JPH1057084A - Microbiological production method from diester to monoester - Google Patents

Abstract

(57) [Problem] To produce a diester into a monoester using a microbiological technique. SOLUTION: The following general formula (I): (In the above general formula (I), R represents a C ₁ -C ₆ alkyl group or a C ₂ -C ₆ alkenyl group, and n is 2, 4 or 6
Is reacted with a cell of a microorganism and / or a treated product of the cell to give a diester represented by the following general formula (II): (Wherein R and n in the above general formula (II) are as defined above) are produced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a microbiological method for producing a diester from a diester to a monoester, and more particularly to a method for producing a monoester from a diester by a microorganism and / or a treated product of the microorganism.

[0002]

2. Description of the Related Art 4-Hydroxybutyl acrylate is a substance useful as an industrial raw material. Conventionally, monoesters are
It was produced by chemical synthesis from raw materials diol and acid. In addition, many methods for biochemically producing a monoester from a diester are known, such as a reaction using a lipase.

[0003]

When a monoester is produced from a raw material diol and an acid in a conventional chemical synthesis, a diester is produced as a by-product, so that a burden is imposed on a purification step. Therefore, there has been a demand for the development of a method for selectively producing a monoester by a biological method. Further, in the conventional biological production method for producing a monoester from a diester, it may be difficult to obtain a large amount of a lipase as a catalyst, and a more effective method for producing a monoester has been demanded.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to provide a novel method for producing a monoester.
A microorganism capable of producing a monoester from a diester has been found, and the present invention has been completed. That is, the present invention relates to the following general formula (I)

[0005]

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(In the above general formula (I), R represents a C ₁ -C ₆ alkyl group or a C ₂ -C ₆ alkenyl group, and n represents 2, 4 or 6) Characterized by the following general formula (II):

[0007]

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(In the above general formula (II), R and n are as defined above.) Note that two Rs in the general formula (I) represent the same group at the same time.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the present invention, the diester represented by the above general formula (I) is used as a raw material, and the cells of the microorganism and / or the processed product of the cells are allowed to act on the diester represented by the above general formula (II). Produce monoester.

In the present invention, any microorganism can be used as long as it has a capability of converting the diester represented by the above general formula (I) into a monoester. It is preferable to use cells of microorganisms selected from microorganisms belonging to the cells or processed cells thereof. As a microorganism belonging to the genus Brevibacterium, for example, Brevibacterium linens (Bre
vibrantium linens),
Specifically, Brevibacterium linens (Brevi)
(bacterium linens) IFO12141
(Fermentation Research Institute).

[0011] The microorganism may be UV-irradiated, N-methyl-N
-Mutant strains by nitrosoguanidine (NTG) treatment, ethyl methanesulfonate (EMS) treatment, nitrite treatment, acridine treatment, etc., or recombinant strains induced by genetic techniques such as cell fusion or gene recombination. Any strain may be used. In the production method of the present invention, cells of the above-mentioned microorganisms and / or treated cells are used. Specifically, the cells obtained by culturing the microorganisms as they are or those obtained by culturing the cells obtained by a known method, that is, those treated with acetone, those subjected to freeze-drying, A treated product of cells, such as one obtained by physically or enzymatically crushing cells, can be used. Also,
From these cells or processed cells, the above-mentioned general formula (I)
It is also possible to take out and use an enzyme fraction having the ability to act on the diester represented by and convert it to the monoester represented by the general formula (II) as a crude product or a purified product. Furthermore, the cells obtained in this manner,
It is also possible to use a product obtained by immobilizing a treated bacterial cell, an enzyme fraction or the like on a carrier such as polyacrylamide gel or carrageenan gel. Therefore, in this specification, the term "microbial cells and / or processed cells" is used as a concept containing all of the above-mentioned microbial cells, processed microbial cells, enzyme fractions, and immobilized products thereof.

Next, the production method of the present invention will be specifically described. In the production method of the present invention, the microorganism is usually
It is used after culturing, and this culturing can be performed in a usual manner. The medium used for culturing the present microorganism contains a carbon source, a nitrogen source, inorganic ions, and the like that can be used by the present microorganism. As the carbon source, carbohydrates such as glucose, alcohols such as glycerol, organic acids and the like are appropriately used. As the nitrogen source, NZ amine, tryptose, yeast extract, polypeptone and the like are appropriately used. As the inorganic ion, a phosphate ion, a magnesium ion, an iron ion, a manganese ion, a molybdenum ion and others are appropriately used as needed. Furthermore, it is effective to add inositol, pantothenic acid, nicotinamide and other vitamins as necessary. The culture is carried out under aerobic conditions at a pH of about 6-8 and a temperature of about 20-35.
The reaction is performed for 15 to 100 hours while controlling the temperature in an appropriate range of ° C.

As a method for producing a monoester by allowing the above-mentioned microorganism to act on the diester represented by the above-mentioned general formula (I), the present microorganism is cultured, and the obtained cell suspension is mixed with the above-mentioned general formula (I) A method in which a monoester is obtained by mixing and reacting with a diester represented by the following formula, a method in which a medium and a diester represented by the above general formula (I) are mixed and culture and reaction are carried out simultaneously, or The above general formula (I)
And a method of further reacting the mixture by mixing with the diester represented by the formula (1). The reaction temperature is preferably from 15 to 40 ° C, and the pH is in the range of from 5 to 10. The above general formula (I)
Is preferably in the range of 0.1 to 10%, and if necessary, the diester represented by the above general formula (I) is added during the reaction. In some cases, the reaction may be promoted by coexisting a metal ion if necessary. The monoester obtained by the culture and the reaction can be collected by a conventional separation / purification method such as solvent extraction and chromatography.

[0014]

The present invention will be described in more detail with reference to the following examples. However, ordinary modifications in the technical field of the present invention can be made without departing from the gist of the present invention. <Example 1> Peptone 10.0 g / L, meat extract
5.0 g / L, dipotassium phosphate 1.0 g / L (pH
7.0) was added to 400 mL of a liquid medium having the composition of Brevibacterium linens.
m. linens) IFO12141 and inoculated at 28 ° C.
For 48 hours. The obtained culture was centrifuged, and the cells were collected and washed twice with physiological saline. This is 10
A 200 mM potassium phosphate buffer (pH 8.0) containing 0 mM 1,4-butanediol diacrylate was added to make a total volume of 400 mL, and reacted at 28 ° C. for 24 hours. After completion of the reaction, 4-hydroxybutyl acrylate was fractionated by liquid chromatography. 4 at the end of the reaction
-Hydroxybutyl acrylate formation accumulation amount is 71.3.
mM.

Example 2 Glucose 20.0 g /
L, saccharose 10.0 g / L, meat extract 10.
0g / L, Peptone 5.0g / L, Yeast extract
2.0 g / L, sodium chloride 3.0 g / L, dipotassium phosphate 4.0 g / L, potassium phosphate 2.0 g / L
L, magnesium sulfate heptahydrate 0.2 g / L (pH
7.0) was added to 400 mL of a liquid medium having the composition of Brevibacterium linens.
m. linens) IFO12141 and inoculated at 28 ° C.
After aerobically culturing for 48 hours, 1,4-butanediol diacrylate (100 mM) was added to the culture solution, and shaking was further continued for 24 hours. After completion of the reaction, 4-hydroxybutyl acrylate was fractionated by liquid chromatography. The amount of 4-hydroxybutyl acrylate produced and accumulated at the end of the reaction was 75.0 mM.

Example 3 An enzyme catalyzing this reaction was ultrasonically crushed from the cells cultured in the medium shown in Example 2,
EAE-Toyopearl (manufactured by Tosoh Corporation), D
EAE-Sepharose (Pharmacia), Q
-Sepharose (Pharmacia), Phen
yl-Sepharose (Pharmacia), Su
Purdex (Pharmacia), Mono Q (Pharmacia), Resource Q (Pharmacia), and Superose 12 (Pharmacia) columns were used in this order for purification.
This purified enzyme (0.2 unit) and 1,4-butanediol diacrylate (10 mM) were reacted at 30 ° C. for 6 hours in 1 ml of 100 mM potassium phosphate buffer (pH 7.5). After the reaction was completed, 4-hydroxybutyl acrylate was fractionated by liquid chromatography. After the reaction for 6 hours, the amount of 9.5 mM 4-hydroxybutyl acrylate was produced.

<Examples 4 to 12> 0.2 unit of the purified enzyme obtained in Example 3 was added to 100 mM potassium phosphate buffer 1
10 mM and 3 mM of various diesters per ml (pH 7.5).
The reaction was performed at 0 ° C. for 60 minutes. Table 1 shows the results.

[0018]

[Table 1] ─────────────────────────────────── Concentration of diester formed monoester ────── ───────────────────────────── Example 4 1,4-butanediol diacrylate 8.2 mM Example 5 Ethylene glycol diacetate 2.2 mM Example 6 Ethylene glycol dimethacrylate 2.6 mM Example 7 Ethylene glycol dibutyrate 4.7 mM Example 8 1,4-butanediol diacetate 7.3 mM Example 9 1,4-butanediol dimethacrylate 0. 71 mM Example 10 1,6-hexanediol diacetate 0.35 mM Example 11 1,6-hexanediol diacrylate 4.0 mM Example 12 1,6-hexanediol dimeta Acrylate 0.69 mM ───────────────────────────────────

[0019]

According to the present invention, a monoester compound can be easily produced from a diester compound using a microorganism.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:13)

Claims (3)

[Claims] 1. A compound represented by the following general formula (I) (In the above general formula (I), R represents a C ₁ -C ₆ alkyl group or a C ₂ -C ₆ alkenyl group, and n is 2, 4 or 6
Represents Wherein the cells of the microorganism and / or the treated cells are allowed to act on the diester represented by the following general formula (II): (In the above general formula (II), R and n are as defined above.) 2. The method according to claim 1, wherein the microorganism is a microorganism belonging to the genus Brevibacterium. 3. The method according to claim 1, wherein the microorganism is Brevibacterium linens.