JPH0724588B2 - Method for producing esters - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a fatty acid ester by reacting a higher fatty acid with an alcohol using an immobilized enzyme according to a comprehensive method.

[Prior Art] It is already known that a hydrolase such as lipase performs a reverse reaction, that is, a synthetic reaction of ester. However, in this reaction, the yield of fatty acid ester is low, the enzyme is unstable, and it is difficult to separate and recover the enzyme after the reaction, so that the enzyme cannot be reused. Have Therefore, the fact is that a method for producing a fatty acid ester, which is excellent in stability, productivity, and economical efficiency and can be industrially carried out using an enzyme, has not yet been found.

In addition, methods for immobilizing enzymes include adsorption method, covalent feeding method, microcapsule method, encapsulation method, etc. Among them, encapsulation method has less enzyme leakage, is easy to immobilize, and is easy to industrialize. The soft immobilization condition is characterized by less enzyme deactivation.

[Problems to be Solved by the Invention] Therefore, an attempt was made to synthesize fatty acid esters using an enzyme in which a hydrolase such as lipase was immobilized by the encapsulation method, but the immobilized carrier became a barrier. Therefore, there is a problem that the permeation / diffusion of the substrate is deteriorated, and the synthetic reaction rate becomes extremely slow.

[Means for Solving Problems] As a result of intensive studies to solve the above-mentioned problems, the present inventors have used hydrolase entrapped and immobilized by a specific photocurable resin. They have found that the problem can be solved by reacting a higher fatty acid with an alcohol, and completed the present invention.

Thus, according to the present invention, in a method for producing an ester from an acid and an alcohol by an enzymatic reaction, a hydrolase as an enzyme and at least two ethylenically unsaturated bonds in one molecule and a hydrophilic parent. Provided is a method for producing an ester, which comprises using an immobilized enzyme obtained by irradiating a mixture with a photocurable resin having an oily balance (hereinafter abbreviated as "HLB") of 11 or less with active light. .

Hereinafter, the method of the present invention will be described in more detail.

Photocurable resin In the method of the present invention, the immobilizing carrier has at least two ethylenically unsaturated bonds in one molecule, and
A photocurable resin having an HLB of 11 or less, preferably 9 or less is used.

The higher fatty acid which is the substrate of the reaction in the present invention is hydrophobic, but alcohols generally have various types from hydrophobic to hydrophilic, and therefore, the most suitable photo-curing agent depending on each substrate is used. It is necessary to select a functional resin. That is, it is extremely important to select a photocurable resin having a well-balanced hydrophilicity and hydrophobicity.

There are two methods for adjusting the HLB of the photocurable resin: one method using one resin and one method using two or more resins. When one type of resin is used, the ratio is adjusted by arbitrarily changing the ratio of the hydrophobic group and the hydrophilic group in the resin. When two or more kinds of resins are used, the HLB may be adjusted to 11 or less by mechanically mixing the hydrophobic photocurable resin and the hydrophilic photocurable resin in an arbitrary ratio.

Hereinafter, typical photocurable resins used in the present invention will be described. These resins include hydrophilic resins having an HLB of 11 or more, but as mentioned above, they are used in combination with a hydrophobic resin in the range where the HLB is 11 or less.

The HLB of the photocurable resin is measured by the following method.

30 ml of mixed solvent of dioxane: benzene = 96: 4 (weight ratio)
Dissolve 1 g of the photocurable resin in it, and titrate this with water to obtain ml (number of water) of water at the point where it begins to become cloudy. Separately, the HLB of a resin is determined from the relationship between the HLB of known HLB-active agents and the number of water [See Seibundou Shinkosha (1962), Yoshio Ishii, Nonionic Surfactants, pages 145-146. ] (I) Unsaturated polyesters; at least one unsaturated polyvalent carboxylic acid such as maleic anhydride, maleic acid, fumaric acid, itaconic acid, itaconic anhydride, or these and trimellitic acid, trimellitic anhydride, The acid value obtained by esterification of a polyvalent carboxylic acid component composed of at least one saturated polycarboxylic acid such as pyromellitic acid and pyromellitic dianhydride with a polyhydric alcohol is less than 200 (acid value 100 or less HLB11 or less) unsaturated polyester (number average molecular weight is about 1,000 or more, preferably about 3,000)
Above); at least one unsaturated polyvalent carboxylic acid such as maleic anhydride, maleic acid, fumaric acid, itaconic acid, and itaconic anhydride, and at least 5 polyhydric alcohols having more than 3 hydroxyl groups in one molecule. Unsaturated polyesters (number average of 200 or less (acid value 100 or less corresponds to HLB 11 or less)) obtained by reacting an acid anhydride with the hydroxyl group in the esterified product or polyhydric alcohol containing 100% by weight Molecular weight of about 1,000 or more, preferably about 30,
00 or more).

(Ii) Unsaturated epoxides; Epicoat 828,1001,1004 (manufactured by Shell Chemical Co., having nmole of glycidyl group,
Product name) and other polyvalent glycidyl compounds and (n-2) moles of carboxyl group-containing polyvalent carboxylic acid such as maleic acid, adipic acid, trimellitic acid and 2 moles of (meth)
Unsaturated epoxides with an acid value of less than 200 (acid value of 100 or less corresponds to HLB 11 or less) obtained by adding an acid anhydride to the residual hydroxyl group of the addition reaction product with an unsaturated carboxyl compound such as acrylic acid; n A compound obtained by adding an acid anhydride to a hydroxyl group remaining in the addition reaction product of the polyvalent glycidyl compound having a mole of glycidyl group and the polyvalent carboxylic acid having a (n + 2) mole of a carboxyl group to (meth) acryl Acid value less than 200 (acid value 100 or less is HLB 1 by reacting unsaturated glycidyl compound such as glycidyl acid
1 or less) unsaturated epoxides.

(Iii) Anionic unsaturated acrylic resins; the anionic unsaturated acrylic as used herein is a copolymer of (meth) acrylic acid and (meth) acrylic acid ester, and C + 5P + 10S = A (1) [here C is the concentration of the carboxyl group in the resin (mol / k
g), P is the concentration of phosphoric acid groups in the resin (mol / kg), and S is the concentration of sulfonic acid groups in the resin (mol / kg). ] A in the formula (1) is less than 5.0 (mol / kg) (1.0 or less is HLB 11
The following applies), and refers to a resin in which the concentration of the photopolymerizable ethylenically unsaturated group in the resin is 0.1 to 5 (mol / kg). The copolymer of (meth) acrylic acid and (meth) acrylic acid ester is synthesized by a known method. To introduce a carboxyl group into the resin, an unsaturated carboxyl compound such as (meth) acrylic acid is used. To introduce a phosphoric acid group, Phosmer M, Phosmer cl (both are products of Fats and Oil Products Co., Ltd.) In order to introduce a sulfonic acid group, unsaturated phosphoric acid ester of (meth) acrylic acid-2-sulfoethyl, (meth) acrylic acid-3-sulfopropyl, and other unsaturated sulfonic acid ester copolymerizable ethylene It is possible to introduce an unsaturated unsaturated group by reacting an unsaturated glycidyl compound such as glycidyl (meth) acrylate with a carboxyl group, a phosphoric acid group or a sulfonic acid group present in the copolymer.

(Iv) Cationic unsaturated acrylic resins: 2-diethylaminoethyl (meth) acrylate, -tert-butylaminoethyl (meth) acrylate, unsaturated amino compounds such as vinylpyridine in any proportion (10% by weight). Below is HL
(Under B 11) Unsaturated acrylic resin obtained by reacting (meth) acrylic acid ester copolymer containing unsaturated glycidyl compound such as glycidyl (meth) acrylate, after chloromethylation of polystyrene, unsaturated amino compound Examples thereof include unsaturated acrylic resins quaternized with and adducts of polyethyleneimine and unsaturated glycidyl compounds.

(V) Polyesters of polyalkylene glycol and (meth) acrylic acid; polypropylene glycol (meth) having a molecular weight of 400 to 10,000 and an ethylene oxide group (55% by weight or less corresponds to HLB 11 or less) in an arbitrary ratio.
Diesters of unsaturated monocarboxylic acids such as acrylic acid.

(Vi) Urethane-added product of polyalkylene glycol and 2-hydroxyethyl (meth) acrylate; diisocyanates such as n moles of tolylene diisocyanate, xylylene diisocyanate, hexamethylene diisocyanate and isophorone diisocyanate, and n-1 mole of optional. Ethylene oxide group (55% by weight or less is HLB
(Applicable to 11 or less) and urethane compounds with 2 mol of unsaturated monohydroxy compounds such as 2-hydroxyethyl (meth) acrylate; 1 mol of trifunctional hydroxy compounds such as trimethylolpropane and 4 mol Diisocyanate, 2 moles of polypropylene glycol containing an ethylene oxide group (55% by weight or less corresponds to HLB 11 or less) at an arbitrary ratio, and 2 moles of an unsaturated monohydroxy compound such as 2-hydroxyethyl (meth) acrylate. Examples thereof include carboxylated urethane compounds obtained by reacting an acid anhydride such as maleic anhydride or succinic anhydride with the urethane compound.

Other examples include unsaturated celluloses and unsaturated polyamides.

Among these photocurable resins, those which can be particularly advantageously used in the present invention include the polyesters of the above (v) polyalkylene glycol and (meth) acrylic acid and (vi) the polyalkylene glycol ( Examples thereof include urethanized adducts with 2-hydroxyethyl (meth) acrylate.

Further, a photopolymerization initiator (photosensitizer) is used in combination with the above-mentioned photocurable resin for the purpose of promoting the photopolymerization reaction.
The photopolymerization initiator that can be used is decomposed by irradiation with light to generate a radical, which is a polymerization initiation species to cause a crosslinking reaction between resins having a polymer unsaturated group,
For example, α-carbonyl alcohols such as benzoin, acetoin, α-hydroxyisobutylphenone; benzoin methyl ether, benzoin propyl ether,
Acyloin ethers such as anisoin ethyl ether and piroparoin ethyl ether; polycyclic aromatic compounds such as naphthol and hydroxyanthracene; α-substituted acyloins such as methylbenzoin and α-methoxybenzoin; 2-cyano-2 Azoamide compounds such as butylazoformamide; metal salts such as lauryl nitrate and ferric chloride; mercaptans; disulfides; halogen compounds; dyes and the like.

These photopolymerization initiators are usually used alone or in combination of two or more kinds.
It can be used at a rate of 0.01 to 10 PHR (Parts hundred Resin).

Enzymes Enzymes are hydrolases, especially lipases (enzyme number, 3.1.
1.3) is used. The lipase used may be of animal origin or microbial origin and is not particularly limited. For example,
Aspergillus, Rhizopus, Pseudomonds, Enterobacterium, C
hromobacterium, Geotrichum, Penicillium, Mucor, Candid
Examples thereof include lipase derived from genus a. These enzymes do not necessarily have to be isolated and used, and for example, crude enzymes such as buncreatin can be used as they are, or commercially available enzyme preparations containing lipase can be used as they are.

Preparation of Liquid Composition Composed of Enzyme and Photocurable Resin The components of the photocurable resin, photopolymerization initiator and enzyme described above can be sufficiently mixed with each other to form a liquid composition. The enzyme may be dispersed as a bulk powder, or may be dispersed and used as an enzyme aqueous solution. Further, it is also possible to disperse and use those in which the enzyme has been adsorbed in advance, such as Celite, quartz sand, clay, activated carbon, colloidal silica, and alumina.

When the resulting liquid composition has a high viscosity (10 poise or more), the photocurable resin can be dissolved or dispersed, but it is convenient for handling to dilute the immobilization product with a solvent that does not dissolve it. Examples of these solvents include
Water; organic solvents such as petroleum benzine, heptane, octane, hexane, petroleum ether, ethyl acetate, alcohol;
Fatty acids such as oleic acid, linoleic acid and linolenic acid;
Examples include oils and fats such as olive oil, palm oil, and coconut oil. These may be used alone or as a mixed solution of two or more kinds. Preferably, it is desirable to dilute with the substrate in the reaction.

The use ratio of the constituent components of the liquid composition is not strictly limited and can be varied over a wide range according to the type of each component, etc., but generally, the photocurable resin 100
It is appropriate to use the photopolymerization initiator and the enzyme in the following proportions based on parts by weight. (The range in parentheses is a suitable range.) Photoinitiator: 0.1 to 5 parts by weight (0.5 to 2 parts by weight) Enzyme: 0.001 to 50 parts by weight (0.01 to 20 parts by weight) Diluting the photocurable resin Solvent that can be used: 0 to 1,000 parts by weight (0 to 200 parts by weight) Molding of liquid composition The liquid composition prepared as described above is molded into a lump, a sheet, or a granule. The lumps, sheets, and granules are molded by the following method. Lumped; mainly molded by molding. Sheet: Molded by pouring a liquid composition into a polypropylene film and sandwiching the top and bottom with the film. Granular: Generally, since the liquid composition has a large hydrophobicity, it is molded by a method as described in Japanese Patent Application No. 61-6815. When it is preferable to increase the hydrophilicity of the photocurable resin depending on the substrate to be used, the method described in JP
It may be molded by the method described in Japanese Patent No. 59-11182.

Photocuring The molded product produced as described above is irradiated with an actinic ray to cure the photocurable resin in the molded product,
Gel into lumps, sheets, or granules. As a result, an immobilized product of the enzyme having high mechanical strength can be obtained.

The wavelength of the actinic ray that can be used for the above-mentioned photo-curing varies depending on the type of the photo-curable resin contained in the molded product, etc., but generally a light source that emits light having a wavelength in the range of about 250 to about 600 nm is used. Advantageously, it is used for irradiation. Irradiation time can be generally within the range of about 0.5 to about 10 minutes. Higher fatty acid, higher fatty acid as a substrate for alcohol ester synthesis, saturated or unsaturated fatty acid having 8 to 22 carbon atoms is suitable. . Examples are as follows. Caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid,
Stearic acid, arachidic acid, behenic acid, caproleic acid, lindelic acid, myristoleic acid, palmitoleic acid, oleic acid, caderic acid, erucic acid, decagenic acid, linoleic acid, hiragoic acid, linolenic acid, eicosatrienoic acid, docosatrienoic acid , Hexadecatetraenoic acid,
Stearidonic acid, arachidonic acid, docosatetraenoic acid,
Eicosapentaenoic acid, sardine acid, sabinic acid, iprolic acid, yarapinolic acid, ricinoleic acid, ferronic acid, etc.

In addition, the following are listed as main alcohols.

Monohydric alcohol; methanol, ethanol, 1-propanol, isobutanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-decanol, 1-dodecanol, oleyl alcohol, benzyl alcohol, Keraniol, citronellol, farnesol, 3-pentanol, 2-hexanol, 3-hexanol, l-menthol. Dihydric alcohol; ethylene glycol, trimethylene glycol, 1,
4-butanediol, 1,5-pentadiol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol. Trihydric alcohol; glycerin, trimethylolethane, polyhydric alcohol; diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, heptaglycerin, octaglycerin, nonaglycerin, decaglycerin sugar and sugar alcohol; glucose, fructose, galactose , Maltose, sucrose, cellobiose, lactose, maltotriose, cellotriose, dextrin, cyclodextrin, mannan, cellulose, arabitol, sorbitol, xylitol, mannitol, lactitol, etc.

Reaction method The immobilization enzyme obtained by the above method is added to higher fatty acids and alcohols mixed at an arbitrary ratio and incubated at 15 to 70 ° C, preferably 30 to 40 ° C. In order for lipase to carry out the ester synthesis reaction, it is preferable that there is a small amount of water. At this time, if water is added to the substrate, the equilibrium proceeds in the direction of hydrolysis, and the final synthesis rate decreases. Therefore, if water is added to the liquid composition in advance or the photocured immobilized enzyme is immersed in water, the reaction rate can be increased without lowering the final synthesis rate.

[Action and effect] By using a photo-effect resin of HLB 11 or less and immobilized with a hydrolase such as lipase, permeation and diffusion of higher fatty acid and alcohol in the photo-curable resin are facilitated. It is possible to provide a place for reaction. Further, the enzyme is kept stable, the enzyme can be separated and recovered, and can be used repeatedly. Furthermore, the esters can be continuously synthesized in high yield, and application to a bioreactor becomes possible.

EXAMPLES Next, the present invention will be further described with reference to examples.

Example 1 Polypropylene glycol 2,000 having a number average molecular weight of about 4,000
100 parts by weight of a photo-curable prepolymer (HLB: 5) consisting of 1 mol (222 g) of isophorone diisocyanate and 1 mol (130 g) of 2-hydroxyethyl methacrylate, and α-
Add 0.5 parts by weight of hydroxyisobutylphenone and 5 parts by weight of enzyme lipase (as the bulk powder) and disperse well. The photocurable resin-enzyme mixture obtained was dropped into a 1% by weight polyethylene glycol octylphenyl ether aqueous solution and then irradiated with actinic rays to obtain a particulate immobilized enzyme. Further, this granular immobilized enzyme was immersed in water for 16 hours to absorb water in the resin.

To a mixed solution of 100 parts by weight of oleic acid and 41 parts by weight of 1-heptanol, 5 parts by weight of the above particulate immobilized enzyme was added, and the mixture was heated at 30 ° C. for 500
When the reaction was performed in 5 hours at pm, 63%, 81% in 20 hours,
90% of oleic acid heptanol ester was obtained in 45 hours.

Example 2 Polypropylene glycol 2,000 having a number average molecular weight of about 4,000
67 parts by weight of a photocurable prepolymer (HLB: 5) consisting of 1 mol (222 g) of isophorone diisocyanate and 1 mol (130 g) of 2-hydroxyethyl methacrylate, 2,000 g of polyethylene glycol having a number average molecular weight of about 4,000, and isophorone. 33 parts by weight of a photocurable (resin) prepolymer (HLB: 17) consisting of 1 mol (222 g) of diisocyanate and 1 mol (130 g) of 2-hydroxyethyl methacrylate are thoroughly mixed. To 10 parts by weight of the photocurable resin mixture, 5 parts by weight of oleic acid and 5 parts by weight of glycerin were added and mixed well. To 20 parts by weight of this liquid, 1 benzoin ethyl ether
Add parts by weight and 8 parts by weight of enzyme lipase and disperse well.
The resulting photocurable resin-enzyme mixture was molded into a sheet having a thickness of 0.5 mm and irradiated with actinic rays to obtain a film-shaped immobilized enzyme. This membrane-shaped immobilized enzyme was used after being cut into a size of 2 mm × 2 mm and immersed in water for 12 hours.

When 10 parts by weight of the above-mentioned membrane-immobilized enzyme was added to a mixed solution of 100 parts by weight of oleic acid and 100 parts by weight of glycerin, and the reaction was carried out at 30 ° C. and 300 rpm, 65% in 24 hours and 91% in 72 hours Got

In addition, repeated use was performed 5 times under the above conditions. As a result, the reaction rate of the 5th time was about 92% as compared with the 1st time, and repeated use was possible.

Example 3 Polypropylene glycol 1,700 having a number average molecular weight of about 4,000
g, polyethylene glycol 300 g, isophorone diisocyanate 1 mol (222 g) and 2-hydroxyethyl methacrylate 1 mol (130 g) 100 parts by weight of a photocurable prepolymer (HLB: 8) and 30 parts by weight of oleic acid were added and mixed well. To do. To 10 parts by weight of the above photocurable resin mixture, 0.5 parts by weight of α-hydroxyisobutylphenone and 1.5 g of an enzyme solution of 8% by weight were added and dispersed well. The resulting photocurable resin-enzyme mixture is molded into a 0.5 mm thick sheet,
By irradiation with actinic rays, a membrane-shaped immobilized enzyme was obtained. This membrane-shaped immobilized enzyme was used after being cut into a size of 2 mm × 2 mm.

10 parts by weight of the above-mentioned membrane-immobilized enzyme was added to a mixed solution of 100 parts by weight of linoleic acid and 100 parts by weight of diglycerin, 30 ° C, 500 rpm
The reaction was carried out at. As a result, 40% in 24 hours, 100 hours
77% ester was obtained.

10 parts by weight of the membranous immobilized enzyme was added to a mixed solution of 100 parts by weight of linoleic acid and 100 parts by weight of hexaglycerin, and the mixture was heated at 30 ° C. for 500
When the reaction was performed at rpm, 35% in 24 hours, 6 in 100 hours
8% ester was obtained.

The lipases used in Examples 1 to 3 were all Candid
It is Lipase OF (trade name, manufactured by Meito Sangyo Co., Ltd.) derived from a cylindracea.

Claims (3)

[Claims] 1. A method for producing an ester from an acid and an alcohol by an enzymatic reaction, which comprises a hydrolase as an enzyme and at least two ethylenically unsaturated bonds in one molecule, and a hydrophilic lipophilic balance ( A method for producing an ester, comprising using an immobilized enzyme obtained by irradiating a mixture with a photocurable resin having an HLB of 11 or less with an actinic ray. 2. The method for producing an ester according to claim 1, wherein the hydrolase is a lipase. 3. The method for producing an ester according to claim 1, wherein the acid is a higher fatty acid and the alcohol is a monohydric or polyhydric alcohol, a sugar alcohol or a sugar component.