JPS6058086A - Production of ester - Google Patents

Abstract

PURPOSE:To separate and recover an immobilized enzyme easily and stably, and to obtain an ester in high yield, by reacting an acid with an alcohol in the presence of a small amount of water and an immobilized enzyme obtained by immobilizing lipase to a polymer particles dispersed in water. CONSTITUTION:An acid such as a fatty acid is made to react with an alcohol such as glycerol at 3-11pH and 20-50 deg.C in the presence of (A) 0.01-30wt% water and (B) an immobilized enzyme prepared by (1) dispersing a dispersible polymer particles having an average particle diameter of 0.03-2mum and obtained e.g. by the coplymerization of a vinyl monomer and a polyfunctional internal crosslinking monomer, in water, and (2) reacting a polyamine with a dialdehyde in the dispersion in the presence of a lipase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing carboxylic acid esters, and more specifically, the present invention relates to a method for producing carboxylic acid esters, and specifically, the presence of an immobilized enzyme in which lipase is immobilized on water-dispersed polymer particles having an average particle diameter of 0.03 to 2 μm. The present invention relates below to a method for producing carboxylic acid esters by reacting acids and alcohols.

It has been known for a long time that hydrolytic enzymes such as lipases have a catalytic effect on the reverse reaction, which is the production of carboxylic acid esters. However, according to conventional literature, this reaction generally has a low yield of carboxylic acid esters, and the enzyme is unstable, making it difficult to separate and recover after the reaction, as well as deactivating the enzyme. There are problems such as not being able to withstand reuse due to the significant amount of oxidation, and therefore, a method with excellent stability, productivity, and economic efficiency that can be implemented industrially has not yet been found.

The present inventors conducted extensive research to solve the above-mentioned problems in the production of carboxylic acid esters using lipase, and as a result, lipase was immobilized on water-dispersed polymer particles with an average particle size of 0.03 to 2 μm. By reacting the immobilized enzyme with acid and alcohol in the presence of as little water as possible, it is possible to stably and easily separate and recover the immobilized enzyme from the reaction mixture and achieve a high yield. The present invention was achieved by discovering that it is possible to obtain esters that

The method for producing carboxylic acid esters according to the present invention uses as little water as possible and an immobilized enzyme in which lipase is immobilized on water-dispersed polymer particles having an average particle size of 0.03 to 2 μm. It is characterized by reacting an acid and an alcohol in the presence of alcohol to produce a corresponding ester.

In the present invention, some of the water-dispersed polymer particles used as carriers for immobilized enzymes are, for example,
This is already known from Publication No. 0380 and the like. In the present invention, a first vinyl monomer such as styrene or its derivative, (meth)acrylic acid ester, (meth)acrylonitrile, or a mixture thereof; (meth)acrylic acid, (
It is possible to use water-dispersed polymer particles obtained by emulsion copolymerization with a second vinyl monomer having a reactive functional group such as glycidyl meth)acrylate or (meth)acrylamide, or a mixture thereof. However, preferably, in addition to the above monomers, a water-dispersed polymer obtained by copolymerizing a polyfunctional internal crosslinking monomer such as divinylbenzene, poly(meth)acrylate of a polyhydric alcohol, etc. The polymer particles are preferably used because they do not swell and dissolve in the acid or alcohol present in the reaction system. Note that it is preferable not to use an emulsifier in this emulsion copolymerization. This is because when an emulsifier is present, there is a risk that the emulsifier will be mixed into the resulting water-dispersed polymer particles and deactivate the enzyme during enzyme immobilization. However, if there is no risk of this happening,
Of course, emulsion copolymerization may be carried out in the presence of an emulsifier.

The average particle diameter of the water-dispersed polymer particles is o, 03-2
μm, especially o, 07 to 1 μm
It is preferable that The average particle diameter of the polymer particles is 0.0
When it is smaller than 3 μm, it becomes difficult to separate the immobilized enzyme with lipase as a carrier from the substrate and reaction products after the ester production reaction is carried out by dispersing the immobilized enzyme on which lipase is immobilized in the reaction system. When the average particle diameter exceeds 2 μm, it becomes difficult to disperse the particles in the reaction system, and the surface area per unit volume of the particles is small, resulting in a relative decrease in the enzyme activity of the immobilized enzyme, which hinders the reaction. It becomes less efficient.

The lipase used is not particularly limited, but preferably, for example, a lipase derived from Rhizopus derma or Aspergillus is used.

The method of immobilizing lipase on the water-dispersed polymer particles is not particularly limited, and lipase is immobilized by conventionally known physical adsorption methods, ion adsorption methods, entrapment methods, crosslinking methods, covalent bonding methods, etc. An immobilized enzyme can be used. However, in the method of the present invention, in particular, by dispersing water-dispersible polymer particles in water and reacting polyamine and dialdehyde in the presence of lipase,
It is desirable to use an immobilized enzyme in which the enzyme is deposited and immobilized together with a polymer consisting of a Schiff base of polyamine and dialdehyde. When an enzyme is immobilized in a polymer made of such a Schiff base by a deposition method, the Schiff base has a hydrophobic -CH=N- bond, so it has a low affinity for hydrophobic substrates such as fatty acids. Because of its high properties, the reaction efficiency is high, the reaction proceeds smoothly, and esters can be obtained in high yields.

Furthermore, in the immobilization of an enzyme by this deposition method, part of the enzyme is entrapped in the Schiff base polymer in a free state, and part of the enzyme is incorporated into the Schiff base polymer through covalent bonds. Therefore, if the water-dispersed polymer particles have amino groups from the beginning, or if amino groups are introduced onto the polymer particles using a functional reactive group derived from the monomer used for emulsion copolymerization. , or when formed, the enzyme is advantageously cross-linked by the dialdehyde and also covalently immobilized on the polymer particles, thus making the immobilization of the enzyme on the particles even stronger.

The above-mentioned polyamine used in the immobilization of the enzyme by the above-mentioned deposition method is a monomer or polymer having two or more amino groups in the molecule that can react with an aldehyde group,
Aromatic, aliphatic or alicyclic diamines and triamines are used. Specific examples include phenylenediamine, xylylene diamine, triaminobenzene, triaminotoluene, ethylenediamine, hexamethylenediamine, and the like. Moreover, polyethyleneimine can be mentioned as a specific example of the polymer. As the dialdehyde, aromatic or aliphatic dialdehydes are used, and specific examples thereof include glutaraldehyde, succinic dialdehyde, glyoxal, maleic dialdehyde, and terephthalaldehyde.

The acids and alcohols used as substrates in the method of the present invention are not particularly limited, and as acids, saturated and unsaturated acids, -basic acids and polybasic acids can be used, and
Saturated and unsaturated alcohols, -hydric and polyhydric alcohols can also be used as alcohols. For example, specific examples of acids include acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid, palmitic acid, stearic acid, oleic acid, ricinoleic acid, cyclohexanecarboxylic acid, cyclohexyl Examples include acetic acid, gluconic acid, sebacic acid, speric acid, pimelic acid, malic acid, and succinic acid.

Specific examples of alcohol include monohydric alcohols such as methanol, ethanol, propatool, phthanol, pentanol, hexanol, hexanol, occuenol, dodecanol, oleyl alcohol, benzyl alcohol, geraniol, citrononeol, farsonellol, phytol, and ethylene. Glycol, propanediol, l.

4-butanediol, 1,5-bentanediol, 1,
6-hexanediol, 1.8-octanediol, ■
, 1°-decanediol, varaxylylene diol, glycerin, and other polyhydric alcohols. Although the reason is not clear, it is generally preferable to react alcohol in excess of 50 mol % or more relative to acid in order to increase reaction efficiency.

However, it is not limited to this.

The method of the present invention can be particularly suitably applied to the production of fatty acid esters of glycerin. 'In the method of the present invention, as little water as possible is present in the reaction system, and the reaction system is maintained at a pH range in which lipase is active, that is, pu 3
It is desirable to maintain the temperature between 11 and 11. The amount of water in the reaction system is usually 0.0% of the total weight. O1 to 30% by weight, preferably 0.1 to 20% by weight. When no water is present in the reaction system, the enzyme activity is low, and when the amount of water is too large, the hydrolysis reaction of esters becomes significant, and the reaction efficiency is similarly low. The reaction can also be carried out using as little water and acid as possible and a large excess of alcohol as the reaction medium. The above-mentioned immobilized enzyme is dispersed in such a reaction system containing a substrate and water, and the reaction is usually carried out under stirring. The reaction temperature is a temperature range in which the enzyme retains its activity, and is usually 20 to 50°C. The amount of lipase-immobilized particles used is not particularly limited, but when converted to the amount of enzyme, it is usually r! The amount is about 0.1 to 50 g per 11 moles. Further, the time required for the reaction is usually about 0.5 to 24 hours under the above conditions.

After the reaction is completed, for example, the immobilized enzyme is separated by membrane separation using a microfiltration membrane or ultrafiltration membrane, centrifugation, etc., and then the ester, which is the reaction product, is concentrated and crystallized from the liquid phase.
It can be separated by methods such as extraction. Moreover, the separated immobilized enzyme can be reused.

According to the method of the present invention, water-dispersed polymer particles with an average particle diameter of 0.03 to 2 μm are used as carriers for immobilized enzymes, so that they can move in the reaction system in the same manner as free enzymes. Therefore, the reaction efficiency is high. In particular, according to the method using an immobilized enzyme by the above-mentioned deposition method, the reaction proceeds smoothly because the immobilized enzyme has a high affinity for the substrate, and esters can be obtained in high yield. .

Furthermore, according to the method of the present invention, it is easy to separate and recover the immobilized enzyme.

The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 +l) Production of immobilized lipase Methyl methacrylate 47.5 g, acrylonitrile 7
．． 5 g of acrylic acid, 2.5 g of triethylene glycol dimethacrylate were added to 230 g of distilled water, and an aqueous polymerization initiator solution prepared by dissolving 0.3 g of potassium persulfate in 10 g of water was added at a temperature of 70° C. under a nitrogen stream. rp
Polymerization was carried out for 8 hours while stirring at m, and the average particle size was 0.36.
An aqueous dispersion of water-dispersible polymer particles of μm size was obtained. After centrifuging and washing the polymer particles with distilled water,
ml to obtain an aqueous dispersion with a solid content of 20%.

3 mmol of m-xylylenediamine and 1-cyclohexyl-3-(2-
Add 7.5 mmol of morpholinoethyl)carbodiimide-meth-p-toluenesulfonic acid, and dilute with IN hydrochloric acid.
After adjusting H to 5.0, the reaction was carried out at a temperature of 10° C. overnight. After repeating centrifugation and water washing of the polymer particles with amino groups formed on their surfaces in this way, 50ml of distilled water was added.
was redispersed.

Next, 4 ml of a 0.1% by weight m-xylylene diamine aqueous solution and lipase AP were added to 5 ml of the aqueous dispersion of the above polymer particles.
6 (manufactured by Ohno Pharmaceutical @, Aspergillus origin) 100m
Add an aqueous solution of g dissolved in 6 ml of water, and add 0 while stirring.
．． Add 5 ml of 1% by weight aqueous gluturaldehyde solution,
After reacting at ℃ for 5 hours, centrifugation and water washing were repeated.
Thereafter, it was again dispersed in a buffer solution so that the solid content was 60% by weight.

(2) Production of oleic acid glyceride Glycerin 9 ml (123 mn+ol), oleic acid 0.5 ml (1.6 mmol), 0.1 M, p
0.5 ml of H7 phosphate buffer and 1 ml of the aqueous dispersion of immobilized lipase obtained above were mixed and stirred. The ester yield after 10 hours of reaction at room temperature was determined by alkaline titration of unreacted acid in the reaction mixture, and was 60%.
%Met. When the product was examined by thin layer chromatography, elemental analysis, etc., it was found that most of the product was triglycerides, with small amounts of mono- and diglycerides.

Example 2 Glycerin 9 ml, oleic acid 0.5 ml, 0.
1M.

Mix 0.5 ml of pn 7 phosphate buffer and 1 ml of the aqueous dispersion of immobilized lipase obtained above, add a predetermined amount of water to this, and make the total amount of water in the reaction system as shown in the table. The mixture was reacted for 24 hours at room temperature with stirring. The ester yield is shown in the table. However, Experiment No. 5 is a comparative method, and shows the results using 1.0 ml of 0.5% by weight free lipase in water instead of immobilized lipase.

The lower the amount of water present in the reaction system, the higher the yield of ester. Furthermore, as is clear from a comparison of Experiment Nos. 5 and 2, the method according to the present invention has a higher yield of ester than when using free lipase. This appears to be because the immobilized lipase according to the present invention has a high affinity for the Schiff base polymer substrate.

That is, even when the immobilized enzyme obtained in Example 1 was heated at 80°C for 30 minutes to inactivate it and then placed under the same conditions as in Example 1, oleic acid in the reaction system was consumed over time. It was observed that This apparent consumption of oleic acid is thought to be based on the fact that oleic acid is adsorbed onto the polymer particles because the polymer particles have an affinity for oleic acid.

In addition, when the immobilized lipase that has not been inactivated in this way is used, the consumption rate of oleic acid over time, which is obtained by subtracting the above-mentioned apparent consumption amount of oleic acid, is also shown in the drawing.

[Brief explanation of the drawing]

The drawing shows the apparent consumption rate of oleic acid when the polymer particles in which the immobilized lipase used in the method of the present invention has been deactivated are placed under reaction conditions, and the immobilized lipase used in the method of the present invention compared to the above. It is a graph showing the consumption rate of oleic acid obtained by subtracting the above-mentioned apparent consumption rate under the same reaction conditions. Female employee (hr)

Claims (1)

[Claims]
A method for producing esters, which comprises reacting an acid and an alcohol to produce a corresponding ester in the presence of an immobilized enzyme in which lipase is immobilized on water-dispersed polymer particles. (2) Claim 1, wherein the immobilized enzyme is formed by immobilizing lipase together with a polymer consisting of a Schiff base of polyamine and dialdehyde on water-dispersed polymer particles. A method for producing esters. (3) The method for producing esters according to claim 2, wherein the water-dispersed polymer particles have an amino group. (4) The method for producing esters according to claim 1, wherein the acid is a fatty acid and the alcohol is glycerin.