JPH09234093A - Production of organic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject compound useful as an intermediate for medicines, agrochemicals, etc., by treating a specific aldoxime compound with the cells (treated products) of a microorganism having an ability for converting an aldoxime into an organic acid. SOLUTION: An aldoxime compound of formula I [R is an alkyl, an aromatic ring or a heterocyclic ring which may have one or more substituents; (n) is 0, 1; but when R is the alkyl, (n) is 0] (e.g. phenylacetaldoxime) is treated with the cells of a microorganism [e.g. Rhodococcus SP YH3-3 (FERM P-15478)] and/or the treated products of the cells having an ability for converting an aldoxime into an organic acid to efficiently obtain the objective organic acid of formula II (e.g. phenylacetic acid) useful as an intermediate for medicines, agrochemicals, etc., in mild conditions.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an organic acid. Specifically, the present invention relates to a microbial cell, and / or
Alternatively, it relates to a method for producing an organic acid by using the treated product of the cells. Organic acids are useful as intermediate raw materials for medicines and agricultural chemicals.

[0002]

2. Description of the Related Art A method for enzymatically producing an organic acid from a nitrile compound is described in C.I. D. Mathew, T .; Nagasaw
a, M. Kobayashi, H .; Yamada (Ap
pl. Environ. Microbiol. , 54,
1030 (1988)), but it is sometimes difficult to synthesize a nitrile compound as a raw material for production.
There has been a demand for a more effective method for producing an organic acid. Also,
Report of conversion of indole-3-acetoaldoxime to indoleacetic acid by enzymes of higher plant origin (J. Helmlinger, T. Rausch, W. et al.
Hilgenberg (Phytochemistry)
26, 3 (1987)), but this was a biochemical study and was not aimed at industrially producing an organic acid.

[0003]

The conventional method for producing organic acids by chemical synthesis requires harsh reaction conditions, so that its applicable range is limited. An object of the present invention is to provide a method for producing an organic acid under industrially mild conditions.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found a microorganism capable of producing an acid from an aldoxime compound and completed the present invention. That is, the present invention provides a compound represented by the general formula (I):

[0005]

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(In the formula, R represents an alkyl group which may have a substituent, an aromatic ring or a heterocycle, and n is 0 or 1. However, when R is an alkyl group, n is 0. A microbial cell having the ability to convert aldoxime to an organic acid, and / or a treated product of the microbial cell is allowed to act on the aldoxime compound represented by the general formula (II).

[0007]

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(In the formula, R and n have the same meanings as in formula (I)). Hereinafter, the method of the present invention will be described in detail.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, an aldoxime represented by the formula (I) is used as a raw material, and a microbial cell of the above-mentioned specific microorganism or a treated product of the microbial cell is allowed to act on the aldoxime to obtain the compound of the formula (II) An organic acid represented by (Raw materials and products) Regarding the aldoximes represented by the formula (I), when R is an alkyl group, the number of carbon atoms of the alkyl group is not particularly limited, but is usually 1 to 5, preferably 1
~ 3. The alkyl group may be linear, branched or cyclic, and examples of the linear alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group and a hexyl group, and a branched alkyl group. Examples of the group include isopropyl group, isobutyl group, sec-butyl group, ter
Examples thereof include t-butyl group, and examples of the cyclic alkyl group include cyclopropyl group and cyclobutyl group. Then, specific examples of the corresponding aldoximes include acetoaldoxime, propionaldoxime, n-butylaldoxime, isobutylaldoxime, n-barrelaldoxime, isobarrelaldoxime, capronaldoxime, and the like.

Further, examples of the aromatic ring which may have a substituent include phenyl, tolyl, hydroxyphenyl, methoxyphenyl, chlorophenyl, bromophenyl, fluorophenyl, nitrophenyl and the like. Specific examples include benzaldoxime, tolualdoxime, salicylaldoxime, methoxybenzaldoxime, chlorobenzaldoxime, bromobenzaldoxime, fluorobenzaldoxime, nitrobenzaldoxime, and phenylacetoaldoxime. To be

Further, examples of the heterocyclic ring which may have a substituent include a furfuryl group, a pyridyl group, an indole group and the like, and specific examples of the corresponding aldoximes include 2-furfuryl group. Examples thereof include aldoxime, indole-3-aldoxime, pyridine-3-aldoxime and the like.

The organic acid produced by the present invention is a compound represented by the formula (II), and specific examples thereof include organic acids corresponding to the above-mentioned aldoximes, such as benzoic acid and methylbenzoic acid. , Salicylbenzoic acid, methoxybenzoic acid, chlorobenzoic acid, bromobenzoic acid, fluorobenzoic acid, nitrobenzoic acid, phenylacetic acid, 2-
Furfuryl acetic acid, indole-3-acetic acid, nicotinic acid and the like can be mentioned.

(Microorganisms) In the present invention, the formula (I)
Any microorganism can be used as long as it has the ability to convert the aldoxime group of the aldoxime group represented by Particularly preferred microorganisms include microorganisms belonging to the genus Rhodococcus or microorganisms belonging to the genus Bacillus. Rhodococcus (Rho) used in the present invention
The microorganism belonging to the genus dococcus is, for example, Rhodococcus sp.
H3-3 can be mentioned. Examples of the microorganisms belonging to the genus Bacillus used in the present invention include Bacillus sp OxB-1. Rhodococcus sp.
s sp) YH3-3 and Bacillus
sp) OxB-1 is a bacterium isolated from the natural soil by the present inventors, and each is found in the Institute of Biotechnology, National Institute of Industrial Science and Technology, Biotechnology Research Institute No. 15478 (FERM P).
-15478) and Institute for Biological Science and Technology No. 15477 (FE
RM P-15477). Rhodococcus sp. Y
H3-3 and Bacillus sp Ox
The mycological properties of B-1 are as follows.

Identification Results of YH3-3 Strain 1. Morphological Properties On a normal agar medium, microscopic observation was carried out at 30 ° C. after 5 to 24 hours and 6 days after culturing. In the YH3-3 strain, the cells were unevenly elongated in the early stage of the culture, and some branched mycelial cells were also observed. After that, the cells are divided (fr
Aggregation), resulting in a short rod shape to a non-uniform rod shape. The short rod-shaped cells are in the form of double to short chains and have no motility. No aerial hyphae, spores and sporangia were observed.

2. Cultural Properties ISP [International Streptomyces Project (International Strep
on the defined medium, 3
The properties of the YH3-3 strain after culturing at 0 ° C. for 10 days are shown in Table 1.

[0016]

[Table 1] Table 1 ─────────────────────────────────── Cultivated land item YH3-3 ─ ────────────────────────────────── Yeast / malt agar G Good, smooth, light orange (ISP No. 2 ) AM not formed SP not produced Oatmeal agar G Very poor, smooth, light orange (ISP No. 3) AM not formed SP not produced, starch / inorganic salt agar G Very poor, smooth, light orange (ISP No. 3) 4) No AM formation No SP production Glycerol / G Poor, smooth, light orange asparagine agar No AM formation (ISP No. 5) No SP production ─────────────── ──────────────────── G: Growth, AM: Aerial mycelium, SP: Soluble pigment

3. Physiological properties Table 2 shows the physiological properties of the YH3-3 strain.

[0018]

[Table 2] Table 2 ─────────────────────────────────── YH3-3 ───── ────────────────────────────── (1) Growth temperature range 5 to 37 ℃ (2) Liquefaction of gelatin- (3) Hydrolysis of starch + (4) Coagulation of skim milk and peptone formation- (5) Formation of melanin-like pigment- (6) Utilization of carbon source (cultivation on Pridham-Gotlieve agar medium at 30 ° C for 7 days) ──── ─────────────────────────────── The YH3-3 strain shows some growth even in the control medium containing no carbon source. Therefore, the relative assimilation with negative control as the third
It is shown in the table.

[0019]

[Table 3] Table 3 ──────────────────────────────── YH3-3 ───────── ──────────────────────── L-arabinose-D-fructose + D-glucose + inositol-D-mannitol + raffinose ± L-rhamnose ± sucrose + D-xylose-───────────────────────────────── +: Use, ±: Use weakly, −: Do not use

4. Chemotaxonomic properties The chemical taxonomic properties of the YH3-3 strain are shown in Table 4.

[0021]

[Table 4] Table 4 ────────────────────────────────── YH3-3 ────── ──────────────────────────── (1) Dibasic amino acid of cell wall peptidoglycan meso-diaminopimelic acid (2) Presence or absence of mycolic acid ( 3) Cellular fatty acid C16: 0 C16: 1 10Me-C19: 0 ───────────────────────────────────

5. Taxonomic consideration 1) Higher-order classification YH3-3 strain forms a light orange colony, and as a cell morphology, from mycelial cells that are unevenly elongated in the early stage of culture,
The Rod-Coccus cycle is shown as a rod-like shape that is then divided with time.
In addition, the dibasic amino acid of cell wall peptidoglycan is me
So-diaminopimelic acid. Cellular fatty acids are straight-chain
It is monounsaturated and contains 10 methyl-C19: 0, and has mycolic acid which is a cell wall lipid component. The above features are the features of the Burjay's Manual of Systematic Bacteriology [[Bergey's Manu
al of Systematic Bacterio
2], pp. 1472-1481, 1986.
Rhodococcus (Rhodococcus)
cus) and the characteristics of the genus
Rhodococcus sp.
sp. ).

Identification Results of OxB-1 Strain Each property of the OxB-1 strain is shown in Tables 5 to 7. 1. Morphological properties

[0024]

[Table 5] Table 5 ───────────────────────────────── OxB-1 ─────── ────────────────────────── (1) Cell shape Long bacillus (2) No cell polymorphism (3) Motility, Periflagellate (4) Presence or absence of spores, no spore expansion of cells ────────────────────────────────── 2. Cultural properties

[0025]

[Table 6] Table 6 ─────────────────────────────────── OxB-1 ───── ────────────────────────────── (1) Meat broth agar plate culture (30 ℃, 7 days) Colony size: Diameter 2-5mm Colony characteristics: cream color, translucent, smooth, flat, full edge, glossy (2) Broth liquid culture growing on the surface (3) Meat juice gelatin stab culture (20 ° C, 30 days) No liquefaction ( 4) Litmus milk (30 ° C, 7 days) No change ──────────────────────────────────── 3 ． Physiological properties

[0026]

[Table 7] Table 7 ──────────────────────────────── OxB-1 ───────── ──────────────────────── (1) Gram stain positive (2) Reduction of nitrates and nitrites + (3) Denitrification reaction (4) MR test- (5) VP test- (6) Production of indole- (7) Production of hydrogen sulfide- (8) Hydrolysis of starch- (9) Utilization of citric acid Christians medium + shimmons medium- (10) Pigment Production- (11) Urease- (12) Oxidase + (13) Catalase + (14) Growth range: Temperature 15-45 ° C pH pH 6-9 (15) Attitude toward oxygen Aerobic (16) OF test reaction ──────────────────────────────────

[0027]

[Table 8] Table 7 (continued) ──────────────────────────────── OxB-1 ────── ─────────────────────────── (17) Production of Acid from Sugar L-arabinose-D-xylose-D-glucose-D- Mannose-D-galactose-maltose-sucrose-lactose-trehalose-D-sorbitol-D-mannitol-inositol-glycerin-starch- (18) βgalactosidase + (19) DNase + (20) hydrolysis of hippuric acid- ( 21) Casein hydrolysis- (22) Tyrosine decomposition + (23) Vitamin requirement + (24) Salt tolerance NaCl 5% + ────────────────────── ───────────

4. Taxonomic consideration As a result of the above, the present bacterium is 1) aerobic Gram + bacillus, 2) shows motility, and 3) forms spores. Therefore, the Burjay's Manual of Systematic Bacteriology [[Bergey's Manual o
f Systematic Bacterialog
y] Volume 2, 1104-1139, 1986], it was found to be included in the genus Bacillus. In addition, from the "Guide for Medical Bacterial Identification, 3rd Edition, page 97, 1993", Bacillus is characterized by the fact that it does not generate acid from many sugars including glucose.
Brevis (Bacillus brevis), Bacillus sphaeri (Bacillus sphaeri)
cus and Bacillus Bacillus
However, the three types did not agree with each other in some properties such as cell swelling by spores, galactosidase activity, and hydrolysis of hippuric acid. Therefore, the OxB-1 strain of this bacterium is used as a Bacillus sp.
Illus sp).

The above-mentioned microorganism may be any strain such as a mutant strain or a recombinant strain derived by a genetic technique such as cell fusion or gene recombination. In the production method of the present invention, one type or two or more types of the above-mentioned microorganisms are used as the cells and / or the treated cells. Specifically, the bacterial cells obtained by culturing the above-mentioned microorganism as they are, or those obtained by treating the bacterial cells obtained by culturing by a known method, that is, acetone-treated, freeze-dried, Treated cells such as those obtained by physically or enzymatically disrupting the cells can be used. In addition, an enzyme fraction having the ability of acting on the oxime group of the oxime represented by the formula (I) and converting the oxime group of the oxime represented by the formula (I) into an organic acid represented by the formula (II) is obtained as a crude product. Alternatively, it can be taken out and used as a purified product. Furthermore, it is also possible to use the thus obtained cells, treated cells, enzyme fraction, etc. immobilized on a carrier such as polyacrylamide gel or carrageenan gel. Therefore, in the present specification, the term “bacterial body and / or processed product of the bacterial body” is used as a concept including all of the above-described bacterial cell, processed product of bacterial body, enzyme fraction, and immobilized products thereof.

(Manufacturing Method) Next, the manufacturing method of the present invention will be specifically described. In the production method of the present invention, the microorganism is usually used by culturing, but this culturing can be performed by a standard method. The medium used for culturing the present microorganism contains a carbon source, a nitrogen source, inorganic ions, and the like that can be utilized 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 a nitrogen source,
NZ amine, tryptose, yeast extract, polypeptone and the like are used appropriately. 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 to 8 and at a temperature of about 20 to 35 ° C. while controlling within an appropriate range.
Perform for 0 hours.

As a method for producing an organic acid by allowing the above-mentioned microorganism to act on the oxime represented by the formula (I), the microorganism is cultivated, and the microbial cell suspension thus obtained is subjected to the oxime represented by the formula (I). To obtain an organic acid by reacting the compounds with each other, adding an oxyl represented by the formula (I) to the medium to carry out the culture and the reaction simultaneously, or adding the oxime represented by the formula (I) after the completion of the culture. Then, a method of carrying out further reaction can be used. The reaction temperature is preferably from 15 to 40 ° C, and the pH is in the range of from 5 to 10. The concentration of the oxime represented by the formula (I) is preferably in the range of 0.1 to 10%, and if necessary, the oxime represented by the formula (I) may be used during the reaction.
Addition is added. The reaction may be promoted by adding a metal ion as needed. The organic acid obtained by the culturing and the reaction can be collected by a usual separation / purification method such as solvent extraction and chromatography.

[0032]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but ordinary modifications in the technical field of the present invention can be made without departing from the scope of the invention.

Example 1 Disodium malonate 5.0 g / l, yeast extract 0.5 g / l, dipotassium phosphate 2.0 g / l, N
aCl 1 g / l, magnesium sulfate heptahydrate 0.02
g / l, syn / anti-phenylacetoaldoxime 1 g / l, vitamin mixture solution (Table 8) 10 ml, trace elements (Table 9) 10 ml, ammonium phosphate 2.
Liquid medium 400 having a composition of 0 g / l (pH 8.0)
Rhodococcus sp.
us sp) YH3-3 was inoculated and cultured aerobically at 30 ° C for 24 hours. The obtained culture was centrifuged, and the cells were collected and washed with a 50 mM potassium chloride aqueous solution. 10m to this
A 100 mM potassium phosphate buffer solution (pH 7.0) containing M syn / anti-phenylacetoaldoxime was added to make the total volume 40 ml and reacted at 30 ° C. under aerobic conditions for 2 hours. The amount of phenylacetic acid produced and accumulated at the end of the reaction is 10
It was mM. Example 2 Disodium malonate 5.0 g / l, yeast extract 0.5 g / l, dipotassium phosphate 2.0 g / l, N
aCl 1 g / l, magnesium sulfate heptahydrate 0.02
g / l, phenylacetoaldoxime 1 g / l, vitamin mixture solution (Table 8) 10 ml, trace elements (Table 9) 1
0 ml, ammonium phosphate 2.0 g / l (pH 8.
Bacillus sp. OxB-1 was inoculated into 400 ml of a liquid medium having the composition of
The cells were cultivated aerobically at 24 ° C for 24 hours. The obtained culture was centrifuged, and the cells were collected and washed with a 50 mM potassium chloride aqueous solution. A 100 mM potassium phosphate buffer solution (pH 7.0) containing 10 mM syn / anti-phenylacetoaldoxime was added thereto to make the total volume 40 ml, and the reaction was carried out at 30 ° C. under aerobic conditions. During the reaction, syn / anti-
Phenylacetaldoxime was sequentially added to give a final addition amount of 250 mM. The amount of phenylacetic acid produced and accumulated at the end of the reaction was 230 mM.

[0034]

[Table 9] Table 8 ─────────────────────────────── Vitamin mixture stock solution ───────── ─────────────────────── Biotin 20 μg Thiamine hydrochloride 4 mg Calcium pantothenate 4 mg Inositol 20 mg Pyridoxine hydrochloride 4 mg Nicotinic acid 4 mg Paraaminobenzoic acid 2 mg Riboflavin 2 mg Folic acid 100 μg Distilled water 1000 ml ────────────────────────────────

[0035]

[Table 10] Table 9 ─────────────────────────────── Stock solution of trace elements ────────── _{─────────────────────── Titrplex 0.5g H 3 BO 4 0.03g} FeSO 4 · 7H 2 O 0.2g CoCl 2 · 6H 2 O 0 _{.02g ZnSO 4 · 7H 2 O 0.01g} CuCl 2 · 2H 2 O 0.001g MnCl 2 · 4H 2 O 0.003g NiCl 2 · 6H 2 O 0.002g Na 2 MoO 4 · 2H 2 O 0.003g distilled 1000 ml of water ─────────────────────────────────

[0036]

INDUSTRIAL APPLICABILITY According to the present invention, an organic acid can be easily produced from an oxime derivative by utilizing a microorganism.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:07) (C12N 1/20 C12R 1:01) (C12N 1/20 C12R 1:07)

Claims (2)

[Claims] 1. A compound of the general formula (I) (In the formula, R represents an alkyl group which may have a substituent, an aromatic ring or a heterocycle, and n is 0 or 1.
However, when R is an alkyl group, n is 0. ) The aldoxime compound represented by the formula (II) is treated with a microbial cell having the ability to convert an aldoxime to an organic acid, and / or a treated product of the microbial cell. (In the formula, R and n have the same meanings as in formula (I).) A method for producing an organic acid represented by the formula. 2. The method for producing an organic acid according to claim 1, wherein the microorganism is a microorganism belonging to the genus Rhodococcus or the genus Bacillus.