JPH03168090A - Production of saccharide fatty acid monoester - Google Patents

Abstract

PURPOSE:To improve yield of saccharide fatty acid monoester by reacting specific saccharides with fatty acids and lipase in the presence of an organic solvent containing no water. CONSTITUTION:1mol saccharides selected from 5-7C monosaccharides having no substituent group, disaccharides consisting of hexose and 4-5C saccharide alcohol is reacted with 1-50mol fatty acids selected from 6-22C (un)saturated fatty acid and ester of the above-mentioned fatty acid and 1-3C lower alcohol in the presence of a heat resistant lipase of 0.1-10000 pts.wt. based on 100 pts.wt. fatty acids in an organic solvent (e.g. 2,4-dimethyl-3-pentanol) containing no water at >=40 deg.C for about 24hr.

Description

[Detailed description of the invention] Published by Keio Sha and Tatemi The present invention is capable of selectively combining monoesters when producing saccharide fatty acid esters from saccharides and fatty acids through an enzymatic reaction using lipase. The present invention relates to a method for producing sugar fatty acid monoester.

BACKGROUND OF THE INVENTION Previously, it is known that lipase is an enzyme that hydrolyzes esters of fats or higher fatty acids, but lipase also undergoes the reverse reaction of hydrolysis under appropriate conditions. It is known to combine esters and perform transesterification reactions.

However, when these enzymatic reactions are carried out in an aqueous solution, in the case of ester synthesis reactions, the reverse reaction, ester hydrolysis, takes precedence. Furthermore, in the case of transesterification, a hydrolysis reaction of the ester of the raw material 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. 62-10779;
J. Am. Chem. Soc. ,108,5638
(1986); J. Am. Chem. Soc. ,110
, 584 (1988)). In this case, 2- The proposal of JP-A No. 61-268192 uses alkaline lipase derived from a microorganism, and the proposal of JP-A No. 62-1077
In the proposal of Publication No. 9, Candida cylindrace (Ca.
A lipase mutagenized from ndida cylindracea is used. 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 was not possible to obtain a sugar fatty acid ester having a high monoester content with a sufficient reaction rate.

In addition, conventionally, a method of esterifying sugars using a -3-acylating agent such as acid chloride according to a chemical method (Chem. Pharm. Bull, 27 (
1 1), 2661 (1979)), but this method also produces a large amount of polyester as a by-product even if equimolar amounts of acylating agent and saccharide are used, making it impossible to selectively obtain monoester. .

The present invention was made in view of the above circumstances, and enables efficient ester aggregation or transesterification.
The object of the present invention is to provide a method for producing sugar fatty acid monoesters that can selectively synthesize monoesters at a high reaction rate.

In order to achieve the above-mentioned object, the present invention uses a saccharide selected from unsubstituted monosaccharides having 5 to 7 carbon atoms, disaccharides consisting of hexoses, and sugar alcohols having 4 to 6 carbon atoms. , 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 an organic solvent that does not substantially contain water. When producing a sugar fatty acid monoester, the raw fatty acid is used at a molar ratio of more than 1 mole to 1 mole of the raw sugar.

That is, as a result of various studies, the inventors of the present invention found that by using molar excess of fatty acids relative to sugars,
Monoesters can be obtained preferentially, and in particular, the applicant has found that sugar fatty acid monoesters can be obtained with a high synthesis rate and content by using a thermostable immobilized lipase as a lipase in the above reaction system. Provided a method for producing monoester (Japanese Patent Application No. 1-210495)
However, in this method, the inventors discovered that if a molar excess of fatty acids relative to sugars was used, only monoesters could be obtained in higher yields in a shorter time, leading to the present invention. .

The present invention will be explained in more detail below.

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

-5- 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.
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, and lauric acid. Methyl, ethyl laurate, provil laurate, methyl myristate, ethyl myristate, provil monog-tate, methyl palmitate, ethyl palmitate, provil palmitate, methyl stearate, ethyl stearate, provil stearate, Methyl oleate, ethyl oleate, probyl oleate, methyl linoleate, ethyl linoleate, probyl linoleate, methyl linolenate, ethyl linoleate, probyl linolenate, methyl eicosanoate, methyl arachidonate, methyl docosanoate, docosenoic acid Examples include methyl.

Furthermore, 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 of the monosaccharide having 7 carbon atoms include 6-deo-7-xymannose and 2-deoxygalactose, and examples of the monosaccharide having 7 carbon atoms include alohepulose, sedoheptulose, mannoheptulose, and glucoheptulose.

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

Furthermore, examples of the sugar alcohol include erythritol, ribitol, xylitol, allitol, sorbitol, mannitol, galactitol, and the like.

In this case, when reacting the fatty acids and sugars, the present invention provides a molar ratio of more than 1 mole of fatty acids per mole of sugars, preferably more than 1 mole of fatty acids and less than 50 moles of fatty acids per mole of sugars. range, more preferably 1.5-20
It is used in a molar amount, particularly in a range of 2 to 8 molar, and thereby sugar fatty acid monoester can be selectively obtained.

The lipase used in the enzymatic reaction of the present invention may be any lipase conventionally used in this type of enzymatic reaction, such as the above-mentioned alkaline lipase derived from microorganisms or mutagenic lipase derived from Candida cylindracea. For example, from the viewpoint of obtaining a monoester, it is recommended to use the immobilized heat-stable lipase disclosed in Japanese Patent Application No. 1-210495.

Here, as a heat-stable lipase, 50 μg of lipase powder was dissolved in 0.4% phosphate buffer (0.1 M, pH 7), and the residual activity after heating at 70°C for 30 minutes was 40%.
Various materials can be used as long as they have a heat resistance of preferably 80% or more, more preferably 95% or more, such as Candida antarctica (Candi antarctica).
thermostable lipase (
sp-382, manufactured by NOVO), thermostable lipase derived from Mucor miehei (
Liρozyme (manufactured by N.O.V.O.) and the like are preferably used, but are of course not limited to these.

Note that these heat-stable lipases may be purified products or crude products, and furthermore, dried products of microorganisms (treated microbial cells, resting or stationary microbial cells) that produce heat-stable lipases 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 polymeric compounds such as starch and gluten, and synthetic polymeric substances such as polyethylene, polypropylene, phenol-formalin resin, acrylic resin, anion exchange resin, and cation exchange resin. However, in the present invention, polymeric materials 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, there is no problem in using various immobilization carriers other than those mentioned above, 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 gram 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. Also, aromatic hydrocarbons such as benzene, toluene, xylene, and phenol, ketones such as acetone and methyl ethyl ketone, ethers such as dimethyl ether, diethyl ether, and dioxane, and aliphatic carbons such as n-hexane, n-octane, and isooctane. Hydrogens, 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 and dimethylformamide, which are good solvents for sugars. , dimethylacetamide, quinoline, and other nitrogen-containing solvents; and dimethyl sulfoxide and other sulfur-containing solvents. 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 rivet 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 a sugar fatty acid monoester by the method of the present invention, for example, a method of filling a column with lipase and passing a substrate liquid through it (packing force ram method), introducing a substrate liquid and lipase into a reaction tank, stirring, shaking, etc. (batch method)
The reaction can be carried out by employing the above-mentioned batch method (continuous stirring tank method), etc., in which the reaction is carried out continuously.

In addition, in the method of the present invention, water or carbon number ■
~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. 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 out of the reaction tank. If these removal methods are appropriately combined with the above-mentioned enzyme reaction apparatus, the reaction can be carried out efficiently.

In addition, although the present invention uses more than 1 mole of fatty acids per saccharide emole as described above, unreacted fatty acids contained in the reaction mixture can be separated, recovered, and reused. can.

In this case, a suitable method for separating and recovering the fatty acids is to dissolve the fatty acids in the reaction mixture, treat the reaction mixture with an organic solvent in which the saccharide fatty acid ester is insoluble, and separate the fatty acid and the saccharide fatty acid ester. will be adopted.

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-methylhebutane, n-octane, and isooctane; Examples include unsaturated hydrocarbons such as -hexene and 2-octene, aromatic hydrocarbons such as benzene, toluene and xylene, and ketones such as acetone and methyl ethyl ketone. It is preferable to transfer fatty acids to these solvents and recover them.

The above-mentioned treatment with an organic solvent can be carried out by removing lipase or unreacted glucose from the reaction system and then distilling off the organic solvent in the reaction mixture, or in some cases, it can be carried out as it is without distilling it off. , an extraction method, a column method, a recrystallization method, and the like may be employed 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.

Sugar fatty acid esters from which fatty acids have been separated can be used as is, but depending on the purpose, recrystallization,
It can be purified by methods such as column methods and crystallization methods. In this case, the purification can be easily carried out since the fatty acids have been separated.

Note that since lipase and saccharides in the reaction mixture precipitate at room temperature or lower temperatures, 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.

In addition, by treating the reaction mixture with water, the saccharides can be transferred to the aqueous phase and separated and recovered, so this method can be adopted or combined with the above-mentioned filtration method to transfer the saccharides to the reaction mixture. It can be removed from

According to the present invention, by using a molar excess of fatty acids with respect to sugars, sugar fatty acid monoesters can be obtained selectively at a high yield through an enzymatic reaction using lipase. This will improve efficiency. below,
The present invention will be specifically explained by showing examples and comparative examples, but
The present invention is not limited to the following examples.

[Example work]

Add 2.5 g of tertiary butyl alcohol to a mixture of 100 g (0.56 mol) of glucose and 515 g (2.78 mol) of methyl cabrate, and further immobilize heat-stable lipase derived from Candeda antarctica to acrylic resin. (immobilized lipase sp-382
, NOVO) Log was added, and then Molecular Sieves 5A500 was added as a -17-demethanol agent.
The mixture was heated to reflux with stirring for 24 hours.

Next, 0.5 mQ of the reaction solution was placed in a 5 ml screw tube, 2.5 ml of pyridine was added, and 10 ml of n-tetradecane was added as an internal standard substance. 1 was added thereto, mixed thoroughly, filtered, and reacted at 60° C. for 30 minutes. 1 mQ of acetic anhydride was added as an acetylating agent to the furnace solution.

The reaction solution IIIft was analyzed by gas chromatography to measure the weight percent of the produced glucose fatty acid ester.

As a result, 98% glucose monopurate ester
It was obtained with a purity of 95% and a yield of 95%.

[Examples 2 to 8] Sugar fatty acid esters were produced in the same manner as in Example 1 using the raw materials shown in Table 1 and under the conditions shown in the table.

The results are shown in the same table.

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 per mole of raw sugar when producing sugar fatty acid monoester using lipase in the presence of an organic solvent that does not substantially contain water. A method for producing a sugar fatty acid monoester, characterized in that raw fatty acids are used at a molar ratio of more than 1 mole.