JP4565447B2 - Method for producing carboxylic acid - Google Patents

Description

  The present invention relates to a nitrilase-producing microorganism having resistance to organic solvents and a method for producing carboxylic acid from nitrile using the nitrilase-active substance of the microorganism.

As a method for producing carboxylic acid, there is a method in which nitrile is hydrolyzed using a nitrilase enzyme. Nitrilase is a useful enzyme capable of converting nitrile to carboxylic acid, and is produced from various microorganisms. For example, microorganisms belonging to the genus Arthrobacter have been reported as microorganisms producing nitrilase active substances (Patent Documents 1 and 2).
In general, an enzyme reaction exhibits high activity in an aqueous solution, but in the presence of an organic solvent, the activity may be significantly reduced or may not show any activity. Actually, when a nitrile having low water solubility is used as a raw material, it is desirable to dissolve in advance using an organic solvent. However, the aforementioned nitrilase derived from the genus Arthrobacter does not have resistance to an organic solvent. Therefore, known nitrilase enzymes derived from microorganisms belonging to the genus Arthrobacter have not been suitable as means for producing carboxylic acids using nitriles with low water solubility as raw materials.
On the other hand, Non-Patent Document 1 reports that a nitrilase derived from a microorganism belonging to the genus Pseudomonas exhibits resistance to alkanes having 6 to 16 carbon atoms and alkanols having 6 to 11 carbon atoms. It is unclear whether it exhibits resistance to water-soluble organic solvents.
JP-A-11-341979 JP 2003-274933 A Applied and Environmental Microbiology, 69 (8), 4359-4366 (2003).

  An object of the present invention is to provide a nitrilase-producing microorganism that is highly resistant to an organic solvent, and to provide a method for efficiently producing a carboxylic acid from a nitrile using the nitrilase activity of the microorganism.

  As a result of intensive studies, the present inventors have found that a novel microorganism belonging to the genus Arthrobacter exhibits not only high nitrilase activity but also desirable organic solvent resistance. Further, the present inventors have found that the microorganism maintains a high nitrilase activity in the presence of an organic solvent and is useful as a means for producing a carboxylic acid from a nitrile having low water solubility, and has reached the present invention. Specifically, the present invention is as follows.

1. Earth Lobacter SP F-73.
2. A method for producing a carboxylic acid, comprising contacting an Arthrobacter sp. F-73 strain or an enzyme active substance having nitrilase activity derived from the strain with a nitrile in an aqueous reaction solution to recover the carboxylic acid produced from the nitrile.
3. An aqueous system in which an organic solvent is added to a microorganism belonging to the genus Arthrobacter having a nitrilase activity of 10% or more when treated at 20 ° C. for 60 minutes in the presence of 40% acetone, or an enzyme active substance having a nitrilase activity derived from the microorganism. A method for producing a carboxylic acid, wherein the carboxylic acid produced from the nitrile is recovered by contacting with a nitrile in a reaction solution.
4). 4. The production method according to 3 above, wherein the concentration of the organic solvent added to the aqueous reaction solution is 60% (V / V) or less.
5). 5. The production according to 3 or 4 above, wherein the organic solvent is at least one selected from alcohols, ketones, amides, dimethyl sulfoxide, esters, hydrocarbons or ethers optionally substituted with halogen. Method.
6). The alcohol is methanol, ethanol, propanol, 1-octanol, 1,3-propanediol, or ethylene glycol, or a combination thereof;
Ketones are acetone,
Amides are dimethylformamide,
The ester is ethyl acetate,
The hydrocarbon optionally substituted with halogen is toluene, cyclohexane, octane or chloroform,
6. The production method according to 5 above, wherein the ether is 1,4-dioxane or tetrahydrofuran.
7). The manufacturing method in any one of said 2-6 whose nitrile is alpha-hydroxy nitrile.
8). The method for producing a carboxylic acid according to any one of 2 to 6 above, wherein the nitrile is mandelonitrile in which a hydrogen atom on the benzene ring may be substituted with a halogen atom.
9. 8. The production method according to 7 above, wherein α-hydroxynitrile is produced from an aldehyde and a cyanate added to the aqueous reaction solution.
10. The production method according to 9 above, wherein the organic solvent added to the aqueous reaction solution is an organic solvent capable of forming two layers with the aqueous solvent.
11. The production method according to 9 above, wherein the aldehyde is benzaldehyde in which a hydrogen atom on a benzene ring may be substituted with a halogen atom.
12. The production method according to 10 above, wherein the organic solvent is ethyl acetate.
13. The production method according to any one of 2 to 12 above, wherein the microorganism belonging to the genus Arthrobacter is the Arthrobacter sp. F-73 strain.

  According to the present invention, not only can a carboxylic acid be efficiently produced using a water-soluble nitrile as a raw material, but also a nitrile that is low in water solubility and not suitable for conventional enzyme reaction can be efficiently hydrolyzed to produce a corresponding carboxylic acid. It becomes possible to do.

  The present invention provides a microorganism belonging to the genus Arthrobacter that can produce nitrilase activity exhibiting resistance to organic solvents, specifically, the Arthrobacter sp. F-73 strain. As described above, the nitrilase is an enzyme that hydrolyzes a nitrile to convert it into a carboxylic acid. The nitrilase of this strain exhibits resistance to organic solvents including water-soluble organic solvents not found in conventional nitrilases. Therefore, it can be efficiently converted into the corresponding carboxylic acid using a nitrile that is hardly soluble in water as a substrate. Therefore, by utilizing the nitrilase activity of this strain, it is possible to industrially produce the corresponding carboxylic acid from a wide range of substrate compounds such as a water-soluble nitrile to a water-insoluble nitrile.

  Earth Lobacter SP F-73 strain is a new microbial strain isolated from the soil collected from the campus of Gifu University, and was deposited as “FERM P-20349” at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. Yes. This strain can be obtained from the trustee based on this accession number.

  The medium for culturing the microorganism used in the present invention is not particularly limited as long as the microorganism can grow. For example, any carbon source that can be used by the microorganism can be used as the carbon source. Specifically, sugars such as glucose, fructose, sucrose and dextrin, alcohols such as sorbitol and glycerol, organic acids such as fumaric acid, citric acid, acetic acid and propionic acid and salts thereof, hydrocarbons such as paraffin, Toluene, cresol, benzoic acid, or the like or a mixture thereof can be used.

Examples of nitrogen sources include ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate, and ammonium phosphate, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, meat extract, yeast extract, corn steep liquor, and casein hydrolysis. Products, inorganic organic nitrogen-containing compounds such as urea, or mixtures thereof. In addition, nutrient sources used in normal culture, such as inorganic salts, trace metal salts, vitamins, and the like can be appropriately mixed and used. If necessary, a factor that promotes the growth of microorganisms, a factor that enhances the ability to produce the target compound of the present invention, or a substance such as CaCO ₃ that is effective for maintaining the pH of the medium can be added.

  As the culture method, the medium pH is 3 to 11, preferably 4 to 8, the culture temperature is 15 to 60 ° C., preferably 20 to 45 ° C., and anaerobic or aerobic conditions suitable for the growth of the microorganism. The culture is performed for about 5 to 240 hours, preferably about 12 to 120 hours.

  The present invention provides a method for producing a carboxylic acid from a nitrile using the above-mentioned Arthrobacter sp. F-73 strain or a microorganism belonging to the genus Arthrobacter having an organic solvent resistance nitrilase activity equivalent to the strain. .

  The nitrilase possessed by the above-mentioned Arthrobacter sp. F-73 strain has a nitrilase activity of 10 after treating acetone at 30 ° C. in the presence of 0 to 60 v / v% in a buffer solution containing a substrate as shown in the Examples described later. % Or more remained. Therefore, the organic solvent resistant nitrilase activity equivalent to that of Arthrobacter sp. F-73 is, for example, a nitrilase activity of 10% or more when treated at 20 ° C. for 60 minutes in the presence of 40% acetone. It can be left. If nitrilase activity remains at 10% or more when treated at 20 ° C. for 60 minutes in the presence of 40% acetone, more preferably in the presence of 60% acetone, it is suitably used for production at an industrial level. Can do.

  Isolation of a strain of the genus Arthrobacter having nitrilase activity can be performed, for example, based on a method as shown in the Examples. First, the presence of a microorganism having a target nitrilase activity can be identified by culturing a test sample containing an Arthrobacter microorganism in a nitrile-containing medium and measuring the carboxylic acid accumulated in the culture. The test sample can be a material collected from soil, rivers, or lakes. For a method for isolating and identifying microorganisms belonging to the genus Arthrobacter, for example, “Bergey's Manual of Determinative Bacteriology, 9th Edition” (Edited by John G. Holt, Williams & Wilkins, Baltimore) can be referred to.

  Next, the evaluation of the nitrilase activity of the isolated microorganism belonging to the genus Arthrobacter can be performed as follows. The enzyme preparation is added to 50 mM potassium phosphate buffer (pH 7.0) containing 10 mM 2-thiopheneacetonitrile as a substrate. Instead of the enzyme preparation, a microbial cell or a crudely purified enzyme may be used. After adding the enzyme, react at 30 ° C. for 10 minutes. The reaction is stopped by vigorously shaking the reaction with 3N hydrochloric acid and analyzing the product. The reaction product can be analyzed by HPLC. Based on this measurement method, 1 U of nitrilase is the amount of enzyme that produces 1 μmol of 2-thiopheneacetic acid per minute at 30 ° C. in the standard reaction solution composition.

  Whether or not the nitrilase activity of the isolated microorganism belonging to the genus Arthrobacter exhibits resistance to the organic solvent as described above is determined by adding an organic solvent at a concentration before or after the above reaction to the reaction solution for measuring the nitrilase activity. It can be determined by measuring the residual rate of nitrilase activity.

  In the production method of the present invention, the above-mentioned microorganism may be used as it is added to the reaction solution as it is, or may be used after being treated with an enzyme active substance having nitrilase activity derived from the microorganism. . Enzyme-active substances broadly mean those that can retain the nitrilase activity derived from the microorganism, and purified enzymes naturally change the permeability of cell membranes by treatment with surfactants, organic solvents such as acetone or toluene. Or a cell-free extract obtained by crushing the cells by glass beads or enzymatic treatment, a cell-free extract, or a partially purified product thereof. The nitrilase itself may be a crude product or a purified product obtained by an ordinary enzyme purification method. In addition, microbial cells and purified nitrilase bound to an insoluble carrier or a water-soluble carrier molecule, an immobilized enzyme obtained by comprehensively immobilizing enzyme molecules, and the like are also included in the enzyme active substance of the present invention.

In the production method of the present invention, the nitrile as a raw material is not particularly limited, and saturated mononitriles, saturated dinitriles, α-amino nitriles, nitriles having a carboxyl group, unsaturated nitriles, aromatic nitriles and α -Widely applicable to hydroxy nitriles and the like. These specific compounds are illustrated below.
Saturated mononitriles: acetonitrile, propionitrile, butyronitrile, isobutyronitrile, valeronitrile, isovaleronitrile, capronitrile, etc. Saturated dinitriles: malonitrile, succinonitrile, glutaronitrile, adiponitrile, etc. α-amino nitriles: α-amino Propionitrile, α-aminomethylthiobutyronitrile, α-aminobutyronitrile, aminoacetonitrile, etc. Nitriles having a carboxyl group: Cyanoacetic acid, etc. β-Aminonitriles: Amino-3-propionitrile, etc. Unsaturated nitriles : Acrylonitrile, methacrylonitrile, allyl cyanide, crotonnitrile, etc. Aromatic nitriles: benzonitrile, o-, m- or p-chlorobenzonitrile, o-, m- or p-fluoroben Zonitrile, o-, m- or p-nitrobenzonitrile, p-aminobenzonitrile, 4-cyanophenol, o-, m- or p-tolunitrile, 2,4-dichlorobenzonitrile, 2,6-dichlorobenzonitrile 2,6-difluorobenzonitrile, anisonitrile, α-naphthonitrile, β-naphthonitrile, phthalonitrile, isophthalonitrile, terephthalonitrile, benzyl cyanide, phenylacetonitrile, etc. α-hydroxynitriles: α-hydroxy- n-propionitrile, α-hydroxy-n-butyronitrile, α-hydroxy-isobutyronitrile, α-hydroxy-n-hexonitrile, α-hydroxy-n-heptyronitrile, α-hydroxy-n-octonitrile , Α, γ-dihydroxy-β, β-dimethyl Rubutyronitrile, acrolein cyanohydrin, methacrylaldehyde cyanohydrin, 3-chlorolactonitrile, 4-methylthio-α-hydroxybutyronitrile, α-hydroxy-α-phenylpropionyl, mandelonitrile, 2-chloromandelonitrile , 3-chloromandelonitrile, 2-thiophenecarboxaldehyde cyanohydrin, 2-pyridinecarboxaldehyde cyanohydrin, 2-pyrrolecarboxaldehyde cyanohydrin, 2-furaldehyde cyanohydrin or 2-naphthylaldehyde cyanohydrin Such

  Among the above nitriles, α-hydroxynitriles can be mentioned as particularly preferred nitriles, and most preferred examples include mandelonitrile, 2-chloromandelonitrile, 3-chloromandelonitrile and the like on the benzene ring. Examples thereof include mandelonitrile in which the hydrogen atom may be substituted with a halogen atom.

  When the above α-hydroxynitrile is used as a raw material, it is not always necessary to use an isolated and purified material as a raw material, and the α aldehyde produced in the reaction solution by adding the corresponding reaction solution of aldehyde and cyanate as it is. -Hydroxynitrile may be used as a raw material. As the cyanate, alkali metal or alkaline earth metal cyanate is used, and preferred examples include sodium cyanate and potassium blue oxide. As the aldehyde, benzaldehyde in which the hydrogen atom on the benzene ring may be substituted with a halogen atom, such as benzaldehyde, 2-chlorobenzaldehyde, and 3-chlorobenzaldehyde, can be mentioned as a preferable example. Aldehyde, 3-pyridinecarboxaldehyde, 4-pyridinecarboxaldehyde, 2-tolualdehyde, 3-tolualdehyde, 4-tolualdehyde, 2-furaldehyde, 3-furaldehyde, 2-thiophenaldehyde, 3-thiophenaldehyde, 3 It can be similarly applied to indole carboxaldehyde, 2-pyrrole carboxaldehyde, 3-pyrrole carboxaldehyde, 2-pyrazine carboxaldehyde and the like.

  The reaction liquid for carrying out the method for producing a carboxylic acid of the present invention may be an aqueous reaction such as water or a buffer, or an aqueous reaction liquid to which an organic solvent is added. As described above, the Arthrobacter sp. F-73 strain of the present invention or an equivalent Arthrobacter microorganism has nitrilase activity, and the nitrilase activity is resistant to organic solvents. Therefore, the production can be performed using an aqueous reaction solution such as water or a buffer solution, or can be performed using an aqueous reaction solution to which an organic solvent is added. Therefore, when the nitrile serving as the substrate is difficult to dissolve in the aqueous reaction solution, the reaction can be performed in a state where the organic solvent is added and the solubility of the substrate is increased using the aqueous reaction solution. This makes it possible to industrially produce carboxylic acids starting from nitriles which are difficult or inefficient to produce and which are difficult or inefficient to produce water-insoluble nitriles.

Examples of the organic solvent that can be added to the aqueous reaction solution include water-soluble alcohols, water-soluble ketones, amides, and dimethyl sulfoxide specifically exemplified below.
Water-soluble alcohols: methanol, ethanol, propanol, 1,3-propanediol, ethylene glycol and other water-soluble ketones: acetone and other amides: dimethylformamide, etc.

Alternatively, a two-layer system of an organic solvent and an aqueous reaction solution may be used by adding an organic solvent that is difficult to dissolve in water. Examples of the organic solvent that hardly dissolves in water include esters exemplified below, hydrocarbons optionally substituted with halogen, higher alcohols, ethers, and the like.
Esters: Hydrocarbons optionally substituted with halogen such as ethyl acetate or butyl acetate: Higher alcohols such as toluene, n-hexane, cyclohexane, octane or chloroform: Ethers such as 1-octanol: 1,4-dioxane, Tetrahydrofuran etc.

  The organic solvent may be appropriately added within a range that does not impair the hydrolysis reaction of the present invention. Usually, an amount of 60% (V / V) or less can be added to the aqueous reaction solution. For example, in the case of acetone, methanol, dimethyl sulfoxide, hexane or the like, 10 to 50% (V / V) or less, preferably 20 to 40% (V / V) can be added. In the case of dimethylformamide, ethyl acetate, tetrahydrofuran, 1,3-propanediol, 5 to 30% (V / V) or less, preferably 10 to 20% or less can be added. In addition, these organic solvents may be added in a mixture of two or more as long as the enzyme activity is not impaired.

  When the corresponding aldehyde and cyanate are used as raw materials instead of the purified α-hydroxynitrile described above, it is preferable to use a two-layer system in which an aqueous reaction liquid and an organic solvent that forms two layers are added. In the aqueous solvent, which is the site of the enzyme reaction, the concentration of compounds that may be toxic to the enzyme, such as aldehydes and nitriles, can be lowered, and the reaction can proceed more effectively. In the case of using aldehyde and cyanate as raw materials, preferred organic solvents include ethyl acetate and toluene.

  The concentration of the substrate compound in the reaction solution is not particularly limited. For example, in the case of α-hydroxynitrile, it can be usually 0.1 to 10% by weight, preferably 0.2 to 5.0% by weight. The substrate can be added all at once at the start of the reaction, but it is desirable to add the substrate continuously or discontinuously so that the substrate concentration in the reaction solution does not become too high.

  Furthermore, in the hydrolysis reaction of the present invention, a surfactant may be added to the reaction solution. As the surfactant, 0.1 to 5.0% by weight of Triton X-100 or Tween 60 is used.

  By setting the nitrilase active substance of the present invention to an enzyme activity amount of, for example, 1 mU / mL to 100 U / mL, preferably 100 mU / mL or higher, the enzyme reaction can be efficiently advanced. When microbial cells are used as the enzyme active substance, the amount of microorganisms used relative to the substrate is preferably 0.01 to 5.0% by weight as dry cells. Enzymes and enzyme active substances such as bacterial cells can be brought into contact with the substrate by dissolving or dispersing them in the reaction solution. Alternatively, an enzyme active substance immobilized by a technique such as chemical bonding or inclusion can be used. Furthermore, although the substrate can permeate, it can be reacted in a state where the substrate solution and the enzyme active substance are separated by a porous membrane that restricts permeation of enzyme molecules and bacterial cells.

  The reaction is usually carried out at a freezing point to 50 ° C, preferably 10 to 30 ° C for 0.1 to 100 hours. The pH of the reaction solution is not particularly limited as long as the enzyme activity can be maintained, but it may be appropriately set in the range of usually 5 to 10, preferably 6 to 9.

  When the nitrile and the microorganism of the present invention or the enzyme active substance thereof are contacted in the reaction solution, the nitrile is hydrolyzed to produce the corresponding carboxylic acid and accumulate in the reaction solution. The carboxylic acid accumulated here can be recovered from the reaction solution by a known method and purified. Specifically, for example, it can be recovered and purified by combining ordinary methods such as ultrafiltration, concentration, column chromatography, extraction, activated carbon treatment, and distillation.

Next, the present invention will be described in more detail with reference to examples. The standard measurement method used in the following examples will be described.
Method for measuring enzyme activity:
The standard method for measuring nitrilase activity in the examples is as follows. The basic reaction solution composition of the enzyme reaction was as follows: Total volume 2.00 ml: 2-Thiopheneacetonitrile 10 mM, KPB (pH 7.0) 50 mM, enzyme solution 0.10 ml, 0.85% (w / v) NaClaq 0.90 ml.
The enzymatic reaction was started by adding 2-thiopheneacetonitrile as a substrate compound, and was performed at 30 ° C. for 10 minutes. 0.1 ml of 3N hydrochloric acid was added and shaken vigorously to stop the reaction. The 2-thiopheneacetic acid produced | generated in the reaction liquid was quantified by HPLC shown below, and the nitrilase activity was computed.
Conditions for HPLC analysis of the reaction solution:
Column: Wakosil-II 5C18 RS (4.6 × 150 nm),
Mobile phase: 50 mM K ₂ HPO ₄ (pH 2.8) / CH ₃ OH = 7/3 (v / v)
-Flow rate: 1.0 ml / min. , Detection: UV 235 nm, column temperature: 40 ° C.
In the calculation of the nitrilase activity, nitrilase 1U was defined as the amount of enzyme that produced 1 μmol of 2-thiopheneacetic acid per minute at 30 ° C. in the standard reaction solution composition.

[Example 1] Isolation of a nitrilase-producing microorganism having resistance to organic solvents Soil collected from a premises of Gifu University as a strain having a particularly high decomposing ability among nitrile-degrading bacteria after accumulating in a medium containing various nitriles The more isolated F-73 strain was selected.

  When DNA was extracted from this strain, the base of 16S rDNA corresponding to 16S rRNA was amplified by PCR, and the homology of the first 1469 bases was compared in the database of known type strains of various types of Arthrobacter and MicroSeq. The species with the highest value is Arthrobacter prototype protein DSM 20618, and the homology of 16S rDNA with it was 95.71%, so this bacterium was finally identified as Arthrobacter sp. Was identified.

In the following examples, the strain was cultured as follows unless otherwise specified. 5 ml of a preculture medium having the following composition was dispensed into a test tube (25 × 200 mm), sealed with a silicon stopper, and sterilized by an autoclave. After standing to cool, the strain was inoculated with one platinum ear and cultured with shaking at 28 ° C. for 1 day. Next, the culture after pre-culture was transferred to 20 ml of the main culture medium having the following composition which was dispensed into a 500 ml Sakaguchi flask and sterilized by autoclaving. The culture was shaken at 28 ° C. for 5 days.
Pre-culture medium (pH 7.0):
Polypeptone 5.0 g, meat extract 5.0 g, NaCl 2.0 g, yeast extract 0.5 g, distilled water 1.0 L.
Main culture medium (pH 7.0):
2-thiophene acetonitrile 1.5 ml, lactose 1.0 g, yeast extract 1.0 g, MgSO ₄ 7H ₂ O 0.2 g, K ₂ HPO ₄ 1.0 g, distilled water 1.0 L.

[Example 2] Measurement of nitrilase activity Arthrobacter sp. Pre-culture medium A (peptone 0.5 g, meat extract 0.5 g, NaCl 0.2 g, yeast extract 0.05 g, distilled water 100 ml, pH 7.0) 4 ml / test tube for 1 day at 28 ° C. After culturing, the main culture medium B (yeast extract 1.0 g, glutamic acid Na 4.0 g, KH ₂ PO ₄ 1.0 g, MgSO ₄ .7H ₂ O 0.5 g, isovaleronitrile 3 ml, distilled water 1 L, pH 7. 0) The cells were cultured at 28 ° C. for 3 days in a 40 ml / 500 ml Sakaguchi flask.

The culture was centrifuged to collect the cells, and reacted at 30 ° C. in 2 ml / test tube of reaction solution C (substrate 20 mM, potassium phosphate buffer pH 7.0 50 mM). The reaction was stopped by adding 3N hydrochloric acid, and the supernatant was analyzed by HPLC. The total activity was expressed as the production rate per minute (μmol / ml / min) of the product produced by the cells per 1 ml of the culture solution.
2-thiopheneacetonitrile, 1-naphthylacetonitrile, propionitrile, n-butyronitrile, n-valeronitrile, acrylonitrile, crotononitrile, 2-cyanofuran, cyanopyrazine, 3-thiopheneacetonitrile, 3-indoleacetonitrile, benzylcyanide, 3-tolylacetonitrile, 4-tolylacetonitrile, 2-pyridineacetonitrile, 3-pyridineacetonitrile, 2-hydroxy-4-thiomethylbutyronitrile, mandelonitrile and 2-chloromandelonitrile are used as substrates, respectively. The formation of carboxylic acid was measured. The results are shown in Table 1.

[Example 3] Evaluation of resistance to organic solvent Example 2 except that acetone was added to the reaction solution of Example 2 so that the final concentration was 60% (v / v) at maximum so that the reaction temperature was 20 ° C. The reaction was conducted in the same manner. As shown in Table 3, it was found that 10% or more of nitrilase activity remained even at an acetone concentration of 60%. For comparison, the reaction was carried out using Alcaligenes faecalis JM3 cultured in the same manner. As shown in Table 2, the activity was not observed when the acetone concentration was 30% or more.

[Example 4] Evaluation of organic solvent resistance An organic solvent (acetone, methanol, n-hexane or DMSO) was added to the reaction solution of Example 1 so that the final concentration was 20% and 40% (v / v). The reaction was carried out under the same conditions as in Example 3. As shown in Tables 3 to 6, the activity was shown even at an organic solvent concentration of 40%.

[Example 5] 2-Chloromandelic acid production with addition of organic solvent 48 mg of the cultured bacterial cells of Example 2 were added to the reaction solution (2-chloromandelonitrile 40 mM, potassium phosphate buffer (pH 8.0) 100 mM, organic solvent) (20% v / v) The reaction was carried out at 2.0 ° C./test tube for 22 hours at 30 ° C. The results are shown in Table 7. The optical purity increased in a system in which acetone, DMF, 1,3-propanediol, and THF were added.

[Example 6] Production of optically active 2-chloromandelic acid in a system to which DMF was added 580 mg (dried cells) of cultured cells of Example 2 were added to the reaction solution (2-chloromandelonitrile was initially added at 40 mM and successively 20 mM added). The total reaction was 240 mM, bisulfite 20 mM, potassium phosphate buffer pH 8.0 100 mM, organic solvent 20% v / v) 10 ml / reaction in a test tube at 160 rpm and 30 ° C. for 24 hours. (R) -2-chloromandelic acid having a yield of 61.7% and an optical purity of 93.8% ee was obtained.

[Example 7] Production of optically active mandelic acid in a reaction system to which DMF was added In the same manner as in Example 6, 580 mg (dried cells) of cultured cells were added to the reaction solution (chloromandelonitrile was initially added at 40 mM and successively added at 20 mM). 280 mM, potassium phosphate buffer pH 8.0 100 mM, organic solvent 20% v / v) The reaction was carried out for 24 hours under conditions of 10 ml / test tube and 160 rpm at 30 ° C. The yield was 81.1%, and (R) -mandelic acid having an optical purity of 93.4% ee was obtained.

[Example 8] Optically active chlormandelic acid production in a system to which ethyl acetate was added In the same manner as in Example 6, 580 mg (dried cells) of cultured cells were added to the reaction solution (chloromandelonitrile was initially 100 mM, 2.5 hours, After 4.5 hours and 6.5 hours, 100 mM was added in succession for a total of 400 mM, Tris-HCl buffer pH 9.0 50 mM, ethyl acetate 10% v / v) 10 ml / reacted in a test tube at 160 rpm and 30 ° C. for 9 hours. . The yield was 92%, and (R) -2-chloromandelic acid having an optical purity of 98.5% ee was obtained. The reaction-terminated liquid containing 124 mg of (R) -2-chloromandelic acid was subjected to centrifugation at 15000 rpm for 10 minutes under reduced pressure to remove the cells and the remaining substrate. NaHCO ₃ , ethyl acetate and hydrochloric acid were added, and the mixture was extracted into ethyl acetate, followed by desolvation under reduced pressure. As a result, 110 mg of (R) -2-chloromandelic acid having an optical purity of 99.3% ee was obtained in 89% yield. Crystal was obtained. NMR analysis agreed with the standard.

[Example 9] Optically active chloromandelic acid cyanide from 2-chlorobenzaldehyde and KCN in a system to which ethyl acetate was added
2-Chlorobenzaldehyde 200 mM, KCN 200 mM, potassium phosphate buffer (pH 7.0) 100 mM, 20% (v / v) The cultured cells of Example 1 (dry weight 18.0 mg) in a reaction solution containing ethyl acetate As a result of shaking reaction at 20 ° C. for 24 hours, 135 mM (25.2 g / L) of R-2-chloromandelic acid was produced, and the ee thereof was 100%.

[Comparative Example 1] Optically active chlormandelic acid production in an aqueous system to which no organic solvent was added In the same manner as in Example 8, 580 mg (dried cells) of cultured cells were added to the reaction solution (chloromandelonitrile was initially 100 mM for 2.5 hours). The reaction was continued for 21.5 hours under the conditions of 10 ml / test tube at 160 rpm and 30 ° C. with a total of 200 mM added in succession at 100 mM and Tris-HCl buffer pH 9.0 50 mM). (R) -2-chloromandelic acid having a yield of 78% and an optical purity of 85.9% ee was obtained. Compared to the system in which ethyl acetate was added in Example 7, the accumulated amount, yield, and optical purity were low.

Comparative Example 2 Optically active chloromandelic acid hydrocyanic acid from 2-chlorobenzaldehyde and KCN in a system without addition of ethyl acetate The reaction was carried out in the same manner as in Example 9 except that ethyl acetate was not added. As a result, almost no 2-chlorobenzaldehyde was obtained. The reaction hardly proceeded without dissolving in the reaction system. The amount produced was 2 mM, and the optical purity was 73% ee.

  By utilizing the organic solvent-resistant nitrilase microorganism of the present invention, a reaction solution that can be used for the reaction is an aqueous reaction solution, an aqueous reaction solution to which a water-soluble organic solvent is added, or an organic solvent that is not dissolved in the aqueous reaction solution. As a result, the nitrile that can be used as a substrate can be used in a wide range from a water-soluble nitrile to a nitrile that is hardly soluble in water. Therefore, by using the microorganism of the present invention, it becomes possible to industrially produce more carboxylic acid including carboxylic acid, which has conventionally been difficult to produce using nitrilase.

Claims (8)

Arthrobacter sp. F-73 strain represented by accession number FERM P-20349 . An Arthrobacter sp. F-73 (Arthrobacter sp. F-73) strain represented by accession number FERM P-20349 or an enzyme active substance having nitrilase activity derived from the strain is contacted with nitrile in an aqueous reaction solution A method for producing a carboxylic acid, which recovers a carboxylic acid produced from a nitrile, wherein the nitrile is 2-thiopheneacetonitrile, n-valeronitrile, acrylonitrile, 2-cyanofuran, 3-thiopheneacetonitrile, 3-indoleacetonitrile, benzyl A process selected from the group consisting of cyanide, 3-tolylacetonitrile, 4-tolylacetonitrile, 2-pyridineacetonitrile, 3-pyridineacetonitrile, mandelonitrile, 2-chloromandelonitrile . Accession number FERM ground represented by P-20349 Arthrobacter sp F-73 (Arthrobacter sp. F -73) strain or the enzymatic active substance having nitrilase activity derived from the strain, the aqueous reaction solution an organic solvent is added A carboxylic acid produced from the nitrile by contacting with the nitrile in the process, wherein the nitrile is 2-thiopheneacetonitrile, acrylonitrile, 2-cyanofuran, 3-thiopheneacetonitrile, 3-indoleacetonitrile Benzylcyanide, 3-tolylacetonitrile, 4-tolylacetonitrile, 2-pyridineacetonitrile, 3-pyridineacetonitrile, mandelonitrile, 2-chloromandelonitrile, the organic solvent is an alcohol or ketone Amides, dimethyl sulfoxide, ethanol Ethers, comprising at least one selected from may also hydrocarbons substituted with halogen or ether, and,
The alcohol is methanol, ethanol, propanol, 1-octanol, 1,3-propanediol, or ethylene glycol, or a combination thereof;
Ketones are acetone,
Amides are dimethylformamide,
The ester is ethyl acetate,
The hydrocarbon optionally substituted with halogen is toluene, cyclohexane, octane or chloroform,
A process wherein the ethers are 1,4-dioxane or tetrahydrofuran . The manufacturing method of Claim 3 whose addition density | concentration of the organic solvent to an aqueous reaction liquid is 60% (V / V) or less. The method for producing a carboxylic acid according to any one of claims 2 to 4 , wherein the nitrile is mandelonitrile or 2-chloromandelonitrile . 6. The production method according to claim 5 , wherein mandelonitrile or 2-chloromandelonitrile is produced from benzaldehyde or 2-chlorobenzaldehyde and a cyanate respectively added to the aqueous reaction solution. The manufacturing method of Claim 6 whose organic solvent added to the aqueous reaction liquid is an organic solvent which can form a two-layer with an aqueous solvent. The production method according to claim 7 , wherein the organic solvent is ethyl acetate.