JPH03168093A - Purification of sugar-fatty acid ester - Google Patents

Abstract

PURPOSE:To readily recover fatty acids in reusable state by producing sugar- fatty acid ester with reacting sugars and fatty acids in an organic solvent using lipase, treating reacted mixture with an organic solvent dissolving fatty acids and not dissolving sugar-fatty acid ester. CONSTITUTION:Sugars (e.g. glucose) selected from 5-7C monosaccharide without substituted group, disaccharide composed of hexose and 4-6C sugar-alcohol, fatty acids (e.g. stearic acid) selected from 6-22C (un)saturated fatty acid and ester of the fatty acid and 1-3C lower alcohol and lipase are added to an organic solvent not containing water. Then, sugar-fatty acid ester is produced from sugars and fatty acids by enzyme reaction with lipase. Next, reacted mixture is treated with an organic solvent (e.g. toluene) dissolving fatty acids and not dissolving sugar-fatty acid ester to separate fatty acids and sugar-fatty acid ester. Thus, sugar-fatty acid ester is purified and simultaneously fatty acids are separated, then recovered for reuse.

Description

[Detailed Description of the Invention] The present invention relates to the production of saccharide fatty acid esters from saccharides and fatty acids by an enzymatic reaction using lipase. The present invention relates to a method for purifying sugar fatty acid ester and separating it from sugar fatty acid ester.

Problems to be Solved by the Prior Art and the Invention Conventionally, lipase is known to be an enzyme that hydrolyzes fats or esters of higher fatty acids, but lipase can also perform the reverse reaction of hydrolysis under appropriate conditions. It is known to synthesize esters and carry out transesterification reactions.

However, when these enzymatic reactions are carried out in an aqueous solution, in the case of ester synthesis or reaction, the reverse reaction, ester hydrolysis, takes precedence. Furthermore, in the case of transesterification, a hydrolysis reaction of the esters of the raw materials and the raw material occurs, resulting in a decrease in the reaction rate.

Therefore, it is desirable to perform the reaction in an organic solvent system that contains virtually no water, and to avoid hydrolysis of the ester, the esterification or transesterification reaction is carried out using an enzymatic method in an organic solvent that contains almost no water. It is proposed that
Publication No. 1-268192: Publication No. 62-■0779;
J. Am. Chem. Soc,,108,563
8 (1986); J. Am. Chem. Soc. ,11
0,584 (1988)). In this case, JP-A-61-
The proposal in JP-A No. 268192 uses alkaline lipase derived from microorganisms, and the proposal in JP-A-62-10779 uses Candida cylindracea.
cylindracea). In the above-mentioned documents, an active ester with a complex structure is used to improve the reaction rate.

However, in these known methods, for example, the method described in the above-mentioned literature uses a special substrate to increase the monoester content, and ordinary fatty acids or their lower alcohol esters cannot give a sufficient reaction rate. I can't do it,
Furthermore, the method described in JP-A No. 6,126,8192 cannot selectively obtain monomers, and the two conventional methods described above involve reacting ordinary fatty acids or their lower alcohol esters with sugars. In this case, it is not possible to obtain glycofatty acid esters with a high monoester content at a sufficient reaction rate, and the reaction product contains not only monoesters but also polyesters such as diesters and 1-lyesters in considerable amounts. In addition, unreacted raw material fatty acids and saccharides are also present. It is therefore necessary to separate the components in this reaction mixture.

In addition, the present applicant has previously proposed a saturated or unsaturated fatty acid having 6 to 22 carbon atoms or an ester of the fatty acid and a lower alcohol having 1 to 3 carbon atoms, and a monosaccharide having 5 to 7 carbon atoms without a substituent. , 2v consisting of hexose! and carbon number 4-6
A sugar monofatty acid ester that can be synthesized at a high synthesis rate and content by allowing a heat-stable immobilized lipase to act on a mixture of sugars selected from sugar alcohols in the presence of an organic solvent. proposed a manufacturing method (Patent Application No. 1-2 1.0 4.9 5), but
Even in this method, since unreacted raw materials and the like still exist in the reaction product, it is desirable to separate them from the reaction mixture and purify the sugar fatty acid ester.

4 The present invention was made in view of the above circumstances, and when producing sugar fatty acid esters from sugars and fatty acids by an enzymatic reaction in an organic solvent using lipase, the raw fatty acids and the product are produced from the reaction mixture obtained. To provide a method for purifying sugar fatty acid esters, by which sugar fatty acid esters can be efficiently and easily separated, and the separated fatty acids can be recovered and reused.

In order to achieve the above-mentioned object, the present invention provides sugars selected from unsubstituted monosaccharides having 5 to 7 carbon atoms, disaccharides consisting of hexoses, and sugar alcohols having 4 to 6 carbon atoms. and fatty acids selected from saturated and unsaturated fatty acids having 6 to 22 carbon atoms, and esters of the fatty acids and lower alcohols having 1 to 3 carbon atoms, using lipase in the presence of a substantially water-free organic solvent. After producing the sugar fatty acid ester as described below, the resulting reaction mixture is treated with an organic solvent in which the fatty acid ester is insoluble, and the fatty acid and the sugar fatty acid ester are separated. It is.

According to the present invention, raw fatty acids and raw sugar fatty acid esters can be easily and reliably separated using an organic solvent, and the recovered fatty acids can be reused, and sugar fatty acid esters can also be separated from fatty acid esters. Since it is separated and removed, its production is very easy.

The present invention will be explained in more detail below.

In the present invention, the sugar fatty acid ester is produced using a fatty acid or its ester and a saccharide as starting materials, and utilizing an enzymatic reaction by lipase.

Here, the fatty acid used in the present invention is a saturated or unsaturated linear or branched fatty acid having 6 to 22 carbon atoms, and such a fatty acid has a hydroxyl group, a carbonyl group,
It may also be substituted with a phenyl group or the like. in particular,
Fatty acids include caproic acid, sorbic acid, caprylic acid, cabric acid, lauric acid, myristic acid, palmitoleic acid, palmitic acid, stearic acid, isostearic acid,
Oleic acid, linoleic acid, linolenic acid, eicosanoic acid, docosanoic acid, docosenoic acid, arachidonic acid, ricinoleic acid, dihydroxystearic acid, etc. can be used.

Furthermore, as the fatty acid ester, the above-mentioned carbon number 6-22
It uses esters of fatty acids and lower alcohols having 1 to 3 carbon atoms, such as methanol, ethanol, and propanol, specifically methyl caproate, ethyl caproate, methyl caproate, ethyl caproate,
Methyl laurate, ethyl laurate, provil laurate, methyl myristate, ethyl myristate, provil myristate, methyl palmitate, ethyl palmitate, provil palmitate, methyl stearate,
Ethyl stearate, probyl stearate, methyl oleate, ethyl oleate, probyl oleate, methyl linoleate, ethyl linoleate, probyl linoleate, methyl linolenate, ethyl linolenate, probyl linolenate, methyl eicosanoate, arachidonic acid Methyl,
Examples include methyl docosanoate and methyl docosenoate.

7 The saccharide used in the present invention is one or more selected from unsubstituted monosaccharides having 5 to 7 carbon atoms, disaccharides consisting of hexoses, and sugar alcohols having 4 to 6 carbon atoms.

Here, examples of monosaccharides having 5 carbon atoms include arabinose, ribose, xylose, lyxose, xylulose, ribulose, 2-deoxyribose, etc., and monosaccharides having 6 carbon atoms such as glucose, galactose, fructose, etc. , mannose, sorbose, talose,
2-deoxyglucose, 6-deoxygalactose,
Examples include 6-deoxymannose, 2-deoxygalactose, and monosaccharides having 7 carbon atoms include allohepulose, sedoheptulose, mannohepulose, glucoheptulose, and the like.

In addition, examples of disaccharides consisting of hexose include maltose, sucrose, sophorose, and the like.

Furthermore, examples of the sugar alcohol include erythrol, ribil, xylyl, allitol, sorbitol, mannitol, galactitol, and the like.

The mixing ratio of the fatty acid or its ester and the saccharide is:
0.02 sugars per mole of fatty acid or its ester
Although it is desirable that the amount be 50 mol, particularly 0.1 to 10 mol, monoester can be selectively obtained by using more than 1 mol of fatty acid or its ester per 1 mol of saccharide.

The lipase used in the enzyme reaction of the present invention may be any lipase conventionally used in this type of enzyme reaction, such as alkaline lipase derived from the above-mentioned microorganisms or lipase mutated from Candida cylindracea. However, when obtaining a sugar fatty acid ester with a high monoester content, Japanese Patent Application No. 1-2
1 0 4. As shown in 9 5 8-, it is recommended to use immobilized thermostable lipase.

Here, as a heat-resistant lipase, 50 μl of lipase powder was mixed with 0.4 ntQ phosphate buffer (0.1 M, pH 7).
The residual activity after heating at 70°C for 30 minutes was 4.
0% or more, preferably 80% or more, more preferably 95
% or more can be used, such as Candida antarctica (Candida antarctica).
thermostable lipase derived from P. dida antarctica (sp-382, manufactured by NOVO), thermostable lipase derived from Mucor miehei (Lj pozyme, manufactured by NOVO), etc. Of course, it is not limited to these.

In addition, these heat-stable lipases may be purified products or crude products, and furthermore, heat-stable lipase-producing microbial cells (treated microbial cells,
Dried products of resting or quiescent bacterial cells can also be used.

Further, as a method for immobilizing the thermostable lipase, any of the carrier binding method, crosslinking method, and entrapping method may be employed, and the carrier binding method is particularly preferably employed.

In this case, specific examples of the immobilization carrier include activated carbon, porous glass, acid clay, bleaching clay, kaolina 1~, alumina, and silica gel. bentonai 1, hydroxyapatite, calcium phosphate, inorganic substances such as metal oxides, natural polymer compounds such as starch and gluten, polyethylene,
Examples include composite polymer materials such as polypropylene, phenol-formalin resin, acrylic resin, anion exchange resin, cation exchange resin, etc., but in this research, we specifically use composite polymer materials that have porosity as a physical form, For example, porous polyethylene, porous polypropylene, porous phenol formalin resin, and porous acrylic resin are most preferably used. In the present invention, various immobilization carriers other than those described above may be used without any problem as long as they do not inhibit the expression of enzyme activity.

Furthermore, the amount of lipase immobilized on the immobilization carrier is usually 0.1 to 500 μg of protein per 1 g of the immobilization carrier,
Particularly suitable is one in which a protein containing about 2 to 50% lipase is immobilized.

In the present invention, the amount of lipase used is not particularly limited, but 0 parts by weight per 100 parts by weight of the above fatty acid or ester thereof.
．． It can range from 1 to 10,000 parts by weight, preferably from 1 to 2,000 parts by weight.

In the present invention, the enzymatic reaction between the fatty acid or its ester and saccharide using lipase is carried out in the presence of an organic solvent substantially free of water.

The organic solvent is preferably a secondary or tertiary alcohol, such as 2,4-dimethyl-3-pentanol, 2
, 6-dimethyl-4-heptanol, tertiary butyl alcohol, tertiary amyl alcohol, diacetone alcohol, 3-methyl-3-pentanol, 3-ethyl-3-
Pentanol, 3-propyl-3-pentanol, 2-
Methyl 2-hexanol, 2-ethyl-2-hexanol, etc. can be used. In addition, aromatic hydrocarbons such as benzene, toluene, xylene, and phenol, carbons such as acetone and methyl ethyl ketone, ethers such as dimethyl ether, diethyl ether, and dioxane, and n-hexane, n-octane, isooctane, etc. Aliphatic hydrocarbons, alicyclic hydrocarbons such as cyclopentane and cyclohexane, and halogenated hydrocarbons such as carbon tetrachloride, chloroform and methylene dichloride are also preferably used, as well as pyridine, which is a good solvent for sugars. , dimethylformamide, dimethylacetamide, quinoline, etc., and sulfur-containing solvents such as dimethylsulfoside. Note that these solvents may be used alone or as a mixed solvent of two or more.

The amount of the organic solvent used depends on the type of organic solvent, the carbon chain length of the fatty acid or its ester, the reaction temperature, etc., but is preferably 10 to 99% by weight of the entire reaction system, especially 6% by weight of the entire reaction system.
It is 0 to 80% by weight.

When enzymatically reacting the fatty acids or their esters with saccharides using lipase, the reaction conditions can be adjusted as appropriate, and the reaction proceeds even at low temperatures, but in particular when using a heat-resistant immobilized lipase, the reaction rate can be accelerated. , 40℃ or higher, especially 6
It is preferable to carry out the reaction at a temperature of 0 to 120°C, and if the reaction is carried out under this temperature condition, the reaction can be completed in about 24 hours. In this case, the saccharide is preferably used after being dissolved in the organic solvent at a temperature of 60° C. or higher in order to increase its utilization efficiency. In addition, even in such a high temperature reaction, there is no enzyme deactivation due to the use of heat-resistant immobilized lipase.

Furthermore, when producing sugar fatty acid esters by the method of the present invention, for example, lipase is packed in a column and the substrate liquid is passed through it (packing force ram type), the substrate liquid and lipase are introduced into a reaction tank, and the process is carried out by stirring or shaking. A method of carrying out the reaction (batch method), a method of carrying out the reaction continuously in the batch method (continuous stirring tank method)
etc. can be adopted.

In addition, in the method of the present invention, water or carbon number 1
~3 lower alcohol is produced as a by-product, but in this case, the concentration of this by-product in the system is 0.5% by weight or less, particularly 0.1% by weight.
In order to proceed with the reaction efficiently, it is preferable to remove by-products as follows. Methods for removing these byproducts include, for example, zeolite, molecular sieves,
A method of adsorbing and removing Glauber's salt, etc.14 using outside and/or inside the reaction system, introducing dry air or an inert gas into the reaction tank and removing it by evaporation into the gas, or removing the inside of the reaction tank by evaporating it into the gas. Examples include a method of reducing the pressure, evaporating and discharging the material to the outside of the reaction tank.If these removal methods are appropriately combined with the above-mentioned enzyme reaction apparatus, the synthesis reaction can be carried out efficiently.

Accordingly, in the present invention, the reaction mixture thus obtained is treated with an organic solvent in which the fatty acids are dissolved and the sugar fatty acid ester is insoluble, thereby separating the fatty acids and the sugar fatty acid ester.

Here, examples of organic solvents that dissolve fatty acids and insoluble sugar fatty acid esters include saturated hydrocarbons such as n-pentane, n-hexane, n-hebutane, 2-methylehbutane, di-octane, and isooctane; Examples include unsaturated hydrocarbons such as 2-hexene and 2-octene, aromatic hydrocarbons such as benzene, toluene, and xylene, and ketones such as acetone and methyl ethyl ketone. and collect it.

The above-mentioned treatment with an organic solvent can be carried out by distilling off the organic solvent in the reaction mixture, or without distilling it off as the case may be. Treatment methods include washing methods, extraction methods, column methods, recrystallization methods, etc. as appropriate. These methods may be used alone or in combination. The amount of the organic solvent used is also appropriately selected, but is usually 2 to 50 times the solid content of the reaction mixture (weight ratio). Although the treatment can be carried out at room temperature, a heated organic solvent can be used, and the treatment can also be carried out at the reflux temperature of the organic solvent.

The fatty acids thus separated by treatment with an organic solvent can be recovered and reused in the above reaction.

Furthermore, the sugar fatty acid ester from which the fatty acids have been separated can be purified by an appropriate method to separate monoester from polyester such as diester and triester. In this case, the purification can be easily carried out since the fatty acids have been separated.

Note that since lipase and sugars in the reaction mixture precipitate at room temperature or lower, they can be easily separated and removed by filtration. Therefore, it is preferable to cool and filter the reaction mixture to separate lipase and saccharides before the organic solvent treatment. Note that the immobilized lipase and saccharide thus separated can be reused as they are. Furthermore, by treating the reaction mixture with water, the saccharides can be transferred to the aqueous phase, separated and recovered, so this method can be adopted or combined with the filtration method described in It can be removed from the reaction mixture.

According to the present invention, fatty acids and sugar fatty acid esters can be easily and reliably separated from a reaction mixture of an enzymatic reaction using lipase, and the fatty acids can be easily recovered for reuse.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to examples, but the present invention is not limited to the following examples.

〔Example〕

17 Glucose5. Tertiary butyl alcohol 25Id was added to a mixture of OOg (27.64 mmol) and methyl cabrate (5.55 mmol), and heat-resistant lipase derived from Candeda antarctica was immobilized on acrylic resin. After adding 100 μm of immobilized lipase, 10 g of Molecular Sieves 5A was used as a demethanol agent, and the mixture was heated under reflux with stirring for 24 hours.

In addition, the reaction solution was analyzed by gas chromatography,
The weight percent of the raw glucose fatty acid ester was determined. As a result, glucose monopurate ester was 98
% purity and 70% yield.

Next, the reaction mixture was cooled to room temperature and filtered in a furnace to recover immobilized lipase and unreacted glucose.

Next, the solvent was distilled off from the tertiary butyl alcohol solution of the mixture of methyl cabrate and glucose phosphoric acid ester obtained as a filtrate. The resulting methyl cabrate and glucose purinate mixture is dried,
When washed with n-hexane, methyl cabrate was eluted into n-hexane, and 1.26 g of white powder of glucose purinate was obtained as a residue. Further, when n-hexane was distilled off from the furnace liquid, 0.3 g of methyl cabrate was recovered.

Furthermore, 1 g was taken from 1.26 g of the obtained white powder of glucose phosphoric acid ester, dissolved in 20 d of mixed solvent of acel/methanol = 4:1 by heating (over a hot water bath), and then heated to 5°C. It was cooled to obtain 0.68 g of glucose capric acid monoester crystals.

The above reaction was repeated five more times, and instead of washing with n-hexane, washing was performed with cyclohexane, n-octane, 2-hexene, toluene, and acetone, and the same results were obtained for each.

Claims (1)

[Claims] 1. Saccharides selected from unsubstituted monosaccharides with 5 to 7 carbon atoms, disaccharides consisting of hexoses, and sugar alcohols with 4 to 6 carbon atoms, saturated and unsaturated fatty acids with 6 to 22 carbon atoms, and the fatty acids and fatty acids selected from esters of lower alcohols having 1 to 3 carbon atoms using lipase to produce sugar fatty acid esters in the presence of an organic solvent that does not substantially contain water, and then the resulting reaction mixture is A method for purifying saccharide fatty acid esters, which comprises dissolving the above fatty acids and treating with an organic solvent in which saccharide fatty acid esters are insoluble to separate fatty acids and saccharide fatty acid esters.