JPH05168489A - Production of glycolipid - Google Patents

Abstract

PURPOSE:To obtain glycolipid of high purity useful as a surfactant for cosmetics and medicines by allowing glycoglycerol to react with a fatty acid or a derivative thereof in the presence of lipase in an organic solvent which dissolves these reactants, but is inert as such. CONSTITUTION:beta-Galactosidase originating from Escherichia coil is added to a 50mM phosphate buffer (pH7.3) containing lactose or the like, glycerol and magnesium chloride, and the reaction is effected at 40 deg.C for 50 hours to give glycoglycerol such as galactosylglycerol and the product is allowed to react with a fatty acid such as palmitic acid or its derivative in an organic solvent which dissolves these reactants but is inert as such, for example, 2-propanol in the presence of a lipase with 1,3-position specificity, originating from Mucor miehei, etc., to give the objective glycoliped of formula I (R1 is 6-24C aliphatic residue; G is a residue of monosaccharide or disaccharide) or formula II (R2 is same as R1) such as monogalactosyl monopalmityulglycerol.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for synthesizing glycolipids by an enzymatic reaction in an organic solvent.

[0002]

2. Description of the Related Art Glycolipid is not a single substance but a general term for compounds in which sugar and lipid are bound. It is known that glycolipids are particularly abundant in grain seeds such as soybeans, corn, wheat and spinach. In addition, it is abundant in the body as a membrane constituent of plants and microorganisms. Traditionally,
Glycolipids are extracted from these glycolipid-containing raw materials and combined with solvent fractionation and column chromatography, or the acetone concentration method (Japanese Patent Laid-Open No. 62-178596). There are problems such as bad.

On the other hand, in the enzymatic synthesis method, the reaction is selective,
The advantage is that the amount of by-products is small, and a method for obtaining glycolipids by ester synthesis using lipase in a substantially solvent-free manner has been proposed (Agric. Biol. Chem., 55 (8), 2083.
-2089, 1991), however, such a method has problems that the reaction is not uniform, the reaction efficiency is poor, and that it does not react with a fatty acid having a high melting point.

[0004]

DISCLOSURE OF THE INVENTION The present invention aims to solve the above-mentioned problems and provides a method for efficiently synthesizing glycolipids of all kinds.

[0005]

[Means for Solving the Problems] As a result of intensive investigations by the present inventors for the purpose of efficiently obtaining glycolipids with high purity by enzymatic reaction, as a result, sugar glycerol and fatty acid
Alternatively, it was found that by reacting a fatty acid derivative with a specific organic solvent in the presence of lipase, any kind of glycolipid can be efficiently produced with high purity, and the present invention is completed. I arrived.

That is, the method for producing a glycolipid of the present invention is characterized by reacting sugar glycerol with a fatty acid and / or a fatty acid derivative in the presence of a lipase in an organic solvent which dissolves them but does not react with itself. It is what Hereinafter, the method of the present invention will be described in detail. The glycolipid synthesized in the present invention is mainly represented by the following formula, in which the fatty acid residues R ₁ , R ₂ and R ₃ are the same or different and have 6 carbon atoms.
~ 24 saturated or unsaturated fatty acid residues and G is the residue of a mono- or disaccharide.

[0007]

Examples of such fatty acids include caproic acid
(C_{6: 0}), Caprylic acid (C_{8: 0}), Capric acid (C _{10: 0}), Lau
Phosphoric acid (C_{12: 0}), Myristic acid (C_{14: 0}), Palmitic acid
(C_{16: 0}), Palmitoleic acid (C_{16: 1}), Stearic acid (C
_{18: 0}), Oleic acid (C_{18: 1}), Linoleic acid (C_{18: 2}),
Nolenic acid (C_{18: 3}), Arachidonic acid (C₂₀:_Four), Eikosape
Ntaenoic acid (C₂₀:_Five), Behenic acid (C_{22: 0}), Erucic acid (
_{22: 1}), Docosahexaenoic acid (C_{twenty two : 6}), Tetracosate
Raenoic acid (C_{24: 4}) And the like.
Sylose, glucose, mannose, galactose, fur
Neutral monosaccharides such as course, acidic monosaccharides such as glucuronic acid,
Amino monosaccharides such as glucosamine and galactosamine,
Or disaccharides composed of those monosaccharides
It In addition, fatty acid residue R₁, R₂And R₃At least of
One is eicosapentaenoic acid, γ-linolenic acid or ara.
When it is a residue of chidonic acid, these bioactive effects
Can be effectively used.

The high-yield and efficient synthesis of these glycolipids means that long-chain fatty acids such as palmitic acid and eicosapentaenoic acid have a high melting point, and a solvent characteristic of the reaction system cannot be obtained. For this reason, it has never been carried out industrially because many kinds of glycolipids cannot be obtained and the products are not uniform. Therefore, in the present invention, this could be made possible by using a solvent system in which the sugar glycerol used and the fatty acid and / or the fatty acid derivative are easily dissolved.

The organic solvent used in the present invention may be any one as long as it does not use lipase as a substrate.
Acetone, chloroform, dioxane, acetonitrile, methanol, ethanol, n-propanol, 2-
Propanol, n-butanol, 2-butanol, t-
Examples thereof include butanol, octanol, decanol, cyclohexanol, benzyl alcohol, and the like, preferably alcohols, more preferably secondary or tertiary alcohols such as 2-propanol, 2-butanol, and t-butanol.

The lipase used in the reaction can be isolated / purified from the living body or secretory liquid of a plant or microorganism, but is conveniently derived from the microorganism. For example,
In Pseudomonas, Pseudomonas
Lipase derived from Pseudomonas fluorescens (for example, Amano Pharmaceutical Co., Ltd., trade name "Lipase-
P '), in Candida
Lipase derived from Cylindracea (for example, manufactured by Meito Sangyo Co., Ltd., trade name "lipase OF",
Amano Pharmaceutical Co., Ltd., trade name “Lipase-AY”), Candida lipolytica-derived lipase (for example, Amano Pharmaceutical Co., Ltd., trade name “Lipase-L”), etc.
For Penicillium (Penicillium), lipase derived from Penicillium cyclopium (for example, Amano Pharmaceutical Co., Ltd., trade name "Lipase-GT"), and for Alcaligenes (Alcaligenes), Microbacterium Research Institute Contribution No. 3883 Derived lipase (for example, product name "Lipase PL" manufactured by Meito Sangyo Co., Ltd.), Humimicola lanuginosa (Humi
cola lanuginosa) -derived lipase (for example, Amano Pharmaceutical Co., Ltd.
(Lipase-CE), a lipase derived from Mucor miehei in the genus Mucor (Mucor) (for example, Novo Industry, trade name "Lipase-3A")
, Rhizopus, Rhizopus nibeus (Rhzopus)
izopus niveus) -derived lipase and the like. The present invention is not limited to microorganisms or lipases of these genera or species, and lipases from mutant strains, auxotrophic strains or drug resistant strains belonging to these genera or species may be used.

Next, the above lipase can be used as an immobilized product. In general, enzymes are used as immobilized products for the purposes of pH and temperature, improvement of organic solvent stability, activity maintenance, reuse, etc., and as carriers for immobilization, cellulose, dextran, polystyrene, polyacrylamide are used. , Polyvinyl alcohol, ion exchange resin, magnetic material, activated carbon, alumina, photo-crosslinked resin, alginate and the like are used. In the present invention, an enzyme immobilized on these immobilization carriers by adsorption, ionic bond, covalent bond or entrapment can be used. For example, an immobilized lipase derived from Mucor miehei (for example, a product name “Li manufactured by Novo Industry Co., Ltd.
pase IM60)), Candida Antarctica (Can
immobilized lipase from eg dida antarctica)
The product name is “Lipase SP382” manufactured by Industry, etc.

Further, according to the present invention, various products can be obtained depending on the kind of lipase used. That is, for example, 1,3-position specific immobilized lipase derived from Mucor miehei (eg, Novo Industry Co., trade name “Li
The reaction using pase IM60)) gives only the product represented by the above formula (1). In addition, immobilized lipase derived from Candida antarctica (for example, Novo Industry Co., trade name "Lipase
SP382 ”) is used, the above formulas (1) to (4) are mainly used.
The product shown in is obtained.

The sugar glycerol used in the present invention is a glycoside having glycerol as an aglycone, and any kind of sugar may be used, but those which can be obtained in a large amount are preferable, and galactosylglycerol, Examples thereof include glucosyl glycerol and xylosyl glycerol. These can be easily obtained from glycerol and sugar by a glycosyltransferase.

The fatty acid derivative which can be used in place of the fatty acid in the present invention is a fatty acid ester of an alcohol having 1 to 30 carbon atoms such as methyl ester,
Examples thereof include ethyl ester, propyl ester and butyl ester. The temperature of the ester synthesis reaction in the present invention is usually 10 to 70 ° C, preferably 20 to 50 ° C.
Further, since this reaction is an ester synthesis reaction, it is usually preferable that the water content in the reaction system is small, preferably 0 to 20%.

[0016]

EXAMPLES Next, the present invention will be specifically described by showing examples, but the present invention is not limited to the examples.

[0017]

[Example 1] [Synthesis of glycolipid from galactosylglycerol and palmitic acid] 100m of 50mM phosphate buffer (pH 7.3) containing 1g of lactose, 25g of glycerol and 0.045g of magnesium chloride
To l, 100 units of β-galactosidase (manufactured by Sigma Yakuhin Co.) derived from Escherichia coli was added and reacted at 40 ° C for 50 hours to give galactosylglycerol.
0.3 g was obtained. Next, galactosyl glycerol 0.3
g, palmitic acid 0.3 g and molecular sieve 3A
Immobilized lipase derived from Mucor miehei with specificity of 1,3-position in 30 ml of 2-propanol containing 1 g (trade name “Lipase I” manufactured by Novo Industry Co., Ltd.
M60 ″) 100 mg was added and reacted at 40 ° C. for 50 hours. After the reaction was completed, the ester compound was developed by thin layer chromatography, and a densitometer (Shimadzu Corporation, DUAL-W
AVELENGTH FLYING-SPOT SCA
When the purity was measured by NNER), it was confirmed that the ester synthetic product was produced in high purity (99.5%). This product was confirmed to be monogalactosylmonopalmitylglycerol by acid hydrolysis and weak alkali decomposition.

[0018]

[Example 2] [Synthesis of glycolipid from glucosylglycerol and stearic acid] 1 g of maltose and 25 g of glycerol 50
70 units of yeast-derived α-glucosidase (manufactured by Biozyme Laboratories) was added to 100 ml of mM acetate buffer (pH 5.0), reacted at 40 ° C. for 50 hours, and glucosyl glycerol 0.
3 g was obtained. Next, in 30 ml of t-butanol containing 0.3 g of glucosylglycerol, 0.3 g of stearic acid and 1 g of molecular sieve 3A, immobilized lipase derived from Candida antarctica (manufactured by Novo Industry, trade name) "LipaseSP382") 10
0 mg was added and reacted at 40 ° C. for 50 hours. After completion of the reaction, the ester compound was obtained in high yield (9
8%) was confirmed.

Comparative Example 1 In Example 2, t-butanol was not used as a solvent (no solvent), and immobilized lipase derived from Candida antarctica (manufactured by Novo Industry Co., Ltd., Instead of the product name "Lipase SP382"), the reaction was carried out by substituting a lipase derived from Candida cylindracea (manufactured by Meito Sangyo Co., Ltd., product name "Lipase OF") for thin layer chromatography. No product was confirmed by graphy.

[0020]

INDUSTRIAL APPLICABILITY According to the present invention, various glycolipids can be efficiently produced with high purity. The lipid obtained by the method of the present invention is suitable for use as an emulsifier and a surfactant in fields requiring safety, such as cosmetics and pharmaceuticals.

Claims (6)

[Claims] 1. A method for producing a glycolipid, which comprises reacting sugar glycerol with a fatty acid and / or a fatty acid derivative in the presence of lipase in an organic solvent which dissolves them but does not react with itself. 2. The method according to claim 1, wherein the organic solvent is a secondary or tertiary alcohol. 3. The method according to claim 1 or 2, wherein the synthesized glycolipid is mainly a glycolipid represented by the following general formula (1), (2), (3) or (4). (In the formula, R ₁ , R ₂ and R ₃ are the same or different and have 6 to 6 carbon atoms.
24 are saturated or unsaturated fatty acid residues and G is the residue of mono- or disaccharides. ) 4. The method according to claim 1, wherein a 1,3-position-specific lipase is used as the lipase. 5. The method according to claim 4, wherein the synthesized glycolipid is the glycolipid represented by the general formula (1) according to claim 3. 6. The method according to claim 1, wherein at least one of R ₁ , R ₂ and R ₃ is a residue of eicosapentaenoic acid, γ-linolenic acid or arachidonic acid.