JPH11192096A - Production of hydroxyfatty acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a hydroxybattery acid useful as a surfactant, an intermediate for pharmaceuticals and agrochemicals, etc., in a high efficiency using an easily recoverable and reusable catalyst by introducing hydroxyl group into a fatty acid using an immobilized biocatalyst produced by immobilizing a biocatalyst on a solid carrier. SOLUTION: A biocatalyst such as a homogenate prepared by disintegrating young foliage of pea with a homogenizer in 0.2M phosphate buffer is suspended in sterilized 3% aqueous solution of sodium alginate and the suspension is dropped into sterilized 1M Call2 solution under agitation to obtain immobilized biocatalyst having a spherical bead form of about 3 mm diameter. The immobilized biocatalyst is added to 0.2M phosphate buffer (pH 6.0) and a fatty acid (e.g. palmitic acid) is added to the mixture and reacted at 20 deg.C for 10 hr under oxygen gas stream to introduce hydroxyl group into the fatty acid and obtain the objective hydroxyfatty acid useful as a surfactant, its raw material, intermediate for various pharmaceuticals and agrochemicals, etc., in a high efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for introducing a hydroxyl group into a carbon chain of a fatty acid using an immobilized biocatalyst which can be easily recovered and reused and can be used in a continuous method and the like. .

[0002]

2. Description of the Related Art Conventionally, as a method for introducing a hydroxyl group at the α-position of a fatty acid, first, a halogen group is introduced at the α-position,
A chemical method for hydrolyzing the halogen group (J. Or.
g. Chem. , 21 , 1426 (1956)). However, these methods are not suitable for industrialization because these methods involve two steps and use a large amount of harmful hydrogen halide and the like. In addition, no method has been found for efficiently introducing a hydroxyl group into another position of the fatty acid carbon chain. On the other hand, as a biochemical method, a metabolic system conventionally known as α-oxidation or β-oxidation of
A method of introducing a hydroxyl group at the position or the β position was known,
Since these use free cells, microbial cells and enzyme systems, these biocatalysts are disposable in a single reaction, so they are still unsuitable for industrial production. (Biochim, Biophys. Act
a. , 239 , 513 (1971). , Tetrah
dron: Asymmetry, 7 (8), 2287
(1996)). In addition, basic research has been carried out on animal and plant cells and microbial cells that introduce hydroxyl groups at the central part, terminal part (ω1 position, methyl terminal), ω2 position and ω3 position of the fatty acid carbon chain. Similarly, it is not suitable for industrialization because it is disposable in a single reaction in a free form.

[0003]

An object of the present invention is to provide a method for producing a hydroxy fatty acid by efficiently introducing a hydroxyl group into an aliphatic carbon chain.

[0004]

Means for Solving the Problems As a means for producing a hydroxy fatty acid more efficiently than a fatty acid, the present inventors have used an immobilized biocatalyst to solidify a biocatalyst. We succeeded in developing a biocatalytic method, and completed the present invention. This method can be manufactured under milder reaction conditions than the chemical synthesis method. According to the present invention, there is provided a method for producing a hydroxy fatty acid, which comprises introducing a hydroxyl group into a fatty acid using an immobilized biocatalyst obtained by immobilizing the biocatalyst on a solid support.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The biocatalyst of the present invention includes various conventionally known biocatalysts, such as animal cells, food cells, organelles (organelles), microbial cells (live cells or dead sterilized cells), Enzymes and coenzymes (cell and microbial cell extracts, crude enzymes, purified enzymes, etc.) are included. Also, these cells,
Microorganisms may also be used as mutants that have been modified in various ways, including genetic engineering. Furthermore, an antibody catalyst or the like obtained from a living body can be used. By immobilizing these biocatalysts on a solid support, they can be easily recovered and reused in a batch reaction, and can also be used easily in semi-continuous and continuous reactions, greatly extending the life of the catalyst. It has been found that an immobilized biocatalyst is obtained.

[0006] The fatty acid of the reaction raw material used in the present invention is:
It is a natural or synthetic product having 3 to 31 carbon atoms, preferably 8 to 24 carbon atoms, and is a linear or branched, saturated or unsaturated fatty acid. Examples of these fatty acids include propionic, butyric, valeric, caproic, enanthic,
Caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, heptadecylic acid, stearic acid, nonadecanoic acid, arachiic acid, behenic acid, lignoceric acid, serotinic acid, heptacosanoic acid , Montanic acid, mericic acid, straight-chain saturated fatty acids such as laccelic acid, crotonic acid,
Non-linear chains such as isocrotonic acid, undecylenic acid, oleic acid, elaidic acid, setleic acid, erucic acid, brassic acid, sorbic acid, linoleic acid, linolenic acid, arachidonic acid, stearolic acid, palmitoleic acid, γ-linolenic acid, etc. Saturated fatty acids; isobutyric acid, 3-methylpentanoic acid,
5-methylhexanoic acid, 3-ethylpentanoic acid, 4-methylheptanoic acid, 3-methyloctanoic acid, 3-methyldecanoic acid, 4-methyldodecanoic acid, 4-methyltetradecanoic acid, 3-methylhexadecanoic acid, 3-methyl Branched saturated fatty acids such as octadecanoic acid and phytanic acid; 3-methylundecylenic acid, 4-ethyloleic acid, 4-methylelaidic acid, 3-methylerucic acid, 3-ethylbrassic acid, 3-methyllinoleic acid, 3-methyllinoleic acid Ethyl linolenic acid,
And branched unsaturated fatty acids such as 3-methylarachidonic acid. These raw fatty acids may be used alone or in combination. In the case of multiple uses, palm oil, palm oil, palm kernel oil, rapeseed oil, tallow fatty acid mixture and the like are also used.

As the biocatalyst used in the present invention, animal cells, plant cells, microbial cells and mutants thereof, organelles (organelles), enzymes, coenzymes, antibody catalysts and the like can be used. Examples of animal cells and organelles (enzymes and coenzymes) include those that perform α-hydroxylation on cells of the liver, skin, brain or mitochondria, β-position water such as mammalian general cells, mitochondria, cytosol (intercellular sol), etc. Oxidizing substances such as those that oxidize, those that hydroxylate the central part of the carbon chain, such as microsomes, cells of the liver, kidneys, lungs, or microreams
And ω2 hydroxylation. Plant cell organelles, enzymes, coenzymes, pea young leaves,
Homogeneates such as cotyledon or germinating peanut or α-hydroxylation of crude enzyme such as extract, avocado extract or the like which performs β-hydroxylation, Vernonia ant
Examples thereof include those that perform hydroxylation of the central part of the carbon chain and ω1 hydroxylation, such as Helmintica seeds, castor seed cells (microsomes), and broad bean leaf young epidermal cells. Microbial cells include Arthrobacter simp
Lex, Candida utilis, etc., which perform α-position hydroxylation, Mycobacteria, Nocard
ia, Corynebacteria, Candida
rugosa, etc. that perform β-hydroxylation, Ergot Cl
avices purpurea, Pseudomo
Nas, Puccinia graniris (Rust fungus uredospores), etc. that hydroxylate the center of the carbon chain, Ba
CILLUS MEGATERIUM, TORULOP
sis, Candida, and Pseudomonas
And the like, which perform hydroxylation at the ω1, ω2, and ω3 positions. Most of these animals, plant cells, organelles, microbial cells, and the like contain enzyme systems and coenzyme systems required for hydroxylation.
H, NADH and other coenzymes are added. The coenzyme is preferably an immobilized coenzyme immobilized by the following method. When an oxygen source is required, oxygen, H ₂ O ₂ , water and the like are appropriately added. Also, if necessary,
Addition of metal ions (Fe ²⁺ , Fe ^3+, etc.) is also performed. The above-mentioned biocatalyst includes animal and plant extracts and purified cells, in the form of organelles, crude enzymes, crude coenzyme systems, purified enzymes, purified coenzymes, treated cells, resting or quiescent cells, and the like. And these are used as appropriate.

As a method for immobilizing a biocatalyst, (1) a carrier binding method, (2) a cross-linking method, (3) an inclusive method, or a combination method of these methods is used. (1) As a carrier binding method, a physical adsorption method, an ion binding method, or a covalent bonding method is used. The physical adsorption method is a method in which an enzyme or the like is physically adsorbed and immobilized on a water-insoluble carrier, and activated carbon, porous glass, acid clay, bleaching clay, kaolinite,
Made of inorganic substances such as alumina, silica gel, bentonite, hydroxyapatite, calcium phosphate, metal oxides, natural polymers such as starch and gluten, porous synthetic resins, ceramics, ultrafiltration membranes and ultrafiltration membranes Examples include hollow fibers, butyl-hexyl sephadex having a hydrophobic group, and cellulose derivatives using tannin as a ligand. The ion bonding method is a method in which an enzyme or the like is ionically bonded to a water-insoluble carrier having an ion exchange group and immobilized. Examples of the carrier used include polysaccharides having ion exchange groups (DEAE-Sephadex) and derivatives of synthetic polymers such as ion exchange resins. The covalent bonding method is a method in which a water-insoluble carrier and an enzyme are covalently bonded and immobilized, and a functional group involved in binding an enzyme, a cell, a microorganism, or the like to the carrier by this method includes α- or ε-amino group, α-, β- or γ-carboxyl group, sulfhydryl group, hydroxyl group, imidazole group, phenol group and the like. Since these functional groups react with diazonium salts, acid azides, isocyanates, active alkyl halides, and the like, a water-insoluble carrier having these reactive functional groups and a biocatalyst can be used under appropriate conditions. When covalently attached below, an immobilized biocatalyst is obtained.

(2) The cross-linking method is a method in which a reagent having two or more functional groups is used to immobilize the enzyme by cross-linking an enzyme or an enzyme or a microorganism between microorganisms.
This method immobilizes enzymes or microorganisms by covalent bonding, as in the case of the covalent bonding method described above, but differs in that a water-insoluble carrier is not used.
Examples of the crosslinking reagent include glutaraldehyde that forms a Schiff base, an isocyanic acid derivative that forms a peptide bond,
N, N'-ethylenebismaleimide, bisdiazobenzidine which undergoes diazo coupling, N, N'-polymethylenebisiodoacetamide which undergoes alkylation, and the like. The functional groups of the enzymes involved in these various cross-linking reactions include N-terminal α-amino group, lysine ε-amino group, tyrosine phenol group or cysteine sulfhydryl group, and histidine imidazole group.

(3) The entrapment method can be classified into a lattice type in which an enzyme or a microorganism is incorporated into a fine lattice of a polymer gel, and a microcapsule type in which an enzyme or a microorganism is coated with a semipermeable polymer film. . These methods are different from the carrier binding method and the cross-linking method in principle, and do not cause a binding reaction with the enzyme itself in principle. Therefore,
It may be applicable to immobilization of many enzymes, coenzymes, microorganisms, and cell organelles.However, in the case of causing a chemical polymerization reaction, it is easy to deactivate the enzyme, so conditions must be set well. It is necessary to. These include 1) a method of polymerizing a monomer for producing a polymer substance in the presence of an enzyme, a microorganism, or a cell; 2) a method of polymerizing a prepolymer for producing a polymer substance in the presence of an enzyme, a microorganism, or a cell; And 3) a method of gelling a sol-like polymer substance in the presence of an enzyme, a microorganism, or a cell. Lattice type As the polymer compound used in this method, polyacrylamide gel of synthetic polymer substance, polyvinyl alcohol,
Light curable resins and natural polymer substances such as starch, konjac powder, gelatin, alginic acid, carrageenan and the like. Stricter conditions are required for the production of microcapsules than in the case of microencapsulation of ordinary substances, and various methods have been considered so far, but they can be divided into the following three methods. 1) interfacial polymerization, 2) drying in liquid, and 3) phase separation. Interfacial polymerization is a method of coating a biocatalyst by applying the principle that a hydrophilic monomer and a hydrophobic monomer polymerize at the interface. For example, there is a method of forming a nylon film using hexamethylenediamine and sebacoyl chloride. The in-liquid drying method is a method of coating a biocatalyst by emulsifying and dispersing a biocatalyst solution in a polymer compound dissolved in an organic solvent, transferring the solution to an aqueous solution, and drying. Ethyl cellulose,
Polystyrene and the like are used as polymer compounds. In the phase separation method, a polymer compound is dissolved in an organic solvent immiscible with water, and the biocatalyst is emulsified and dispersed in this solution. Next, a non-solvent that causes phase separation is gradually added with stirring, and a concentrated solution of the polymer compound wraps around the biocatalyst solution, followed by deposition of the polymer compound, forming a film, and forming a biofilm. Coat the catalyst. Any of these immobilization methods may be employed, but methods that are not easily affected by the permeability of the starting fatty acid into the carrier and the like are used alone or in combination. In the case of cells, microorganisms and the like, they can be used in the form of an immobilized biocatalyst, an immobilized growth biocatalyst and the like.

A hydroxy fatty acid can be produced using the immobilized biocatalyst prepared as described above. The manufacturing conditions in this case are milder than the chemical method. The pH is usually adjusted using a buffer solution near the optimum pH of the enzyme system used. The reaction temperature is 0 to 120 ° C, especially 5
C. to 80C are preferred. The reaction time is batch method, semi-batch method,
There is no particular limitation depending on the continuous method, but in the case of a batch method (batch method) or a semi-batch method (semi-batch method), 2 to 20
The time is preferably about 4 to 14 hours. In the batch method, the immobilized biocatalyst is repeatedly reused after being collected by filtration. In the semi-batch method, the reaction conditions are continuously added to a reaction solution containing a fixed amount of the immobilized biocatalyst, and after the reaction is completed, the immobilized biocatalyst is recovered and reused. In the continuous method, the raw material solution is added at a constant flow rate, and at the same rate, the reacted liquid is withdrawn and the reaction is continuously performed. The immobilized biocatalyst of the present invention exhibited good reaction durability even in these batch, semi-batch, and continuous processes. In this reaction, in order to improve the durability of the biocatalyst by the immobilization treatment, and to improve the solubility of the raw material fatty acid, a nonionic, anionic, cationic or amphoteric surfactant may be appropriately added. . Similarly, an organic solvent may be added to improve the solubility of the fatty acid. These organic solvents are
Without inhibiting enzyme activity and without adversely affecting the immobilized carrier,
Any solvent can be used as long as it can dissolve the raw material fatty acid. Specific examples include polar solvents such as alcohols, ketones, and ethers; nitrogen-containing solvents such as pyridine, dimethylformamide, dimethylacetamide, and quinoline; sulfur-containing solvents such as dimethylsulfoxide; and aromatic, saturated, and unsaturated hydrocarbons. And the like are used.
These organic solvents may be appropriately added to the buffer solution during the reaction, or the reaction may be performed only in the organic solvent. The immobilized biocatalyst used in the present invention can be used continuously or repeatedly for a long time even when additives harmful to these biocatalysts are used.

Examples of the hydroxy fatty acid produced by the method of the present invention include α-position, β-position, carbon chain center, ω3-position, ω2-position, ω1-position (methyl All of those having a hydroxyl group introduced into the terminal are included, and examples thereof include the following. 2-hydroxycaproic acid, 3-hydroxycaproic acid, 2-hydroxycaprylic acid, 3-hydroxycaprylic acid, 2-hydroxycapric acid, 3-hydroxycapric acid, 2-hydroxylauric acid, 3-hydroxylauric acid, 12- Hydroxy lauric acid, ricinoleic acid, 2-hydroxy myristic acid, 3-hydroxy myristic acid, 14-hydroxy myristic acid, 2-hydroxy pentadecanoic acid, 3-hydroxy pentadecanoic acid,
5-hydroxypentadecanoic acid, 2-hydroxypalmitic acid, 16-hydroxypalmitic acid, 2-hydroxystearic acid, 3-hydroxystearic acid, 2-hydroxypalmitoleic acid, 3-hydroxypalmitoleic acid, 2-hydroxylinoleic acid, 3 -Hydroxylinoleic acid, 2-hydroxylinolenic acid, 3-hydroxylinolenic acid, 2-hydroxy-γ-linolenic acid, 3-hydroxyarachidonic acid, 2-hydroxy-4-methyldodecanoic acid, 18-hydroxy-3-methyloctadecane Acids, 2-hydroxylignoceric acid, 2-hydroxyphytanic acid, 18-hydroxy-4-ethyloleic acid.
These hydroxy fatty acids are often obtained as optically active substances except for the hydroxy form at the ω-position. In such a case, by selecting a biocatalyst, it becomes possible to produce an optically active substance according to the intended use. If the optical purity needs to be improved as required, an optical isomer having a high optical purity can be obtained by an optical resolution method.

[0013]

According to the present invention, it becomes a surfactant (α-hydroxy acid salt, JP-A-2-283799) or a raw material of the surfactant, and further has an anti-wrinkle effect (JP-A-8-2980).
17622), and the ester form has antibacterial properties (Japanese Unexamined Patent Publication No.
325107) and various hydroxy acids which can be easily obtained as various medical and agricultural chemical intermediates,
Since it is possible to efficiently produce a fatty acid that can be easily obtained as a natural or synthetic raw material, an industrially extremely useful production method can be provided. In addition, the hydroxy fatty acid which is a product of the method of the present invention can be a mild surfactant having a salt or a derivative thereof. Therefore, it can be used as a detergent (raw material) for skin and hair, and a food or food such as vegetables and fruits. Not only as a cleaning agent for cosmetics, but also as an additive having a physiological activity, it can be incorporated into various cosmetics such as creams and emulsions. Further, this hydroxy fatty acid can be used as an intermediate of various useful medical and agricultural chemicals (oxygen reaction and precise organic synthesis, P.67 (1984) CMC).

[0014]

Next, the present invention will be described in more detail with reference to examples.

Reference Example 1 200 g of pea young leaves (var. Sativu)
m, 14 days of germination) in 0.2 M phosphate buffer (pH
6, 0.1% Triton X-100 (Rohm & Ha
pulverized with a homogenizer together with 2 liters to prepare a homogenate.

Comparative Example 1 10 g of palmitic acid was added to the above homogenate, and the mixture was stirred at 4 ° C. for about 15 hours under an oxygen stream. After completion of the reaction, insolubles were filtered off, the filtrate was adjusted to pH 3 with 6N hydrochloric acid, extracted with ethyl ether, the ether layer was dried over anhydrous sodium sulfate, and the solvent was distilled off. 4 g of reaction mixture were obtained. A part of this reaction mixture was subjected to methyl esterification with HCl / methanol in a conventional manner, and then analyzed by gas chromatography. The residue contained 20.8% of (R) -2-hydroxypalmitic acid. I understand. (Yield: 18.4%, optical purity: 99% ee or more) The filtration residue and 5 g of palmitic acid were added to the aqueous layer at the time of extraction, and the mixture was reacted for 15 hours under the same conditions as above. No production of 2-hydroxypalmitic acid was observed at all, indicating that the enzyme system was inactivated by one reaction.

Example 1 A 3% aqueous solution of sodium alginate was sterilized in an autoclave and allowed to cool to room temperature. 800 ml of this solution
In addition, 50 m of the homogenate prepared by the method of Reference Example 1 is used.
Add 1 and suspend. The suspension was added dropwise to a sterilized and cooled 0.1 M CaCl ₂ solution with stirring to obtain an immobilized biocatalyst as spherical beads having a diameter of about 3 mm. About 400 g of the immobilized biocatalyst was added to a 0.2 M phosphate buffer (pH 6, 0.1% Triton X-10).
0 liters), 5 g of palmitic acid was added, and the mixture was reacted at 20 ° C. for 10 hours under an oxygen stream. Then, the immobilized biocatalyst was collected by filtration, and the filtrate was treated in the same manner as in Comparative Example 1. Upon processing, about 4.8 g of reaction mixture was obtained.
Analysis of this reaction mixture according to conventional methods revealed that it contained 39.8% of 2-hydroxypalmitic acid. (Yield: 36.0%, optical purity: 99% ee or more) Using the recovered immobilized biocatalyst, a batch reaction was further performed under the same reaction conditions. Even when the reaction was repeated 20 times, the reaction yield was large. No significant decrease was observed, and high durability of the catalyst was observed.

[0018]

[Table 1] * Optical isomer excess

Reference Example 2 Arthrobacter simplex (IFO
3530) was slant cultured for 48 hours (the medium composition was the same as the culture solution), and then one platinum loop of bacterial cells was removed from the medium.
1 liter of culture (1% glucose, 1% peptone,
0.5% yeast extract, pH 7.2)
Shaking culture was performed at 30 ° C. for 24 hours using a Sakaguchi flask.

Comparative Example 2 4 g of stearic acid was added to the above-mentioned flask, followed by shaking culture for further 36 hours, and then the cells were collected by centrifugation.
After washing the cells, crushing the cells with ultrasonic waves (chloroform / methanol = 2/1) and extracting with a solvent, a reaction mixture containing 2-hydroxystearic acid (30% yield) was obtained. . A reaction was attempted under the same conditions using the recovered cell residue, but no production of 2-hydroxystearic acid was observed.

Example 2 After collecting and washing cells from 100 ml of the culture solution containing the cells obtained in Reference Example 2, a cell dilution was prepared by dispersing the cells in 100 ml of physiological food water. 20 ml of the above-mentioned bacterial cell dispersion is added to 500 ml of a sterilized carrageenan solution (4% concentration) at 37 ° C. The carrageenan solution containing the cells was dropped into a 2% KCl solvent at 20 ° C. with stirring, and the diameter was about 4 m.
Thus, immobilized cells were obtained as spherical beads of m. 200 g of the immobilized growth cells were added to 1 liter of the culture solution (see Reference Example 2), and then 4 g of stearic acid was added to add 36 g.
Perform shaking culture for hours. After separating and recovering the immobilized cells, the cells were washed with a solvent (chloroform / methanol 2/1) to obtain a washing solution. Then, the cells grown in the medium are collected, crushed, extracted with a solvent (same as above), combined with the washing solvent, dried and concentrated to give 2-hydroxystearic acid (yield) as a residue. 52%). As shown in the following table, no reduction in the reaction yield was observed even when the immobilized cells were repeatedly reacted under the same reaction conditions.

[0022]

[Table 2]

Claims (2)

[Claims] 1. A method for producing a hydroxy fatty acid, which comprises introducing a hydroxyl group into a fatty acid using an immobilized biocatalyst obtained by immobilizing the biocatalyst on a solid support. 2. The method according to claim 1, wherein the hydroxy fatty acid is an α- or β-hydroxy fatty acid.