]
A phospholipid represented by the following formula () R-OH ... () where R represents a member selected from the group consisting of the following (1) to (3), (1) C ₆ (excluding hexanol) ~ _C26 saturated or unsaturated aliphatic or aromatic hydrocarbon residue, where the hydrocarbon residue is substituted with a substituent selected from halogen, amino, acetyl, and hydroxyl group or the above-mentioned hydrocarbon residue having a bond selected from the group consisting of ether, ester, and amide bonds in the molecule, (2) pregnane steroid compound residue, (3) galactono-γ-lactone , N(2-hydroxyethyl)phthalimide, 2-(3-indole)ethanol, 2-(2-hydroxyethyl)pyridine, pyridoxine, N(2-hydroxyethyl)morpholine, 5-hydroxymethylcytosine, cytidine, uridine, A heterocyclic compound residue selected from the group consisting of arabinocytidine, thiamine, 2-(2-hydroxyethyl)piperazine, adenosine, guanosine and cyclocytidine is reacted with a primary alcohol represented by: in the presence of phospholipase DM. The following formula () is characterized by However, in the formula, A and R have the same meanings as above,
A method for producing a phospholipid primary alcohol derivative represented by