JPH10276792A - Production of hydroxycarboxylic acid and its amide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound that is useful as a pharmaceutical, perfume or pharmaceutical intermediate in high yield by allowing a microorganism capable of hydrolyzing the nitrile group in a specific hydroxynitrile to act on a hydroxynitrile in an aqueous medium. SOLUTION: A microorganism that is capable of hydrolyzing the nitrile group in a hydroxynitrile represented by formula I (R1 and R2 are H, a 1-4 C straight branched chain alkyl or R1 and R2 link to each other to from a cyclic ring; (n) is an integer of 1-3) as 5-hydroxyhexanenitrile such as Corynebacterium nitrilophilus ATCC 21419 or the treatment product of this microorganism is allowed to act on a hydroxynitrile in an aqueous medium to industrially advantageously produce the objective hydroxycarboxylic acid (5-hydroxyhexanoic acid) that is represented by formula II and is important as medicine, perfume or medicinal intermediate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The general formulas [II] and [II
The hydroxycarboxylic acids and amides thereof represented by the formula [I] are an important group of compounds as pharmaceuticals, fragrances or pharmaceutical intermediates. Above all, 3-hydroxy-3-methylbutyric acid is a compound useful as a foodstuff, a pharmaceutical intermediate or an agricultural chemical intermediate, for example, which has recently been reported for various pharmacological activities.

[0002]

2. Description of the Related Art Known synthetic carboxylic acid production methods for hydroxycarboxylic acids include (1) a method in which a lactone is synthesized from a ketone and ketene and then ring-opened [J. Am. Che
m. Soc. , 76, 486 (1954)], (2) a method of reacting a mixture of alcohol, carbon monoxide and hydrogen peroxide in the presence of iron sulfate [J. Am. Chem. S
oc. , 80, 2882 (1958)], (3) a ketone and a halogenated alkyl ester are subjected to a Reformatsky reaction,
Alkali hydrolysis [Zh. Russ. Fiz.
-Khim. O-va. 22, 47 (1890)],
(4) A method of oxidizing a compound having an olefin bond and a hydroxyl group in the same molecule with potassium permanganate [J. Prakt. Chem. , 23, 206 (18
81)], (5) Hydration and oxidation reaction of a primary alcohol having an olefin bond with nitric acid [Khim. Pir.
Soedin. , 3,313 (1991)], (6)
A method of oxidizing 4,4-dimethyl-1,3-dioxane using nitric acid as a catalyst (Arm. Khim. Zh., 44
(7-8), 443 (1991)], (7) oxidizing the terminal acetyl group of a compound having a hydroxyl group and a terminal acetyl group in the same molecule with sodium hypochlorite,
Method for deriving a carboxyl group [US Pat.
No. 70], (8) 3-hydroxy-3-methylbutano-
A method of oxidizing oxygen with a platinum-based catalyst in the presence of water (Japanese Patent Application Laid-Open No. 8-198800) is known. In addition, as a known microbiological production method, (9) Endomycesreses
ii) β- of isovaleric acid by CBS strain 179.60
Synthesis by oxidation reaction [J. Ferment. Tecno
l. , 59, 257 (1981)].

On the other hand, as a microbiological method for producing an amide compound having a hydroxyl group, (10) a hydration reaction using a bacterium belonging to the genus Rhodococcus (Japanese Patent Publication No. 2-291283) is known.

[0004]

As described above, many methods for producing hydroxycarboxylic acids and amides thereof have been reported. However, in the method (1), ketene as a raw material is a gas at normal temperature and normal pressure, which causes difficulties in handling.
Unreacted raw materials remain. The method (2) uses dangerous high-pressure carbon monoxide gas and has a low yield. In (3) and (4), toxic heavy metals which are a problem in industrial disposal in the treatment after the reaction are used, and the raw materials are expensive. In the methods (5) and (6), nitric acid is used as a catalyst for the oxidation reaction, so that the corrosion of the apparatus and
There is a problem such as by-product of nitric oxide. According to the method (7), toxic formalin is formed after the reaction, and thus is not suitable for a production method for food use. In (8), an expensive platinum catalyst must be used, and since it is a pressurized oxygen oxidation reaction, problems such as scale-up remain. The reaction (9) has a low yield and a low productivity of the microbial catalyst. In the reaction of (10), there was no specific disclosure in the examples of the production examples of the hydroxyamide represented by the general formula [II], and the production of the corresponding hydroxyamide was unknown. As described above, all of the above methods (1) to (10) have problems to be solved and are not satisfactory methods for producing hydroxycarboxylic acids and amides thereof.

Under these circumstances, the present inventors aimed at developing an industrially advantageous process for producing hydroxycarboxylic acids and amides thereof, and converted the nitrile group of hydroxynitrile represented by the general formula [I] to acidic or acidic. Various studies were made on methods for producing the hydroxycarboxylic acids represented by the general formulas [II] and [III] or amides thereof by chemically hydrolyzing or hydrating them under basic conditions. However, in any of the general chemical hydrolysis methods, the hydrolysis reaction or the hydration reaction hardly proceeds in any of the methods, and the yield in the production reaction of the target hydroxycarboxylic acid and its amide is extremely low. It turned out to be unsuitable for production.

[0006]

Accordingly, the present inventors have focused on a microbial catalyst that hydrolyzes or hydrates nitrile under mild conditions, and has studied a method for producing hydroxycarboxylic acid and its amide using microorganisms. ,as a result,
The present invention has been found that the use of a microorganism capable of hydrolyzing or hydrating a nitrile group is extremely effective in improving the yield which could not be achieved by a general chemical hydrolysis method. Reached.

That is, the present invention provides a method for treating a hydroxynitrile represented by the following general formula [I] by reacting a microorganism having hydrolytic ability to a nitrile group of the hydroxynitrile or the treated product with the hydroxynitrile in an aqueous medium. A method for producing a hydroxycarboxylic acid, which comprises producing a hydroxycarboxylic acid represented by the formula (II), [Wherein, R ₁ and R ₂ may be the same or different and are formed by bonding a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or R ₁ and R ₂ A cyclo ring, n represents an integer of 1 to 3. Further, a microorganism having a hydrating ability for the nitrile group of the hydroxynitrile represented by the above general formula [I] or the treated product is allowed to act on the hydroxynitrile in an aqueous medium, whereby the following general formula [III] is obtained. A method for producing a hydroxycarboxylic acid amide, which comprises producing the hydroxycarboxylic acid amide shown below. [Wherein, the definitions of R ₁ , R ₂ and n are the same as above. ]

[0008]

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on an enzymatic reaction derived from a microorganism capable of hydrolyzing or hydrating a nitrile group of hydroxynitrile represented by the general formula [I]. The hydroxycarboxylic acid represented by the general formula [I] from the hydroxy nitrile,
Through the production of the hydroxycarboxylic acid amide represented by the general formula [III] directly by the action of nitrilase or by a combination of nitrile hydratase and an amidase, the hydroxycarboxylic acid amide represented by the general formula [III] is converted to the general formula [III] It is thought that it can be obtained from the hydroxy nitrile represented by I) by the action of nitrile hydratase.

It has been reported that the above three kinds of enzymes are mixed in one microbial cell. However, by appropriately selecting the culture conditions and reaction conditions, the enzyme activity for obtaining the desired product can be reduced. It can be expressed predominantly.

The microorganism in the present invention is, for example, Rhodococcus, Brevibacterium, Corynebacterium, Nocardia, Mycobacterium.
obacterium) or micrococcus (Mi)
crococcus).

[0011] Specifically, Rhodococcus rhodochrous
J-1 (FERM BP-1478), Rhodococcus
Erythropolis (Rhodococcus eryth)
ropolis) IFO12538, Rhodococcus sp. SK70
(FERM P-11304), Rhodococcus sp. HT40-6.
(FERM BP-5231), Brevibacterium
Acetylcum (Brevacterium ace)
Tylicum) IAM1790, Corynebacterium nitrophyllus (Corynebacterium)
nitrileophilus ATCC 21419, Corynebacterium sp.
rium. sp. ) KO-2-4 (FERM BP-2)
53), Nocardia Erisropolis (Nocard)
iaerythropolis IFO12539, Mycobacterium sp.
umu. sp. ) AC 777 (FERM BP-23)
52), Micrococcus Luteus
(C. luteus) ATCC383. These strains have been deposited under the above numbers, respectively, and the Institute of Biotechnology and Industrial Technology,
It can be easily obtained from the Institute of Fermentation (IFO), Institute of Applied Microorganisms of the University of Tokyo (IAM), and American Type Culture Collection (ATCC).

The hydroxynitrile represented by the general formula [I] used as a raw material in the present invention can be easily and easily obtained by a reformate ski reaction of a ketone compound or an aldehyde compound with a compound in which an alkyl group is substituted with a halogen or a nitrile. It can be manufactured industrially at low cost [Sy
nthesis (1987), (12), 113
0)].

The hydroxynitrile of formula (I) wherein n = 1 can be easily and industrially produced at low cost by reacting a ketone compound or an aldehyde compound with acetonitrile.
ompt. end. Acad. bulg. Sci. 1
9, 8, 739 (1966), Esti NSVT
ead. Akad. Toim. , Keem. (198
8), 37 (4), 248].

Specific examples of the hydroxynitrile represented by the general formula [I] include 3-hydroxypropionitrile, 4-hydroxybutyronitrile, 3-hydroxybutyronitrile, and 3-hydroxy-3-methylbutyro. Nitrile, 4-hydroxypentanenitrile, 3-hydroxypentanenitrile, 5-hydroxyhexanenitrile, 4-hydroxyhexanenitrile, 3-hydroxyhexanenitrile, 4-hydroxyheptanenitrile,
3-hydroxy-4-methylpentanenitrile, 3-hydroxyheptanenitrile, 3-hydroxy-3-methylpentanenitrile, 4-hydroxyoctanenitrile, 4-hydroxy-4-methylhexanenitrile, 3
-Hydroxy-3-methylhexanenitrile, 4-hydroxy-4-methylheptanenitrile, 3-hydroxy-3,4-dimethylpentanenitrile, 3-hydroxy-3-methylheptanenitrile, 4-hydroxy-4-
Methyloctanenitrile, 3-hydroxy-3, 4-dimethylhexanenitrile, 3-hydroxy-3,4,
4'-trimethylpentanenitrile, 3-hydroxy-
3,5-dimethylhexanenitrile, 4-ethyl-4-
Hydroxyhexanenitrile, 3-ethyl-3-hydroxyhexanenitrile, 4-ethyl-4-hydroxyheptanenitrile, 3-ethyl-3-hydroxy-4-methylpentanenitrile, 3-ethyl-3-hydroxyheptanenitrile, 4- Ethyl-4-hydroxyoctanenitrile, 3-ethyl-3-hydroxy-4-methylhexanenitrile, 3-ethyl-3-hydroxy-4,
4'-dimethylpentanenitrile, 3-ethyl-3-hydroxy-5-methylhexanenitrile, 4-hydroxy-4-propylheptanenitrile, 3-hydroxy-
3-isopropylhexanenitrile, 3-hydroxy-
3-propylheptanenitrile, 4-hydroxy-4-
Propyloctanenitrile, 3-hydroxy-4-methyl-3-propylhexanenitrile, 4-sec-butyl-4-hydroxyheptanenitrile, 3-tert-
Butyl-3-hydroxyhexanenitrile, 3-hydroxy-5-methyl-3-propylhexanenitrile, 3
-Hydroxy-3-isopropylheptanenitrile, 3
-Hydroxy-4-methyl-3-isopropylhexanenitrile, 3-hydroxy-4,4'-dimethyl-3-
Isopropylpentanenitrile, 3-hydroxy-5-
Methyl-3-isopropylhexanenitrile, 4-butyl-4-hydroxyoctanenitrile, 3-sec-butyl-3-hydroxyheptanenitrile, 3-tert
-Butyl-3-hydroxyheptanenitrile, 3-isobutyl-3-hydroxyheptanenitrile, 3-ter
t-butyl-3-hydroxy-4-methylhexanenitrile, 3-isobutyl-3-hydroxy-4-methylhexanenitrile, 3-sec-butyl-3-hydroxy-4-methylhexanenitrile, 5-hydroxy-6
Methylheptanenitrile, 5-hydroxy-6-methyloctanenitrile, 5-hydroxy-5-methyloctanenitrile, 5-hydroxy-5,6,6'-trimethylheptanenitrile, 3-ethyl-3-hydroxypentanenitrile, 5 -Ethyl-5-hydroxyoctanenitrile, 5-ethyl-5-hydroxy-7-methyloctanenitrile, 3-hydroxy-3-propylhexanenitrile, 5-hydroxy-5-propyloctanenitrile, 5-propyl-5-hydroxy -6-methyloctanenitrile, 3-hydroxy-4-methyl-3-isopropylpentanenitrile, 5-hydroxy-6, 6 '
-Dimethyl-5-isopropylheptanenitrile, 3-
Butyl-3-hydroxyheptanenitrile, 5-ter
t-butyl-5-hydroxynonanenitrile, 5-isobutyl-5-hydroxynonanenitrile, 3-sec-
Butyl-3-hydroxy-4-methylhexanenitrile, 5-isobutyl-5-hydroxy-6-methyloctanenitrile, 3-tert-butyl-3-hydroxy-4,4'-dimethylpentanenitrile, 5-tert
-Butyl-5-hydroxy-6,6'-dimethylheptanenitrile, 3-isobutyl-3-hydroxy-5-methylhexanenitrile, 5-isobutyl-5-hydroxy-7-methyloctanenitrile, (1-hydroxycyclohexyl) Examples include acetonitrile, (1-hydroxycyclopentyl) acetonitrile, (1-hydroxycyclohexyl) propionitrile, (1-hydroxycyclopentyl) propionitrile, (1-hydroxycyclohexyl) butyronitrile, and (1-hydroxycyclopentyl) butyronitrile.

The present invention is suitable for producing a hydroxycarboxylic acid represented by the general formula [II]. As the hydroxycarboxylic acid, specifically, 3-hydroxypropionic acid,
4-hydroxybutyric acid, 3-hydroxybutyric acid, 3-hydroxy-3-methylbutyric acid, 4-hydroxyvaleric acid, 3-hydroxyvaleric acid, 5-hydroxyhexanoic acid, 4-hydroxyhexanoic acid, 3-hydroxyhexanoic acid, -Hydroxyheptanoic acid, 3-hydroxy-4-methylvaleric acid, 3-hydroxyheptanoic acid, 3-hydroxy-3-
Methylvaleric acid, 4-hydroxyoctanoic acid, 4-hydroxy-4-methylhexanoic acid, 3-hydroxy-3-methylhexanoic acid, 4-hydroxy-4-methylheptanoic acid, 3-hydroxy-3,4-dimethylvaleric acid Folic acid, 3-hydroxy-3-methylheptanoic acid, 4-hydroxy-4
-Methyloctanoic acid, 3-hydroxy-3,4-dimethylhexanoic acid, 3-hydroxy-3,4,4'-trimethylvaleric acid, 3-hydroxy-3,5-dimethylhexanoic acid, 4-ethyl-4- Hydroxyhexanoic acid, 3-ethyl-3-hydroxyhexanoic acid, 4-ethyl-4-hydroxyheptanoic acid, 4-ethyl-3-hydroxy-4
-Methylvaleric acid, 3-ethyl-3-hydroxyheptanoic acid, 4-ethyl-4-hydroxyoctanoic acid, 3-ethyl-3-hydroxy-4-methylhexanoic acid, 3-ethyl-3-hydroxy-4,4 '-Dimethylvaleric acid, 3-
Ethyl-3-hydroxy-5-methylhexanoic acid, 4-
Hydroxy-4-propylheptanoic acid, 3-hydroxy-3-isopropylhexanoic acid, 3-hydroxy-3-
Propylheptanoic acid, 4-hydroxy-4-propyloctanoic acid, 3-hydroxy-4-methyl-3-propylhexanoic acid, 4-sec-butyl-4-hydroxyheptanoic acid, 3-tert-butyl-3-hydroxyhexane Acid, 3-hydroxy-5-methyl-3-propylhexanoic acid, 3-hydroxy-3-isopropylheptanoic acid, 3-hydroxy-4-methyl-3-isopropylhexanoic acid, 3-hydroxy-4,4′-dimethyl -3-
Isopropylvaleric acid, 3-hydroxy-5-methyl-3
-Isopropylhexanoic acid, 4-butyl-4-hydroxyoctanoic acid, 3-sec-butyl-3-hydroxyheptanoic acid, 3-tert-butyl-3-hydroxyheptanoic acid, 3-isobutyl-3-hydroxyheptanoic acid,
3-tert-butyl-3-hydroxy-4-methylhexanoic acid, 3-isobutyl-3-hydroxy-4-methylhexanoic acid, 3-sec-butyl-3-hydroxy-
4-methylhexanoic acid, 5-hydroxy-6-methylheptanoic acid, 5-hydroxy-6-methyloctanoic acid, 5
-Hydroxy-5-methyloctanoic acid, 5-hydroxy-5,6,6'-trimethylheptanoic acid, 3-ethyl-
3-hydroxyvaleric acid, 5-ethyl-5-hydroxyoctanoic acid, 5-ethyl-5-hydroxy-7-methyloctanoic acid, 3-hydroxy-3-propylhexanoic acid,
5-hydroxy-5-propyloctanoic acid, 5-propyl-5-hydroxy-6-methyloctanoic acid, 3-hydroxy-4-methyl-3-isopropylvaleric acid, 5-hydroxy-6,6'-dimethyl-5 -Isopropylheptanoic acid, 3-butyl-3-hydroxyheptanoic acid, 5-
tert-butyl-5-hydroxynonanoic acid, 5-isobutyl-5-hydroxynonanoic acid, 3-sec-butyl-3-hydroxy-4-methylhexanoic acid, 5-isobutyl-5-hydroxy-6-methyloctanoic acid, 3 -T
tert-butyl-3-hydroxy-4,4′-dimethylvaleric acid, 5-tert-butyl-5-hydroxy-6,
6'-dimethylheptanoic acid, 3-isobutyl-3-hydroxy-5-methylhexanoic acid, 5-isobutyl-5-
Hydroxy-7-methyloctanoic acid, (1-hydroxycyclohexyl) acetic acid, (1-hydroxycyclopentyl) acetic acid, (1-hydroxycyclohexyl) propionic acid, (1-hydroxycyclopentyl) propionic acid, (1-hydroxycyclohexyl) butyric acid, (1-hydroxycyclopentyl) butyric acid and the like can be mentioned. In particular, the present invention is suitable for producing 3-hydroxy-3-methylbutyric acid.

Examples of the hydroxyamide represented by the general formula [III] include 3-hydroxypropanamide, 4-hydroxybutanamide, 3-hydroxybutanamide, 3-hydroxy-3-methylbutanamide, 4-hydroxypentanamide, 3-hydroxypentanamide, 5-hydroxyhexaneamide, 4-hydroxyhexaneamide, 3-hydroxyhexaneamide, 4-hydroxyheptanamide, 3-hydroxy-
4-methylpentanamide, 3-hydroxyheptanamide, 3-hydroxy-3-methylpentanamide, 4
-Hydroxyoctanamide, 4-hydroxy-4-methylhexaneamide, 3-hydroxy-3-methylhexaneamide, 4-hydroxy-4-methylheptanamide, 3-hydroxy-3,4-dimethylpentanamide, 3-hydroxy -3-methylheptanamide, 4-
Hydroxy-4-methyloctanamide, 3-hydroxy-3,4-dimethylhexaneamide, 3-hydroxy-3,4,4′-trimethylpentanamide, 3-hydroxy-3,5-dimethylhexaneamide, 4-ethyl -4-hydroxyhexaneamide, 3-ethyl-3-hydroxyhexaneamide, 4-ethyl-4-hydroxyheptanamide, 3-ethyl-3-hydroxy-4-methylpentanamide, 3-ethyl-3-hydroxyheptanamide , 4-ethyl-4-hydroxyoctaneamide, 3-ethyl-3-hydroxy-4-methylhexanamide, 3-ethyl-3-hydroxy-4,4'-dimethylpentanamide, 3-ethyl-3-hydroxy- 5
-Methylhexanamide, 4-hydroxy-4-propylheptanamide, 3-hydroxy-3-isopropylhexanamide, 3-hydroxy-3-propylheptanamide, 4-hydroxy-4-propyloctaneamide, 3-hydroxy-4 -Methyl-3-propylhexanamide, 4-sec-butyl-4-hydroxyheptanamide, 3-tert-butyl-3-hydroxyhexaneamide, 3-hydroxy-5-methyl-3-propylhexanamide, 3-hydroxy -3-isopropylheptanamide, 3-hydroxy-4-methyl-3-isopropylhexanamide, 3-hydroxy-4,4 '
-Dimethyl-3-isopropylpentanamide, 3-hydroxy-5-methyl-3-isopropylhexanamide, 4-butyl-4-hydroxyoctaneamide, 3-
sec-butyl-3-hydroxyheptanamide, 3-
tert-butyl-3-hydroxyheptaneamide, 3
-Isobutyl-3-hydroxyheptanamide, 3-t
tert-butyl-3-hydroxy-4-methylhexaneamide, 3-isobutyl-3-hydroxy-4-methylhexaneamide, 3-sec-butyl-3-hydroxy-4-methylhexaneamide, 5-hydroxy-6 Methylheptanamide, 5-hydroxy-6-methyloctaneamide, 5-hydroxy-5-methyloctaneamide, 5-hydroxy-5,6,6'-trimethylheptanamide, 3-ethyl-3-hydroxypentanamide, 5 -Ethyl-5-hydroxyoctaneamide, 5-
Ethyl-5-hydroxy-7-methyloctaneamide,
3-hydroxy-3-propylhexanamide, 5-hydroxy-5-propyloctanamide, 5-propyl-5-hydroxy-6-methyloctaneamide, 3-hydroxy-4-methyl-3-isopropylpentanamide, 5- Hydroxy-6,6'-dimethyl-5-isopropylheptanamido, 3-butyl-3-hydroxyheptanamido, 5-tert-butyl-5-hydroxynonanamido, 5-isobutyl-5-hydroxynonanamido, 3-sec- Butyl-3-hydroxy-4-methylhexanamide, 5-isobutyl-5-hydroxy-6-methyloctaneamide, 3-tert-butyl-
3-hydroxy-4,4'-dimethylpentanamide,
5-tert-butyl-5-hydroxy-6,6'-dimethylheptaneamide, 3-isobutyl-3-hydroxy-5-methylhexaneamide, 5-isobutyl-5
Hydroxy-7-methyloctaneamide, (1-hydroxycyclohexyl) acetamide, (1-hydroxycyclopentyl) acetamide, (1-hydroxycyclohexyl) propanamide, (1-hydroxycyclopentyl) propanamide, (1-hydroxycyclohexyl) butanamide, (1-hydroxycyclopentyl) butanamide and the like.

The cultivation of the microorganism used in the present invention may be carried out by using a carbon source (glycerol, glucose, saccharo-
Source, nitrogen source (casamino acid, meat extract, yeast extract,
Inorganic salts (magnesium chloride, calcium chloride, manganese sulfate, iron chloride, zinc sulfate, etc.) essential for the growth of each microorganism, and metal salts required as prosthetic groups of nitrile hydrolases ( Cobalt chloride, etc.)
This is carried out using a normal medium containing the above. Nitriles or amides (nitrile cinnamate, benzyl cyanide, isobutyronitrile, 2-phenylpropionitrile, benzonitrile, 2-cyanopyridine, 3-cyanopyridine) so that growth is not significantly inhibited in the early or middle stage of culture It is preferable to add pyridine, 4-cyanopyridine, o-aminobenzonitrile, 1-cyclohexenylacetonitrile, ε-caprolactam, γ-butyronitrile, urea, butylamide, etc.) since higher enzyme activity can be obtained. The culture is performed aerobically for 1 to 14 days at a pH of the culture solution of 4 to 10 and a culture temperature of 20 to 70 ° C.

In the reaction, the cells of the microorganisms cultured in the above-mentioned method or the treated cells (crushed cells, crude enzyme solution, purified enzyme solution, immobilized cells, immobilized enzyme, etc.) are added to water or buffer solution. The suspension is suspended in an aqueous medium such as that described above, and a hydroxynitrile represented by the general formula [I], that is, a substrate is coexisted therewith. The reaction is carried out at a pH of 4 to 11, preferably 6 to 10. The amount of the substrate in the reaction solution is 0.1 to 10% by weight, preferably 0.1 to 5.0% by weight. The amount of the microorganism used may be 0.01 to 20% by weight as dry cells. The reaction may be performed at a reaction temperature of 5 to 50C, preferably 10 to 30C for 0.1 to 200 hours.

As a method for obtaining hydroxycarboxylic acid or hydroxycarboxylic acid amide from the reaction solution obtained by the microbial reaction, microorganisms are removed by a known method, for example, centrifugation, and if necessary, granule components, proteins, polysaccharides, etc. are obtained by ultrafiltration or the like. Removal of saccharides, or treatment with activated carbon followed by concentration under reduced pressure, extraction with an organic solvent, and the like can be mentioned.

[0020]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 (1) Culture and Preparation of Cell Suspension Corynebacterium nitrophyllus ATCC214
After culturing No. 19 in the following medium A at 30 ° C for 48 hours, the obtained cells were further cultured in medium B at 30 ° C for 96 hours.

[Medium A] 5 mM phosphate buffer (pH
7.5) In 100 mL, glucose 4%, sodium glutamate 2%, Shefton N 0.5%, taste liquid
0.5%, yeast extract 0.6%, sodium sulfate
Medium containing 0.3%, magnesium chloride 0.04%, calcium chloride 0.004%, manganese sulfate 0.003%, iron chloride 0.0006%, zinc sulfate 0.0003% (all w / v).

[Medium B] A medium obtained by adding 0.03% (w / v) of o-aminobenzonitrile to medium A.

The cells were centrifuged (10000 rpm) from the medium.
m, 20 minutes) and a 50 mM phosphate buffer (pH 7.5)
, And suspended in the same phosphate buffer (100 mL) to prepare a cell suspension (OD ₆₃₀ = 40.0).

(2) Hydrolysis of 5-hydroxyhexanenitrile 5-Hydroxyhexanenitrile (0.3 g) was added to the above cell suspension and stirred at 30 ° C for 3 days. After the microorganism was removed from the reaction solution by centrifugation, the obtained aqueous layer was subjected to activated carbon treatment. Further, after performing ethyl acetate extraction, the solvent was distilled off to obtain a concentrated residue (0.25 g). As a result of HPLC analysis using an ODS column, it was confirmed that the concentrated residue was 5-hydroxyhexanoic acid (reaction yield: 71%).

Example 2 (1) Culture and Preparation of Cell Suspension After cultivating Brevibacterium acetylicum IAM1790 in medium A at 30 ° C. for 48 hours, the obtained cells were further cultured in medium B at 30 ° C. for 96 hours. Cultured for hours. The cells were centrifuged (10000 rpm, 20 minutes) from the medium, and 50 mM
After washing once with a phosphate buffer (pH 7.5), the cells were suspended in the same phosphate buffer (100 mL), and the cell suspension (OD ₆₃₀ =
24.6) was adjusted.

(2) Hydrolysis reaction of 5-isobutyl-5-hydroxynonanenitrile 5-isobutyl-5-hydroxynonanenitrile (0.3 g) was added to the above cell suspension and stirred at 30 ° C for 18 hours. did. As a result of analysis by HPLC using an ODS column, 5-isobutyl-5-hydroxynonanoic acid (0.32
g) was confirmed to have been produced (reaction yield 97%).

Example 3 (1) Culture and Preparation of Cell Suspension After culturing Brevibacterium acetylicum IAM1790 in the following medium C at 30 ° C. for 48 hours, the obtained cells were further cultured in medium D at 30 ° C. Incubated for 72 hours.

[Medium C] 5 mM phosphate buffer (pH
7.5) In 1000 mL, glycerol 2.0%, yeast extract 0.6%, sodium sulfate 0.3%, magnesium chloride 0.04%, calcium chloride 0.00
4%, manganese sulfate 0.003%, iron chloride 0.00
Medium containing 0.6% zinc sulfate and 0.0003% zinc sulfate (all w
/ V). [Medium D] Medium C is ε-caprolactam 0.05%
(W / v) Added medium.

The cells were centrifuged (10000 rpm) from the medium.
m, 20 minutes) and a 50 mM phosphate buffer (pH 7.5)
, And suspended in the same phosphate buffer (100 mL) to prepare a cell suspension (OD ₆₃₀ = 9.5).

(2) Hydrolysis of 3-hydroxy-3-methylbutyronitrile 3-Hydroxy-3-methylbutyronitrile (0.5 g) was added to the above cell suspension, and the mixture was added at 30 ° C. for 18 hours. Stirred for hours. As a result of analysis by HPLC using an ODS column,
It was confirmed that 3-hydroxy-3-methylbutyric acid (0.58 g) was produced (reaction yield: 98%).

Example 4 (1) Culture and Preparation of Cell Suspension After cultivating Brevibacterium acetylicum IAM1790 in medium C at 30 ° C. for 48 hours, the obtained cells were further cultured in medium D at 30 ° C. for 72 hours. Cultured for hours. The cells were centrifuged (10000 rpm, 20 minutes) from the medium, and 50 mM
After washing once with a phosphate buffer (pH 7.5), the cells were suspended in the same phosphate buffer (100 mL), and the cell suspension (OD ₆₃₀ =
13.3) was adjusted.

(2) Hydrolysis reaction of 3-hydroxy-3-methylbutyronitrile 3-hydroxy-3-methylbutyronitrile (0.7 g) was added to the above cell suspension and stirred at 30 ° C. Started. Thereafter, 3-hydroxy-3-methylbutyronitrile (0.14 g) was successively added every two hours, and a total of 2.6 g of 3-hydroxy-3-methylbutyronitrile was added later (total addition amount: 3.3 g). ). After 72 hours from the start of stirring, the reaction solution was analyzed by HPLC using an ODS column.
It was confirmed that 3-hydroxy-3-methylbutyric acid (3.3 g) was produced (reaction yield: 89%).

Example 5 (1) Culture and Preparation of Cell Suspension After culturing Brevibacterium acetylicum IAM1790 in medium E at 30 ° C. for 48 hours, the obtained cells were further cultured in medium F at 30 ° C. for 96 hours. Cultured for hours.

[Medium E] 100 distilled water (pH 7.0)
In 0 mL, peptone 0.5%, meat extract 0.5%,
Yeast extract 0.05%, sodium chloride 0.2%,
A medium containing (all w / v).

[Medium F] Distilled water (pH 7.0) 100
In 0 mL, glucose 1.5%, sodium glutamate 0.75%, yeast extract 0.1%, magnesium sulfate heptahydrate 0.05%, potassium dihydrogen phosphate
0.05%, dipotassium hydrogen phosphate 0.05%, ε-
Medium containing caprolactam 0.05% (w / v) (all w / v).

The cells are centrifuged (10000 rpm) from the medium.
m, 20 minutes) and a 50 mM phosphate buffer (pH 7.5)
In washed once and suspended in the same phosphate buffer (100 mL), and adjusted cell suspension (OD ₆₃₀ = 15.8).

(2) Hydrolysis of 4-ethyl-4-hydroxyoctanenitrile 4-ethyl-4-hydroxyoctanenitrile (0.84 g) was added to the above cell suspension, and stirring was started at 30 ° C. did. Thereafter, 4-ethyl-4-hydroxyoctanenitrile (0.17 g) was successively added every two hours, for a total of 2.6.
g of 4-ethyl-4-hydroxyoctanenitrile were added afterwards (total addition 3.4 g). 96 hours after the start of stirring,
When the reaction solution was analyzed by HPLC using an ODS column, 4-ethyl-4-hydroxyoctanoic acid (3.0
g) was confirmed to have been produced (reaction yield 80%).

Example 6 (1) Culture and Preparation of Cell Suspension Rhodococcus rhodochrous J-1 strain was cultured in medium E for 3 hours.
After culturing at 0 ° C for 48 hours, the obtained cells were further cultured in medium F at 30 ° C for 96 hours. The cells were centrifuged (10000 rpm, 20 minutes) from the medium, washed once with 50 mM phosphate buffer (pH 7.5), and washed with the same phosphate buffer (10,000 rpm).
0 mL) and the cell suspension (OD ₆₃₀ = 24.0)
Was adjusted.

(2) Hydrolysis reaction of 3-hydroxy-3-methylbutyronitrile 3-hydroxy-3-methylbutyronitrile (0.5 g) was added to the above cell suspension, and the mixture was added at 30 ° C. for 48 hours. Stirred for hours. As a result of analysis by HPLC using an ODS column,
It was confirmed that 3-hydroxy-3-methylbutyric acid (0.52 g) was generated (reaction yield: 87%).

Example 7 (1) Culture and Preparation of Cell Suspension After culturing Rhodococcus erythropolis IFO12538 strain in medium E at 30 ° C. for 48 hours, the obtained cells were further cultured in medium F at 30 ° C. for 96 hours. Cultured for hours. The cells were centrifuged (10000 rpm, 20 minutes) from the medium, and 50 mM
After washing once with a phosphate buffer (pH 7.5), the cells were suspended in the same phosphate buffer (100 mL), and the cell suspension (OD ₆₃₀ =
10.0).

(2) Hydrolysis of 3-hydroxy-3-methylbutyronitrile 3-Hydroxy-3-methylbutyronitrile (0.3 g) was added to the above cell suspension, and the mixture was added at 30 ° C. for 48 hours. Stirred for hours. As a result of analysis by HPLC using an ODS column,
It was confirmed that 3-hydroxy-3-methylbutyric acid (0.35 g) was produced (reaction yield: 99%).

Example 8 (1) Culture and Preparation of Cell Suspension After culturing Rhodococcus sp. HT40-6 strain in medium E at 30 ° C. for 48 hours, the obtained cells were further added to medium F.
At 30 ° C. for 96 hours. The cells were centrifuged (10000 rpm, 20 minutes) from the medium, washed once with 50 mM phosphate buffer (pH 7.5), and washed with the same phosphate buffer (10,000 rpm).
0 mL) and the cell suspension (OD ₆₃₀ = 10.3)
Was adjusted.

(2) Hydrolysis reaction of 3-hydroxy-3-methylbutyronitrile 3-hydroxy-3-methylbutyronitrile (0.3 g) was added to the above cell suspension, and the mixture was added at 30 ° C for 24 hours. Stirred for hours. As a result of analysis by HPLC using an ODS column,
It was confirmed that 3-hydroxy-3-methylbutyric acid (0.36 g) was produced (reaction yield: 100%).

Example 9 (1) Culture and Preparation of Cell Suspension Rhodococcus sp.
After culturing at 48 ° C. for 48 hours, the obtained cells were further cultured in medium F for 3 hours.
The cells were cultured at 0 ° C. for 96 hours. Centrifuge the cells from the medium (1
0000 rpm, 20 minutes), and washed once with 50 mM phosphate buffer (pH 7.5).
mL) to prepare a cell suspension (OD ₆₃₀ = 20.0).

(2) Hydrolysis of 3-hydroxy-3-methylbutyronitrile 3-Hydroxy-3-methylbutyronitrile (0.3 g) was added to the above cell suspension, and the mixture was added at 30 ° C. for 24 hours. Stirred for hours. As a result of analysis by HPLC using an ODS column,
It was confirmed that 3-hydroxy-3-methylbutyric acid (0.36 g) was produced (reaction yield: 100%).

Example 10 (1) Culture and Preparation of Cell Suspension Nocardia erythropolis IFO12539 strain was cultured in medium E at 30 ° C. for 48 hours, and the obtained cells were further cultured in medium F at 30 ° C. The cells were cultured for 96 hours. The cells were centrifuged (10000 rpm, 20 minutes) from the medium, and 50 mM
After washing once with a phosphate buffer (pH 7.5), the cells were suspended in the same phosphate buffer (100 mL), and the cell suspension (OD ₆₃₀ =
11.8) was adjusted.

(2) Hydrolysis of 3-hydroxy-3-methylbutyronitrile 3-Hydroxy-3-methylbutyronitrile (0.3 g) was added to the above cell suspension, and the mixture was added at 30 ° C for 24 hours. Stirred for hours. As a result of analysis by HPLC using an ODS column,
It was confirmed that 3-hydroxy-3-methylbutyric acid (0.35 g) was produced (reaction yield: 99%).

Example 11 (1) Culture and Preparation of Cell Suspension Corynebacterium sp. KO-2-4 obtained by culturing according to the method described in JP-B-3-2244963.
Centrifuge the strain (10000 rpm, 20 minutes) and
Washed once with M phosphate buffer (pH 7.5), and suspended in the same phosphate buffer (100 mL), cell suspension (OD ₆₃₀
= 7.7).

(2) Hydrolysis reaction of 3-hydroxy-3-methylbutyronitrile 3-hydroxy-3-methylbutyronitrile (0.3 g) was added to the above cell suspension, and the mixture was added at 30 ° C. for 24 hours. Stirred for hours. As a result of analysis by HPLC using an ODS column,
It was confirmed that 3-hydroxy-3-methylbutyric acid (0.29 g) was produced (reaction yield: 79%).

Example 12 (1) Culture and Preparation of Cell Suspension The Mycobacterium sp. AC777 strain obtained by culturing according to the method described in JP-B-3-2244963 was centrifuged (10000 rpm, 20 minutes). , 50 mM
After washing once with a phosphate buffer (pH 7.5), the cells were suspended in the same phosphate buffer (100 mL), and the cell suspension (OD ₆₃₀ =
11.4) was adjusted.

(2) Hydrolysis of 3-hydroxy-3-methylbutyronitrile 3-Hydroxy-3-methylbutyronitrile (0.3 g) was added to the above cell suspension, and the suspension was added at 30 ° C. for 24 hours. Stirred for hours. As a result of analysis by HPLC using an ODS column,
It was confirmed that 3-hydroxy-3-methylbutyric acid (0.35 g) was produced (reaction yield: 99%).

Example 13 (1) Cultivation and Preparation of Cell Suspension Rhodococcus rhodochrous J-1 strain obtained by culturing according to the method described in Japanese Patent Publication No. 2-291283 is centrifuged (10000 rpm, 20 minutes). And washed once with 50 mM phosphate buffer (pH 7.5),
0 mL) and the cell suspension (OD ₆₃₀ = 14.4)
Was adjusted.

(2) Hydration of 3-hydroxy-3-methylbutyronitrile 3-Hydroxy-3-methylbutyronitrile (0.3 g) was added to the above cell suspension. Stirred for hours. As a result of analysis by HPLC using an ODS column,
3-hydroxy-3-methylbutanamide (0.32
g) was confirmed to be formed (reaction yield 90%).

As comparative examples of Examples 1 to 5, the following chemical hydrolysis was carried out under various conditions. Comparative Example 1 Hydrolysis reaction of 5-hydroxyhexane nitrile 35-hydroxyhexane nitrile (0.89 g)
% Hydrochloric acid (1.56 g) was added, and the mixture was stirred at 80 ° C for 4 hours. 35% hydrochloric acid (1.58 g) was added, the mixture was aged at 80 ° C for 2.5 hours, and 35% hydrochloric acid (1.58 g) was further added. Aging was performed at 80 ° C. for 1.5 hours. Under a basic condition (pH 11), unreacted raw materials were extracted and removed with methylene chloride, and the remaining aqueous layer was again made acidic (pH 2) and extracted with methylene chloride. The organic layer extracted under this acidic condition was dried over magnesium sulfate, the solvent was distilled off, and the remaining concentrated residue was analyzed by GLC, but the target 5-hydroxyhexanoic acid was not contained.

Comparative Example 2 Hydrolysis of 3-hydroxy-3-methylbutyronitrile 3-hydroxy-3-methylbutyronitrile (0.89
g), 62% sulfuric acid (7.0 g) was added thereto, and the mixture was stirred at 80 ° C. for 1 hour.
For 5 hours. Under basic conditions (pH 1
1) After extracting and removing unreacted raw materials with methylene chloride, the remaining aqueous layer was returned to acidic (pH 2) again and extracted with methylene chloride. The organic layer extracted under this acidic condition was dried over magnesium sulfate, the solvent was distilled off, and the remaining concentrated residue was analyzed by GLC.
It did not contain 3-methylbutyric acid.

Comparative Example 3 Hydrolysis of 4-ethyl-4-hydroxyoctanenitrile 4-ethyl-4-hydroxyoctanenitrile (0.8
9g) in ethanol (1.73 g) and 35% hydrochloric acid (1.
After 56 g) was added and the mixture was stirred at 20 ° C. for 1 hour, aging was performed at 80 ° C. for 4.5 hours. Water was added and the mixture was stirred at 20 ° C for 1.5 hours. After extracting the unreacted raw materials with methylene chloride under basic conditions (pH 11), the remaining aqueous layer was again returned to acidic (pH 2) and extracted with methylene chloride. The organic layer extracted under this acidic condition was dried over magnesium sulfate, the solvent was distilled off, and the remaining concentrated residue was analyzed by GLC, but it did not contain the desired 4-ethyl-4-hydroxyoctanoic acid.

Comparative Example 4 Hydrolysis reaction of 5-isobutyl-5-hydroxynonanenitrile 5-isobutyl-5-hydroxynonanenitrile (0.
To 83 g), ethanol (17 g) and potassium hydroxide (0.84 g) were added, and the mixture was stirred at 80 ° C. for 2.5 hours. After extracting and removing unreacted raw materials from the reaction solution with methylene chloride, the remaining aqueous layer was adjusted to be acidic and extracted with methylene chloride. The organic layer extracted under this acidic condition was dried over magnesium sulfate, the solvent was distilled off, and the remaining concentrated residue was analyzed by GLC, but it did not contain the desired 5-isobutyl-5-hydroxynonanoic acid.

Comparative Example 5 Hydrolysis of 3-hydroxyheptanenitrile 25% of 3-hydroxyheptanenitrile (0.83 g)
% Potassium hydroxide (1.68 g) and 30% aqueous hydrogen peroxide (1.7 g) were added, and the mixture was stirred at 40 ° C. for 1 hour under a nitrogen atmosphere. Thereafter, the mixture was further stirred at 80 ° C. for 3 hours. After extracting and removing unreacted raw materials from the reaction solution with methylene chloride, the remaining aqueous layer was acidified (pH 2) and subjected to methylene chloride extraction. The organic layer extracted under this acidic condition was dried over magnesium sulfate, and the solvent was distilled off. The remaining concentrated residue was analyzed by GLC, but it did not contain the target 3-hydroxyheptanoic acid.

Comparative Example 6 Hydrolysis of 4-hydroxybutyronitrile 4-hydroxybutyronitrile (0.83 g) was added to water (9
00mg) and manganese dioxide (15mg).
Stirred at 4 ° C. for 4 hours and left overnight. After the reaction solution was filtered through celite, the filtrate was dried to give a residue (15.9 mg). The residue was analyzed by GLC.
Hydroxybutanamide was not included.

Comparative Example 7 Hydrolysis of 4-butyl-4-hydroxyoctanenitrile 4-butyl-4-hydroxyoctanenitrile (0.1
g), ethylene glycol (1.11 g) and potassium hydroxide (0.27 g) were added, and the mixture was stirred at 100 ° C for 1 hour, and then aged at 120 ° C for 4 hours. After extracting and removing unreacted raw materials from the reaction solution with methylene chloride, the remaining aqueous layer was adjusted to be acidic and extracted with methylene chloride. The organic layer extracted under this acidic condition was dried over magnesium sulfate, the solvent was distilled off, and the remaining concentrated residue was analyzed by GLC, but it did not contain the desired 4-butyl-4-hydroxyoctanoic acid.

[0061]

According to the present invention, the substrate is hydrolyzed or hydrated under mild conditions to suppress the formation of by-products and to convert it to hydroxycarboxylic acid or its amide in high yield. The present invention can provide a process for producing the compound which is industrially superior in terms of operation and economy as compared with conventional production processes.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:15) (C12P 7/42 C12R 1: 365) (C12P 7/42 C12R 1:32) (C12P 7/42 C12R 1 : 265) (C12P 13/02 C12R 1:01) (72) Inventor Kouji Tamura 10-1 Ogurocho, Tsurumi-ku, Yokohama-shi, Kanagawa Prefecture Nitto Chemical Industry Co., Ltd. Central Research Laboratory (72) Inventor Yoshimasa Kobayashi 10-1 Daikokucho, Tsurumi-ku, Yokohama, Kanagawa Prefecture Nitto Chemical Industry Co., Ltd.

Claims (3)

[Claims] 1. A microorganism having hydrolytic ability to a nitrile group of a hydroxynitrile represented by the following general formula [I] or a treated product thereof is allowed to act on the hydroxynitrile in an aqueous medium to give the following general formula [II]: A method for producing a hydroxycarboxylic acid, comprising producing the hydroxycarboxylic acid represented by the formula: [Wherein, R ₁ and R ₂ may be the same or different and are formed by bonding a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or R ₁ and R ₂ A cyclo ring, n represents an integer of 1 to 3. ] 2. A microorganism having a hydrating ability for the nitrile group of the hydroxynitrile represented by the general formula [I] according to claim 1 or the treated product is allowed to act on the hydroxynitrile in an aqueous medium to obtain the following: A method for producing a hydroxycarboxylic acid amide, which comprises producing the hydroxycarboxylic acid amide represented by the general formula [III]. [Wherein, the definitions of R ₁ , R ₂ and n are the same as in claim 1. ] 3. The method according to claim 1, wherein the microorganism is Rhodococcus.
cbus), Brevibacterium (Brevivact)
erium), Corynebacterium (Coryneba)
cterium), Nocardi (Nocardi)
a), Mycobacterium (Mycobacterium)
mu) or Micrococcus
3. The method for producing hydroxycarboxylic acid or hydroxycarboxylic acid amide according to claim 1 or 2, which is a microorganism belonging to the genus s).