JPS6222597A - Production of saccharide glycerol and fatty acid - Google Patents

Abstract

PURPOSE:To easily produce a saccharide glycerol or an acyl-saccharide glycerol and fatty acid ester in a short time without heat history, by hydrolyzing a fatty acid of a glyceroglycolipid in the presence of an enzyme for hydrolyzing fats and oils. CONSTITUTION:One or more of glyceroglycolipids shown by formulas contained in lipids derived from natural materials such as micro-organisms, algae, fishes, higher animals, etc., is treated with a thermostable enzyme for hydrolyzing fats and oils. The enzyme is a thermostable lipase active even at >=50 deg.C and the lipase is an immobilized lipase. A reactor is charged with the glycolipid, an organic solvent, the thermostable enzyme, etc., and they are reacted at >=50 deg.C. After the reaction is over, a saccharide glycerol or acylsaccharide glycer ol can be obtained from an aqueous layer and a fatty acid from an oil layer.

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 an oil-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, lipids from microorganisms such as mold, yeast, and bacteria, algae such as seaweed and chlorella, and even fish are in the form of glycolipids (hereinafter simply referred to as glycolipids) and phospholipids (hereinafter simply referred to as phospholipids). These so-called polar lipids are expected to be effectively utilized. Among these, glyceroglycolipids include, for example, mono- or digalactosylglyceride, mono- or diglucosylglyceride in algae,
It exists in molecular structures such as mono-, dimannosylglyceride, and sulfopyranosylglyceride, and its bound fatty acids include eicosapencitric acid and docosahexaenoic acid.

There are many highly unsaturated fatty acids such as γ-lylunic acid and arachidonic acid, and many of them are important biologically active lipids that exist as components of cell membranes and cell 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 acids can be removed by hydrolysis using an acid or alkali catalyst, but with this method, the highly unsaturated fatty acids deteriorate due to the action of the acid or alkali, leading to peroxidation, rancidity, and polymerization.
It causes coloration and makes post-treatment purification extremely difficult.

It is also difficult to control these catalytic actions to obtain products with preferred structural formulas.

On the other hand, in the enzymatic method, lipase is an enzyme that usually hydrolyzes mono-, di-, or triglycerides (all are esters of fatty acids only), and the conversion can be carried out under mild reaction conditions without altering or degrading the substrate. is possible, but
Due to the substrate specificity of lipase, when the substrate has a sugar chain or even a long-chain highly unsaturated fatty acid, the reaction does not proceed at all or the rate of hydrolysis is only small. 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 a long time, enzymes made from such glycolipids have not been applied.

In addition, if one attempts to chemically synthesize glycerosugar esters, it is usually necessary to mix glycerin and the Ws and etherify them by heating, and if necessary, mix them with fatty acids and esterify them by heating. Due to their thermal history, polyunsaturated fatty acids with a large number of sugars and double bonds undergo side reactions such as polymerization, isomerization, oxidation, cyclization, and decomposition due to their thermal history, resulting in complicated purification processes. It is difficult to obtain the high purity sugar glycerols or acyl sugar glycerols that are needed and ultimately desired.

Means for Solving the +d1 Problems In view of the current situation, the present inventors have proposed that glyceroglycolipids existing in living organisms such as natural animals, plants, and even microorganisms can be decomposed by peroxide 1 without harsh reaction conditions. As a result of extensive research aimed at removing fatty acid groups and modifying them into sugar glycerol or acyl sugar glycerol without causing side reactions such as cyclization, we found that heat-stable fat hydrolase can be used in a mild reaction. The present inventors have discovered a method for producing sugar glycerol or acyl sugar glycerol and fatty acids, which allows the fatty acid group to be easily removed by acting under these conditions, and have completed the present invention.

The present invention will be explained in detail below.

The glyceroglycolipids used in the present invention include, among lipids derived from natural products, microorganisms such as molds, yeasts, and bacteria, and algae such as seaweed and chlorella.

It is contained in lipids extracted from living organisms such as freshwater or saltwater fish and higher animals and plants. Its general structural formula is shown in the following formulas (I) to (TV), where fatty acid residues R1 and R2 are both carbons CH20CO-R1 C11-0-G (IV) C1+□-0-G Number is a saturated or unsaturated fatty acid residue of 11 to 23, and G is a residue of 18 or a dimer thereof. Such fatty acids include octane (
Ca+o), laurin 62 (Chi:o), noglumitin full (CI610), palmitooleic acid CCIb5 summer), stearic acid (CIs +.), olein # (Chi, I), linoleic acid (C+s+z),
Rilunic acid (Chi, 3), behenic acid (Czz+o)
, erucic acid (Czz+, +), eicosapentaenoic acid <ct. 8. ), docosahexaenoic acid CCtz=b)
, arachidonic acid (Czo+a).

Examples include tetracosatetraenoic acid (Cz4+4), and examples of sugars include xylose and glucose.

Examples include neutral monosaccharides such as fructose, mannose, galactose, and fucose, acidic monosaccharides such as crylefuronic acid, amino monosaccharides such as glucosamine and galactosamine, and dimers thereof. In addition, when at least one of the fatty acid residues R+ and R2 is a residue of eicosabencitric acid, T-lyrinnic 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 dieicosapentaenoic acid glyceride, and have a melting point of 50 to 60°C. For this reason, fat and oil hydrolase, which has traditionally been used to hydrolyze fats and oils (
Coupled with the fact that they are thermally stable for long periods of time (optimum temperature: 30-45°C), it is difficult to hydrolyze such glycolipids to produce sugar glycerol or acyl sugar glycerol and fatty acids. Although this has never been done, the present invention has made this 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.
However, it is convenient to use microorganism-derived products. For example, in the Pseudomonas genus, Pseudomonas fluorescens
lipase derived from C. fluorescens (for example, manufactured by Ohno Pharmaceutical Co., Ltd., trade name rLipase PJ) +
In the Candida genus, Candida 'cyl 1nd
racea) derived lipase (e.g. Disaccharide Sangyo Co., Ltd.)
manufactured by Ohno Pharmaceutical Co., Ltd., manufactured by the product name "Lypase-0FJ", manufactured by Ohno Pharmaceutical Co., Ltd., manufactured by the product name "LypaseAYJ";
Pase L J ), Penicillium spp.
Penicillium cyclopium (P cillium)
enicillium cyclopium) (e.g. manufactured by Ohno Pharmaceutical Co., Ltd., trade name: rLypag)
eGT (G) J), alkaline earth metal (^lc
aligenes), lipase derived from the strain No. 3783 (for example, produced by Disaccharide Sangyo Co., Ltd., product name
Lypage-PL) + Humicola genus (1 (um
icola) and Humicola lanuginosa (11u
micola Ianuginosa) (for example, manufactured by Ohno Pharmaceutical Co., Ltd., trade name rLypase)
CEJ).

In the genus Mucor, Mucor mino 1
(Mucor m1ehei) (for example, manufactured by Novo Industries, trade name Lypase)
3 A J), and in the Rhizopus genus, Rhizopus chinensis (Rhizopus chinensis).
Lipase derived from P. nensis (Japanese Unexamined Patent Publication No. 59-1562
Heat-resistant lipase prepared by the method described in 82). Note that the present invention is not limited to microorganisms and lipases from one of these genera, 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.
It is also possible to increase heat resistance by increasing the molecular weight, and a heat-resistant lipase obtained by such modified scratching can also be used.

Next, the above thermostable enzyme can also be used as an immobilized product. Generally, enzymes are made into water-insoluble immobilized substances for the purpose of improving pH and temperature stability, maintaining activity, and reuse, but cellulose is used as a carrier for immobilization.

Dextran, polystyrene, polyacrylamide, polyvinyl alcohol, ion exchange resin, magnetic material, activated carbon, alumina, photocrosslinkable resin, alginate, etc. are used. In the present invention, it is possible to use a thermostable enzyme that is adsorbed, ionically bonded, coextensively bonded, or enclosed in these immobilization carriers.

For example, the method of immobilizing thermostable lipase derived from Mucor m1ehei to a porous anion exchange resin described in JP-A No. 60-98984, the method described in JP-A-52-104506, Candida cylinrace
An immobilized enzyme can be used, such as by adsorbing heat-stable lipase derived from A. dracea to diatomaceous earth powder.

Glycolipids are hydrolyzed by the method of the present invention as follows. That is, a glycolipid having a composition of one or more of the above structural formulas (1) to (rV) is placed in a glass or stainless steel container, and if necessary, a minimum amount of an inert organic solvent such as hexane is added. , dissolve the glycolipids in heptane, etc., add an appropriate amount of water, and if necessary, a buffer adjusted to the optimal pH for the enzyme reaction, and a heat-stable enzyme or its immobilized product, and then stir or shake. While blowing an inert gas such as nitrogen gas, the temperature is raised to 50° C. or higher, preferably 50 to 70° C., and the enzyme reaction is carried out in this state.

Furthermore, even if glycolipids are dissolved in the above-mentioned inert organic solvent and a non-thermostable lipase active at room temperature is applied in the presence of water and a non-reactive emulsifier such as polyvinyl alcohol or polyvinylpyrrolidone, the hydrolysis reaction will not occur. proceed. However, this method inevitably 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, in the method of the present invention, a thermostable enzyme is used, and only a heating treatment of 50 to 70''C is sufficient to show activity, and if the melting point of the glycolipid is even higher, it is dissolved at 50 to 70''C. The enzymatic reaction can be easily carried out by using only the minimum amount of solvent necessary for the reaction.The progress of the hydrolysis reaction can be checked by measuring the neutralization value of the liberated fatty acids.Also, the present invention According to the method, the reaction is milder than the method for producing sugar glycerose or acyl sugar glycerol by chemical reaction, and if necessary, an enzyme with substrate specificity, e.g. 1.3-position specificity, e.g. Among the enzymes mentioned above, lipase derived from Mucor mlehei (manufactured by Novo Industries).

The use of lipase derived from Penicillium cyclopium (manufactured by Amano Pharmaceutical Co., Ltd.) has the advantage that the sugar glycerol or acyl sugar glycerol product can be obtained as a highly purified product with a single composition, and furthermore, suitable reaction conditions can be 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 acyl sugar glycerol. In addition, the oil layer contains separated fatty acids, which, as mentioned above, are physiologically active fatty acids, and are treated with adsorption treatment.

Refined high-purity fatty acids can be obtained by performing distillation treatment, solvent fractionation treatment, etc.

(el Examples Example 1 Glycolipids consisting of galactose and eicosapencitrate (Czo+sω-3), monogalactosyl dieicosapencitrate, were extracted from marine chlorella cells, a type of green algae, by solvent fractionation and column chromatography. Acid glyceride (hereinafter abbreviated as MGDEG) was isolated. The melting point was 48 to 52°C. 200 g of MGDII!G was placed in a three-flow flask (11) equipped with a stirrer and a cooling tube, and 0° 1M phosphate buffer was added. Aqueous solution (pH 7,0) 20
0II11 and Pseudomonas 0 fluorescens (
Lipase derived from Pseudomonas fluorescens (manufactured by Amano Pharmaceutical Co., Ltd., trade name: “Lipas”)
After adding 0.4 g of eP J ), 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, the voltage became 8V = 135. As a result, it was confirmed that on average about 1 mol of fatty acid per MHDEG 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. Motojima conducted acid hydrolysis and enzyme analysis of this, and confirmed that it was monogalactosyl glycerin. On the other hand, the oil layer was methyl esterified, and the results of GLC analysis confirmed that highly pure icosabene citric acid was produced.

Example 2 MEDEG prepared in Example 1 200 g, 0.0
5M phosphate buffer (pH 6,5) 50II11. Immobilized lipase with 1,3-position specificity derived from Mucor m1ehei (manufactured by Novo Industries, trade name rLipase3 AJ) 3
5 g and 50 ml of n-hexane in an Erlenmeyer flask (5
00ml) and heated to 65°C while blowing nitrogen gas.
The reaction was carried out by heating to 5 hours and shaking for 5 hours. The AV after the reaction was 65.0, confirming that about 1 mole of fatty acid on a molecular average was hydrolyzed. After the reaction is completed, the reaction solution is
-Butanol/water = 1/1 extraction, and thin layer chromatography of the solvent (n-butanol) layer confirmed that rieicosapentaenoic acid and monogalactosyl monoeicosapentaenoic acid glyceride were produced.

Comparative Example 1 In Example 2, Mucor Mihai (Mucor
-miehei) instead of the thermostable immobilized lipase with 1.3-position specificity, the same 1.3-position lipase derived from Mucor m1ehei
-Replacement with position-specific non-thermostable lipase (manufactured by Novo Industries, trade name: rsp-225J)
When the reaction was carried out at 0°C for 10 hours, the fatty acid decomposition rate calculated from the measured value of the acid value of free fatty acids in the reaction product was 10%, which was 46% (theoretical decomposition rate = 5) in the case of Example 2.
(0%), the reaction proceeded slowly.

Example 3 Filamentous fungus cunningha elegans
mel laeleganse, NRRL 1378)
γ-lylunic acid (C+a
+:+ ω-6) was obtained as a main component. The constituent fatty acids of this glycolipid are linoleic acid, γ-lylunic acid,
oleic acid.

Mixed fatty acids such as stearic acid and palmitic acid,
The constituent sugars were a mixture of 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 0.2M acetate buffer (pH 6.5), 1
0mM calcium chloride and lipase derived from Humicola lanuginosa (manufactured by Ohno Pharmaceutical Co., Ltd., trade name: rLipasec)
EJ) 2 g was added thereto, and the mixture was stirred and heated to 55° C. while blowing nitrogen gas to carry out a reaction. The acid value of the reactant was AV=10 after 5 hours and 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 found to be 5V=5, confirming that the fatty acid was 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 thianium
umtianium + N11L250 2 strains) biosynthesizes arachidonic acid (C2014ω-6) within the bacterial body (Japan Agricultural Chemicals 1981 Conference Abstracts, p1
91).

Using homologous strains, reference materials (Iizuka et al.
E.J.

App18Microbio1. Biotechno
l, +1,173. (1979)), the bacterial cells were extracted with solvent, and the constituent fatty acids were arachidonic acid, lylunic acid, linoleic acid, etc., and the constituent sugars were galactose,
Glycolipids such as glucose (melting point 50-55°C) were obtained. 5 g of this glycolipid was added to n-hexane 3 in a nitrogen atmosphere.
Dissolve in 0.1M phosphate buffer (pH 7,0)
.

Candida cyl
0.7 g of lipase (manufactured by Disaccharide Sangyo Co., Ltd., trade name [Lipase 0FJ)] and 1.0 g of Keosou earth were 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 was AV = 125, indicating that hydrolysis was almost complete by the method described in Example 3.
□Confirmed that progress had been made. 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 by column chromatography according to the method described in the literature (Japanese Biochemistry Complete Edition, Biochemistry Experiment Course 3: Chemistry of Lipids, p572 (1974)). did. Constituent fatty acids are linoleic acid,
The constituent sugars, such as oleic acid, stearic acid, and palmitic acid, were mainly galactose, and mono- and digalactosyl diglycerides were the main components. Using this glycolipid,
The 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 reactants were treated in the same manner as in Example 1 to remove mono- and digalactosyl glycerin from the aqueous layer.
A fatty acid mixture similar to the above was also obtained 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 oil-fat hydrolase. Compared to conventional chemical decomposition methods or synthesis methods, this method has an extremely small thermal history, and therefore almost completely eliminates side reactions such as polymerization, isomerization, and entrainment. 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 the conventional enzymatic reaction, and the reaction can proceed easily and almost completely in a short period of time, making it economical. The benefits are huge.

Furthermore, the method of the present invention makes it possible to separate physiologically active polyunsaturated fatty acids without deterioration.

Claims (5)

[Claims] (1) Sugar glycerol or acyl sugar glycerol and fatty acid, which is characterized by causing a heat-stable fat hydrolase to act on one or more glyceroglycolipids represented by the following formulas (I) to (IV). Manufacturing method. ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (III) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ ( IV) (In the formula, R_1 and R_2 are both saturated or unsaturated fatty acid residues having 7 to 23 carbon atoms, and G is a residue of a monosaccharide or a dimer thereof.) (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) The production method according to claim (1), wherein the heat-stable fat hydrolase is a heat-stable lipase that is active even at 50°C or higher. (5) The production method according to claim (4), wherein the thermostable lipase is an immobilized product.