JPH03168094A - Purification of sugar-fatty acid ester - Google Patents

Abstract

PURPOSE:To simply and surely separate and reuse sugars by producing sugar- fatty acid ester with reacting sugars and fatty acids in an organic solvent using lipase, treating reacted mixture with water and transferring sugars to aqueous phase. 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 water and sugars in the reacted mixture is transferred to aqueous phase. Thus, sugar-fatty acid ester is purified and simultaneously sugars are separated, then recovered for reuse.

Description

[Detailed description of the invention] Production - ■ Awadashi issue The present invention relates to the production of saccharide fatty acid esters from saccharides and fatty acids by an enzymatic reaction using lipase-1. The present invention relates to a method for refining sugar fatty acid esters that can reliably separate, recover, and reuse raw material sugars.

Prior Art and Problems to be Solved by the Invention Conventionally, lipase is known to be an enzyme that hydrolyzes fats or higher fatty acid esters, 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 an ester synthesis 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 products occurs, resulting in a decrease in the reaction rate.

For this reason, it is desirable to perform the reaction in an organic solvent system that contains virtually no water, and in order to avoid hydrolysis of the ester, the ester synthesis and transesterification reactions are 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. 62-10779;
J. Am. Chem. 2 Soc. , 108.5638 (1986); J. Am.
Chem. Soc. , 110, 584 (1988))
．． In this case, the proposal of JP-A No. 61-268192 uses alkaline lipase derived from microorganisms;
The proposal in Publication No. 2-10779 uses a lipase mutated from Candida 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. 61-268192 cannot selectively obtain monomers, and the conventional methods described above all 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 in addition to monoesters, the reactants contain a considerable amount of polyesters such as diesters and triesters. In addition, unreacted raw material fatty acids and saccharides are also present. It is therefore necessary to separate the constituents 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 ↓ to 3 carbon atoms, and a monosaccharide having 5 to 7 carbon atoms without a substituent. , a mixture of disaccharides consisting of hexoses and sugars selected from sugar alcohols having 4 to 6 carbon atoms is reacted with heat-resistant immobilized lipase in the presence of an organic solvent to produce sugar monofatty acid esters with high synthesis rate and We proposed a method for producing sugar monofatty acid esters that can achieve a high content ratio (
Japanese Patent Application No. 1-210495) also uses this method,
Since unreacted raw materials and the like are still mixed in the reaction mixture, it is desirable to separate these 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 enzymatic reaction in an organic solvent using lipase, it is possible to efficiently extract raw material sugars from the resulting reaction mixture. The purpose of the present invention is to provide a method for purifying sugar fatty acid esters that can be easily separated and recovered.

In order to achieve the above-mentioned object, the present invention provides a means and action for determining the above-mentioned objects. Saccharides and fatty acids selected from saturated and unsaturated fatty acids having 6 to 22 carbon atoms, and esters of fatty acids and lower alcohols having 1 to 3 carbon atoms are combined with a substantially water-free organic compound using lipase. After producing a sugar fatty acid ester in the presence of a solvent, the resulting reaction mixture is treated with water, and the saccharides in the reaction mixture are transferred to the aqueous phase, thereby separating the saccharides.

According to the present invention, only saccharides can be easily and reliably separated and recovered by simply treating the reaction mixture with water, and the recovered saccharides can be reused after dehydration and drying. Since sugars are thus reliably removed from the reaction mixture, separation of sugar fatty acid esters from the treated reaction mixture is also facilitated.

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, palmi-I/leic 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, and lauric acid. Methyl, ethyl laurate, provil laurate, methyl myristate, ethyl myristate, provil myristate, methyl palmitate, ethyl palmitate, provil palmitate, methyl stearate, ethyl stearate, provil stearate, methyl oleate, Examples include ethyl oleate, probyl oleate, methyl linoleate, ethyl linoleate, probyl linoleate, methyl linolenate, ethyl linolenate, probyl linolenate, methyl eicosanoate, methyl arachidonate, methyl docosanoate, methyl docosenoate, etc. In addition, the saccharide used in the present invention has 5 to 5 carbon atoms and has no substituents.
7 monosaccharides, 2-7 saccharides consisting of hexoses, and sugar alcohols having 4 to 6 carbon atoms, or two or more thereof.

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 of the monosaccharide having 7 carbon atoms include 6-deoxymannose and 2-deoxygalactose, and examples of the monosaccharide having 7 carbon atoms include alloheptulose, sedoheptulose, mannoheptulose, and glucoheptulose.

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

Furthermore, examples of sugar alcohols include erythritol, ribitol, xylitol, allyl, sorbitol, manniol, galactitol, and the like.

8- The mixing ratio of the above fatty acid or its ester and the above 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, the 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. 1-21
As indicated in No. 049.5, it is recommended to use immobilized thermostable lipase.

Here, as the heat-resistant lipase, 50 mg of lipase powder is used.
0.4ml of phosphate buffer 7-(0.1M, pH 7)
The residual activity after heating at 70°C for 30 minutes was 4.
Various materials can be used as long as they have a heat resistance of 0% or more, preferably 80% or more, 9 more preferably 95% or more, such as Candida antarctica.
thermostable lipase derived from sp-382,
N O V O), Mukor Maihai (Muc
Thermostable lipase (Lipo or miehei)
zyme (manufactured by N O VO) and the like are preferably used, but of course the material is not limited to these.

Note that these heat-stable lipases may be purified products or crude products, and may also be prepared from microbial cells that produce heat-stable lipases (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, kaolinite, alumina, and silica gel. bentonite, hydroxyapatite,
Examples include inorganic substances such as calcium phosphate and metal oxides, natural polymer compounds such as starch and gluten, and polymeric substances such as polyethylene, polypropylene, phenol-formalin resin, acrylic resin, anion exchange resin, and cation exchange resin. However, in the present invention, synthetic polymeric substances having porosity in physical form, such as 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, and other nitrogen-containing solvents; and dimethyl sulfoxide and other sulfur-containing solvents. Incidentally, these solvents may be used alone or as a mixed solvent of two or more thereof.

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 l
~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, especially 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. using outside and/or inside the reaction system, introducing dry air or inert gas into the reaction tank and removing it by evaporation into gas, or reducing the pressure inside the reaction tank. For example, the removal method may be evaporated, evaporated, and discharged outside the reaction tank.If these removal methods are appropriately combined with the above-mentioned enzyme reaction apparatus, the reaction can be carried out efficiently.

Accordingly, in the present invention, the reaction mixture and metal material thus obtained are treated with water, and the unreacted saccharides in the reaction mixture are transferred to the aqueous phase, thereby separating the saccharides.

In this case, when treating with water, if the organic solvent used is water-soluble, it is distilled off, and if it is water-insoluble, the organic solvent is not distilled off, but it is carried out as it is or if necessary, it is distilled off. As a treatment method, an appropriate method such as an extraction method or a washing method may be employed alone or in combination. The amount of water used is also appropriately selected, but is usually 2 to 50 times the solid content of the reaction mixture. Furthermore, although room temperature treatment is sufficient, efficient treatment can be achieved by using hot water heated to 40 to 80°C.

The aqueous phase into which saccharides have been transferred by water treatment in this manner can be dried and recovered to be reused in the above reaction.

In addition, the water-treated reaction mixture can be purified by an appropriate method to separate monoesters from polyesters such as diesters and triesters, but in this case, the water-treated reaction mixture is first purified. n-pentane, n-
Saturated hydrocarbons such as hexane, n-hebutane, 2-methylhebutane, n-octane, isooctane, unsaturated hydrocarbons such as 2-hexene, 2-octene, benzene, 1
・Using organic solvents that dissolve aromatic hydrocarbons such as toluene and xylene, and ketones such as acetone and methyl ethyl ketone, and in which sugar fatty acid esters are insoluble, washing methods, extraction methods, and column methods are used. It is preferable to separate and remove fatty acids from the sugar fatty acid ester by employing an appropriate method such as recrystallization, and then separate and purify the sugar fatty acid ester. At this time, since sugars have been removed from the water-treated reaction mixture, separation of sugar fatty acid esters and fatty acids and recovery of monofatty acid esters of sugars can be easily performed.

Note that the lipase can be separated and recovered from the reaction mixture by filtration and reused. At this time, the lipase can be removed by filtration before the above water treatment, but if the reaction mixture is cooled to room temperature or lower,
Since the saccharides are precipitated, the saccharides precipitated together with the lipase can be removed by the furnace filtration, and the lipase and saccharides can be reused as they are. In this case, water treatment for separating saccharides can be performed on the reaction mixture after the furnace filtration treatment, and the saccharides remaining in the reaction mixture can be separated and recovered.

According to the present invention, sugars can be easily and reliably separated from the reaction mixture of the enzymatic reaction using lipase and 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 25- was added to a mixture of OOg (27.64 mmol) and methyl cabrate (5.55 mmol), and heat-stable lipase derived from Candida antarctica was added to the 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 raw glucose fatty acids was determined. As a result, glucose monopurate was obtained with a purity of 98% and a yield of 70%.

Next, the reaction mixture was cooled to room temperature, and then the tertiary butyl alcohol was distilled off. The obtained residue was washed with 100 d of water at 40° C., and the washing liquid was treated in an oven and dried to obtain 4.2 g of white powder of glucose. At that time, the mixture of immobilized lipase, methyl cabrate, and glucose purinate obtained as a residue from the furnace is dried and washed with n-hexane to obtain a mixture of fixed lipase and glucose purate as a residue. Ta. Further, when n-hexane was distilled off from the furnace liquid, 0.3 g of methyl cabrate was recovered.

Furthermore, by passing this furnace filtration residue through a washing furnace with methanol, glucose phosphate ester can be obtained as a filtrate, and when methanol is distilled off from the furnace filtrate, 1.26 g of white powder of glucose phosphate ester is obtained. It was done.

[Example 2] Glucose5. 25 mQ of tertiary butyl alcohol was added to a mixture of OOg (27.64 mmol) and methyl cabrate (1.03 g (5.55 mmol)), and heat-resistant lipase derived from Candida antarctica was fixed on the acrylic resin. (immobilized lipase) 100■
After adding 10 g of Molecular Sieves 5A as a demethanol agent, 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 glucose fatty acids produced was measured. As a result, glucose monopurate was obtained with a purity of 98% and a yield of 70%.

Next, the reaction mixture was cooled to room temperature, filtered, and the immobilized lipase and unreacted glucose were recovered.

The solvent was distilled off from the tertiary butyl alcohol solution of the mixture of methyl covrate and glucose purinate obtained as a filtrate. the resulting methyl cabrate,
When the glucose-purinate mixture was dried and washed with n-hexane, methyl cabrate was eluted in n-hexane, forming a white powder of glucose-purate 1.
26 g were obtained as a residue. This was washed with 10 parts of water to remove a slight amount of mixed glucose. Furthermore, when n-hexane is distilled off from the furnace solution, methyl cabrate O. a
g was recovered.

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 1. A method for purifying a sugar fatty acid ester, which comprises separating sugars by treating with water and transferring the sugars in the reaction mixture to an aqueous phase.