JPH0528114B2 - - Google Patents

Description

[Detailed description of the invention]

(a) Industrial Application Field The present invention relates to a method for producing sugar glycerol and fatty acids by enzymatically converting glycolipids derived from natural products. More specifically, the present invention relates to a method for producing sugar glycerol or acyl sugar glycerol and fatty acids by partially or completely hydrolyzing fatty acids of glyceroglycolipids in the presence of fat hydrolase. (b) Prior Art Lipids derived from natural products include those collected from higher animals and plants, including glycerides (neutral lipids) containing only fatty acids, and polar lipids containing sugar and phosphoric acid residues. In addition, the lipids of microorganisms such as mold, yeast, and bacteria, algae such as seaweed and chlorella, and even fish are glycolipids (hereinafter simply referred to as glycolipids) and phospholipids (hereinafter simply referred to as phospholipids).
It is expected that these so-called polar lipids can be used effectively. Among these, glyceroglycolipids exist in algae, for example, in molecular structures such as mono- or digalactosylceride, mono- or diglucosylglyceride, mono- or dimanosylglyceride, or sulfopyranosylglyceride, and furthermore, as bound fatty acids. Eicosapentaenoic acid, docosahexaenoic acid, γ-
There are many highly unsaturated fatty acids such as linolenic acid and arachidonic acid, and there are many important biologically active lipids that exist as components of cell membranes and walls. (c) Problems to be Solved by the Invention As a method for producing sugar glycerol or acyl sugar glycerol using such glyceroglycolipids as raw materials, fatty acid removal can be carried out by hydrolysis using an acid or alkali catalyst. In this method, highly unsaturated fatty acids are degraded by the action of acids or alkalis, causing peroxidation, rancidity, polymerization, and coloration, making post-treatment purification extremely difficult. It is also difficult to control these catalytic actions to obtain products with preferred structural formulas. In contrast, in the enzymatic method, lipase is an enzyme that usually hydrolyzes mono-, di-, or doliglycerides (all esters of fatty acids only), and the conversion can be carried out under mild reaction conditions without altering or degrading the substrate. However, due to the substrate specificity of lipase, the reaction does not proceed at all or only has a small hydrolysis rate for substrates with sugar chains or long-chain highly unsaturated fatty acids. It was nothing more than a simple thing. Furthermore, such glycolipids have a higher melting point than ordinary fatty acid triglycerides, and
Since there are almost no enzymes that are thermally stable for long periods of time, such glycolipid enzymes have not been applied. In addition, if a glycerosugar ester is to be synthesized chemically, it is usually necessary to mix glycerin and the saccharide and etherify it by heating, and if necessary, mix it with a fatty acid and esterify it by heating.
Through this synthetic route, polyunsaturated fatty acids with many sugars and double bonds undergo side reactions such as polymerization, isomerization, oxidation, cyclization, and decomposition due to thermal history, resulting in complicated purification steps. As a result, it is difficult to obtain the desired high purity sugar glycerol or sialic sugar glycerol. (d) Means for Solving the Problems In view of the current situation, the present inventors have developed a method for processing glyceroglycolipids existing in living organisms such as natural animals, plants, and even microorganisms without harsh reaction conditions. Does not cause side reactions such as peroxidation, decomposition, and cyclization,
As a result of extensive research aimed at removing fatty acid groups and modifying them into sugar glycerol or acyl sugar glycerol, we found that fatty acid groups can be easily removed by heat-resistant fat hydrolysis under mild reaction conditions. The inventors have discovered a method for producing sugar glycerol or acyl sugar glycerol and fatty acids, which can be eliminated, and have completed the present invention. The present invention will be explained in detail below. The glyceroglycolipid used in the present invention is
Among natural products, it is contained in lipids extracted from living organisms such as microorganisms such as molds, yeast, and bacteria, algae such as seaweed and chlorella, freshwater or saltwater fish, and higher plants and animals. Its general structural formula is shown below () to (), where fatty acid residues R ₁ and R ₂ are both carbon

【formula】

【formula】

【formula】

[Formula] A saturated or unsaturated fatty acid residue having a number of 11 to 23, and G is a residue of a monosaccharide or a dimer thereof. Such fatty acids include octanoic acid ( _C8 : ₀ ), lauric acid ( _C12 : ₀ ), palmitic acid (C16: ₀ ), palmitoleic acid ( _{C16 : 1} ), and stearic acid ( _C18 : ₀₎ . ), oleic acid (C ₁₈ : ₁ ), linoleic acid (C ₁₈ : ₂ ), linolenic acid (C ₁₈ : ₃ ), behenic acid (C ₂₂ : _O ), erucic acid (C ₂₂ : ₁ ), eicosapentaenoic acid ( _C20 : ₅ ), docosahexaenoic acid ( _C22 : ₆ ), arachidonic acid ( _C20 : ₄ ), tetracosatetraenoic acid ( _C24 : ₄ ), and sugars such as xylose, glucose, and fructose. Examples include neutral monosaccharides such as mannose, galactose, and fucose, acidic monosaccharides such as glucuronic acid, amino monosaccharides such as glucosamine and galactosamine, and dimers thereof. In addition, fatty acid residues R ₁ and R ₂
At least one of them is eicosapentaenoic acid, γ
- When the residue is either linolenic acid or arachidonic acid, these physiologically active effects can be effectively utilized. Such glyceroglycolipids generally have a high melting point; for example, glycolipids extracted from certain types of chlorella cells are mainly composed of monogalactosyl dieicosapetenoic acid glyceride, and their melting point is 50 to 60°C.
It is. For this reason, fat and oil hydrolyzing enzymes (optimum temperature: 30-45
Coupled with the fact that there are almost no substances that are thermally stable for long periods of time in the glycolipids (℃), it has never been done industrially to hydrolyze such glycolipids to produce sugar glycerol or sialic sugar glycerol and fatty acids. However, in the present invention, this was made possible by using a thermostable enzyme. One of the features of the present invention is the use of this thermostable enzyme as a glycolipid hydrolase, and any thermostable enzyme may be used as long as it can maintain its activity even at 50°C or higher. Such enzymes can be isolated, purified, and collected from living organisms or secretions of animals, plants, or microorganisms, but enzymes derived from microorganisms are convenient. For example, in the genus Pseudomonas, Pseudomonas fluorescens
fluorescens) derived lipase (e.g. Amano Pharmaceutical Co., Ltd.)
Co., Ltd., trade name "Lipase P"), for Candida, lipase derived from Candida cylindracea (for example, Meito Sangyo Co., Ltd., trade name "Lipase-OF", Amano Pharmaceutical Co., Ltd. In the Penicillium genus, Penicillium cyclopium ) (e.g., Amano Pharmaceutical Co., Ltd., trade name ``LipaseGT(G)''), and in the Alcaligenes genus, lipase derived from the strain No. 3783 of Alcaligenes (e.g., Meito Sangyo Co., Ltd.).
Co., Ltd., product name "Lipase-PL"; for Humicola, lipase derived from Humicola lanuginosa (for example, manufactured by Amano Pharmaceutical Co., Ltd., product name "Lipase CE"); ), lipase derived from Mucor miehei (e.g. Novo Instantly, product name "Lipase3A"), Rhizopus,
Examples include lipase derived from Rhizopus chinensis (heat-resistant lipase prepared by the method described in JP-A-59-156282).
Note that the present invention is not limited to microorganisms and lipases of these genera and species, and thermostable lipases from mutant strains, auxotrophic strains, and drug-resistant strains belonging to these genera or species may also be used. . In addition, lipase is generally a glycoprotein with a molecular weight of tens of thousands to hundreds of thousands of thousands, but it is possible to increase heat resistance by increasing the molecular weight. It can also be used. Next, the above thermostable enzyme can also be used as an immobilized product. In general, enzymes are
For the purpose of improving PH and temperature stability, maintaining activity, and reusing, methods are used to make water-insoluble immobilized substances, but the immobilization carriers include cellulose, dextran, polystyrene, polyacrylamide, polyvinyl alcohol, and ions. Exchangeable resin, magnetic material, activated carbon, alumina, photocrosslinkable resin,
Alginate etc. are used. In the present invention, thermostable enzymes adsorbed, ionically bonded, covalently bonded, or enclosed in these immobilization carriers can be used. For example, the method of immobilizing thermostable lipase derived from Mucor miehei on a porous weak anion exchange resin described in JP-A-60-98984, the method described in JP-A-52-104506, Candida Cylindrace
An immobilized enzyme can be used, such as by adsorbing heat-stable lipase derived from cylindracea to diatomaceous earth powder. Glycolipids are hydrolyzed by the method of the present invention as follows. That is, the above structural formula ()
A glycolipid having a composition of one or more of ~() is taken in a glass or stainless steel container,
If necessary, dissolve the glycolipids in the minimum amount of inert organic solvent such as hexane, heptane, etc., add an appropriate amount of water, and if necessary, add a buffer solution adjusted to the optimal pH for the enzyme reaction, and a heat-resistant solution. After adding the enzyme or its immobilized product, the temperature is heated to 50°C or higher, preferably 50 to 50°C, while stirring or shaking and blowing in an inert gas such as nitrogen gas.
The temperature is raised to 70°C, and the enzymatic reaction is carried out in this state. In addition, glycolipids are dissolved in the above-mentioned inert organic solvent, and in the coexistence with water and a non-reactive emulsifier such as polyvinyl alcohol or polyvinylpyrrolidone,
The hydrolysis reaction proceeds even when a non-thermostable lipase active at room temperature is applied. However, this method unavoidably increases production costs due to the use of organic solvents and emulsifiers, and also requires a long time for the hydrolysis reaction, which is economically disadvantageous. In contrast, the method of the present invention uses a thermostable enzyme, and only requires heating at 50 to 70°C, which is the level at which it shows activity.
In addition, if the melting point of glycolipids is even higher, it is 50 to 70℃.
Enzyme reactions can be easily carried out by using only the minimum amount of solvent necessary for dissolution. The progress of the hydrolysis reaction can be checked by measuring the neutralization value of released fatty acids.
In addition, according to the method of the present invention, the reaction is milder than the method for producing sugar glycerose or sialic sugar glycerol by chemical reaction, and if necessary, substrate specificity, such as 1,3-position specificity, can be obtained. Enzymes, such as lipase from Mucor miehei (manufactured by Novo Industries) among the enzymes mentioned above;
The use of lipase derived from Penicillium cyclopium (manufactured by Amano Pharmaceutical Co., Ltd.) has the advantage that the sugar glycerol or sialic sugar glycerol product can be obtained as a highly purified product with a single composition, and it is also possible to obtain the product by adjusting the appropriate reaction conditions. It is also possible to adjust the composition of these products by setting. After the reaction is completed, the aqueous layer and the oil layer are separated, and the aqueous layer is subjected to adsorption treatment, solvent fractionation treatment, spray drying treatment, etc., as necessary, to obtain sugar glycerol or sialic sugar glycerol. In addition, the oil layer contains separated fatty acids, and as mentioned above, these are physiologically active fatty acids, and it is possible to obtain purified high-purity fatty acids by performing adsorption treatment, distillation treatment, solvent fractionation treatment, etc. can. (e) Examples Example 1 By solvent fractionation and column chromatography,
Galactose and eicosapentaenoic acid (C ₂₀ : ₅ ω
-3) monogalactosyl dieicosapentaenoic acid glyceride (MGDE)
) was isolated. The melting point was 48-52°C. Three-necked flask with stirrer and condenser (1
), add 200 ml of 0.1M phosphate buffer aqueous solution (PH7.0) and lipase derived from Pseudomonas fluorescens (manufactured by Amano Pharmaceutical Co., Ltd., trade name "LipaseP").
After adding 0.4 g, the mixture was heated to 55°C while stirring and blowing nitrogen gas. When the reaction was carried out in this state, the acid value after 3 hours was AV = 71.5,
After 7 hours, AV=135. This results in
It was confirmed that an average of about 1 mol of fatty acid per MEDEG molecule was hydrolyzed in 3 hours, and about 2 mol in 7 hours. After 7 hours of reaction, the aqueous layer and oil layer were separated by centrifugation, and the water in the aqueous layer was distilled off to obtain a viscous liquid. As a result of acid hydrolysis and enzymatic analysis, this product was confirmed to be monogalactosyl glycerin. On the other hand, the oil layer was methyl esterified, and the results of GLC analysis confirmed that eicosapentaenoic acid was produced with high purity. Example 2 200 g of MEDEG prepared in Example 1, 50 ml of 0.05 M phosphate buffer (PH6.5), immobilized lipase with 1,3-position specificity derived from Mucor miehei (Novo Industries) Manufactured by
Product name "Lipase3A") 35g and n-hexane 50g
ml was placed in an Erlenmeyer flask (500 ml), heated to 65°C while blowing nitrogen gas, and shaken for 5 hours to carry out the reaction. AV after reaction = 65.0,
It was confirmed that about 1 mole of fatty acids on average per molecule was hydrolyzed. After the reaction, the reaction solution was extracted with n-butanol/water = 1/1, and eicosapetaenoic acid and monogalactosyl monoeicosapentaenoic acid glyceride were produced by thin layer chromatography of the solvent (n-butanol) layer. It was confirmed. Comparative Example 1 In Example 2, instead of the thermostable immobilized lipase with 1,3-position specificity derived from Mucor miehei, the same 1,3-derived lipase derived from Mucor miehei was used.
Replacement with position-specific non-thermostable lipase (manufactured by Novo Industries, product name “sp-225”)
When the reaction was carried out at 40°C for 10 hours, the fatty acid decomposition rate calculated from the measured acid value of free fatty acids in the reaction product was 10%, which was 46% in Example 2 (theoretical decomposition rate = 50%). ) The reaction proceeded slowly compared to Example 3 The filamentous fungus Cunninghamella elegane (NRRL1378) was cultured, and glycolipids containing γ-linolenic acid (C ₁₈ : ₃ ω-6) as a main component were obtained by solvent extraction from the cells.
The constituent fatty acids of this glycolipid were mixed fatty acids such as linoleic acid, oleic acid, stearic acid, and palmitic acid in addition to γ-linolenic acid, and the constituent sugars were a mixed system such as galactose, mannose, and glucose. The melting point was 50-55°C. Using this glycolipid as a substrate, 50 g of it was placed in the same reaction vessel as in Example 1, and 10 m of 0.2 M acetate buffer (PH6.5) was added.
M calcium chloride and 2 g of lipase derived from Humicola lanuginosa (manufactured by Amano Pharmaceutical Co., Ltd., trade name "LipaseCE") were added, stirred while blowing nitrogen gas, and heated to 55°C.
The reaction was carried out by heating. The acid value of the reactant was AV=107 after 5 hours, AV=110 after 8 hours, and the reaction was stopped after 10 hours. After the reaction was completed, the saponification value of the reactant was measured by a conventional method, and the result was SV=5, confirming that the fatty acids were almost completely hydrolyzed by the enzymatic reaction. The analysis of sugars and fatty acids in the reaction product was carried out in accordance with the method described in Example 1. Example 4 The filamentous fungus Penicillium tianium (No. 250-2 strain) biosynthesizes arachidonic acid (C ₂₀ : ₄ ω-6) within the bacterial body (Collection of lecture abstracts at the 1981 conference of the Japan Agricultural Chemical Society) ,
p191). Using homologous strains, reference (Iizuka et al.
EJAppl.Micrbiol.Biotechnol., 7 , 173,
(1979)), the bacterial cells were extracted with solvent, and the constituent fatty acids were arachidonic acid, linolenic acid,
Glycolipids (melting point 50-55°C) such as linoleic acid whose constituent sugars are caractose and glycol were obtained.
5g of this glycolipid was added to 3g of n-hexane under a nitrogen atmosphere.
Dissolve in 0.1M phosphate buffer (PH7.0), Candida cylindracea (Candida
cylindracea) (manufactured by Meito Sangyo Co., Ltd.)
Product name "Lipase OF") 0.7g and diatomaceous earth
1.0 g was added and the reaction was carried out in the same manner as in Example 3 at 50°C. The acid value after 7.5 hours of reaction is AV = 125,
It was confirmed that hydrolysis proceeded almost completely by the method described in Example 3. Furthermore, analysis of sugars and fatty acids in the reaction product was conducted according to Example 1. Example 5 Glycolipids (melting point 50-60°C) were isolated from rapeseed seeds using column chromatography according to the method in the literature (edited by the Japanese Biochemical Society, Biochemistry Experiment Course 3, Chemistry of Lipids, p.572 (1974)). I let go. The constituent fatty acids were linoleic acid, oleic acid, stearic acid, palmitic acid, etc., the constituent sugars were mainly galactose, and the main constituents were mono- and digalactosyl diglycerides. Using this glycolipid, a reaction was carried out in the same manner as in Example 4, and the fatty acid decomposition rate after about 5 hours was 96%. The reaction product was treated in the same manner as in Example 1 to obtain mono- and dicalactosyl glycerin from the aqueous layer and the same fatty acid mixture as above from the oil layer. (f) Effects of the Invention The present invention easily decomposes fatty acids of glyceroglycolipids extracted from natural living organisms under mild conditions using a heat-stable fat hydrolase to convert them into sugar glycerol or acyl sugar glycerol. This is a reforming method that has an extremely small thermal history compared to conventional chemical decomposition or synthesis methods, and therefore almost completely suppresses side reactions such as polymerization, isomerization, cyclization, and decomposition during the reaction. Therefore, the sugar glycerol or acyl sugar glycerol produced is of excellent quality and is highly safe for use in cosmetics, pharmaceuticals, etc. It is suitable for use as an emulsifier or surfactant in fields where it is required. Furthermore, the method of the present invention does not require auxiliary raw materials such as organic solvents and emulsifiers compared to reactions using conventional enzyme methods, and the reaction can proceed easily and almost completely in a short period of time. , the economic benefits are great. Furthermore, the method of the present invention makes it possible to separate physiologically active polyunsaturated fatty acids without deterioration.

Claims (1)

[Scope of Claims] 1. Sugar glycerol or acyl sugar glycerol and acyl sugar glycerol, characterized in that a heat-stable fat hydrolase is made to act on one or more glyceroglycolipids represented by the following formulas () to (). Method for producing fatty acids. [Formula] [Formula] [Formula] [Formula] (wherein R ₁ and R ₂ are both saturated or unsaturated fatty acid residues having 7 to 23 carbon atoms, and G is the residue of a monosaccharide or its dimer. 2. The production method according to claim 1, wherein at least one of R ₁ and R ₂ contains a residue of eicosapentaenoic acid, γ-linolenic acid, or arachidonic acid. 3. The production method according to claim 1, wherein the glyceroglycolipid is contained in lipids derived from natural products such as microorganisms, algae, fish, higher animals and plants. 4. Claim 1, wherein the heat-stable fat hydrolase is a heat-stable lipase that is active even at temperatures above 50°C.
Manufacturing method described in section. 5. The production method according to claim 4, wherein the thermostable lipase is an immobilized product.