JP4884863B2 - Nitrilase and method for producing carboxylic acid using nitrilase - Google Patents

Description

  The present invention relates to a nitrilase having specific physicochemical properties and a method for producing a carboxylic acid from a nitrile compound using the nitrilase.

  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 that can convert nitrile to carboxylic acid.

  Conventional nitrilases include, for example, a nitrilase derived from Rhodococcus rhodochrous J1 (Non-patent Document 1), an enzyme derived from Rhodococcus rhodochrous K22 (Non-patent Document 2), an enzyme derived from Alcaligenes faecalis JM3 (Non-patent document 3) Enzyme (patent document 1), Pseudomonas DSM11387 (non-patent document 4), Synechocystis sp. PCC6803 (nonpatent literature 5) etc. are reported. In addition, an enzyme derived from Pseudomonas fluorescens (Non-patent Document 6), Synechosystis sp. A derived enzyme (Non-patent Document 7) has also been reported.

  Regarding the Rhodococcus and Alcaligenes enzymes (Non-Patent Documents 1, 2, 3, and Patent Document 1), the resistance to organic solvents is not known or is low, so the nitrile is added using a reaction solution to which an organic solvent is added. There was room for improvement in commercial use such as conversion to produce carboxylic acid. Known nitrilases are relatively sensitive to organic solvents. In addition, Pseudomonas enzyme (Non-patent Document 4) is reported to be resistant to C6-C16 alkanes or to C6-C11 alkanols, but resistant to highly hydrophilic organic solvents. There is no report about. Synechocystis enzyme (Non-Patent Document 5) uses purified enzymes to compare reactions in various organic solvents, but the decrease in activity when exposed to organic solvents is unclear, and in terms of resistance to organic solvents It is unknown. In addition, Non-Patent Document 3, Non-Patent Document 5, Non-Patent Document 6, and Non-Patent Document 7 are known as nitrilases having high activity with respect to acetonitrile having an allyl group. Non-Patent Documents 5 and 7 differ from the enzyme of the present invention in molecular weight and pH stability.

  Furthermore, various nitrilase enzymes were reacted in a reaction solution containing methanol, isopropanol, toluene or hexane (10%, 25% for methanol, 10% for isopropanol, 40% for toluene, 70% for hexane). However, comparison of reactivity is not disclosed (Patent Document 2).

For example, microorganisms belonging to the genus Arthrobacter have been reported as microorganisms that produce nitrilase active substances (Patent Documents 3 and 4).
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.

As described above, conventionally, the resistance of nitrilase to an organic solvent is unknown. However, since it is necessary to add an organic solvent for hydrolysis of a nitrile compound that does not dissolve in water, it has been desired to provide a nitrilase that is resistant to organic solvents.
JP-A-4-341185 US Published Patent No. 20040002147 JP-A-11-341979 JP 2003-274933 A Eur. J. et al. Biochem. , 182, 349-356 (1989) J. et al. Bacteriol. 172, 4807-4815 (1990) Eur. J. et al. Biochem, 194, 765-772 (1990) Biotechnology Letters, 20 (4), 329-331 (1998) Applied and Environmental Microbiology, 69 (8), 4359-4366 (2003). J. et al. Mol. Catal. B. 5,467-474 (1998) Arch. Microbiol. 184, 407-418 (2006)

  An object of the present invention is to provide a nitrilase having nitrile hydrolysis activity and high organic solvent resistance and a method for producing a carboxylic acid using the nitrilase.

  As a result of intensive studies, the present inventors have found that a novel enzyme having a specific physicochemical property, preferably a purified enzyme, derived from the Arthrobacter sp. F-73 strain, has extremely high organic solvent resistance and nitrile hydrolysis activity. It was found to show. Further, the present inventors have found that the enzyme maintains 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, leading to the present invention. Specifically, the present invention is as follows.

[1] Nitrilase derived from Arthrobacter sp. F-73 and having the following physicochemical properties;
[1] Action:
Acts on the nitrile group of the nitrile compound, oxidizing the nitrile group to a carboxyl group,
[2] Substrate specificity:
Acts on 2-thiophene acetonitrile but does not act on acetonitrile or benzonitrile,
[3] Optimum pH:
The oxidation activity of nitrilase is optimal at pH 6.0 to 8.0 (reaction temperature 30 ° C.),
[4] Optimum temperature:
The nitrile group oxidation action shows maximum activity at 40-45 ° C.,
[5] pH stability:
pH 5.0-11.0 is the stable region, and [6] Molecular weight:
The molecular weight by gel filtration is about 530 kDa,
It is separated into subunits having a molecular weight of about 44 kDa by SDS-polyacrylamide gel electrophoresis.
[7] Organic solvent resistance:
Treatment with an aqueous solution containing 20% (V / V) of any organic solvent selected from the group consisting of methanol, ethanol, acetone, DMSO and 2-propanol at 20 ° C. for 60 minutes gives a residual activity of 50% or more. Show.
[2] The nitrilase according to [1], wherein the nitrilase is a purified enzyme.
[3] The nitrilase according to [2], wherein the purified enzyme is obtained through a purification process including the following steps;
(1) A step of preparing a cell-free extract derived from Arthrobacter sp. F-73 strain under the condition that dithiothreitol is present as a reducing agent and the temperature of the solution containing the enzyme is 5 ° C. or lower;
(2) a step of subjecting the cell-free extract to ammonium sulfate fractionation under the condition that dithiothreitol is present as a reducing agent and the pH of the solution containing the enzyme is 7.0 ± 0.3;
(3) performing anion exchange chromatography under the condition that dithiothreitol is present as a reducing agent and the enzyme concentration is not less than 100 μg / ml; and (4) dithiothreitol is present as a reducing agent, A step of performing hydrophobic chromatography under conditions where the concentration does not fall below 100 μg / ml.
[4] A method for producing a carboxylic acid, comprising a step of bringing the nitrilase according to any one of [1] to [3] into contact with a nitrile compound in an aqueous solution containing an organic solvent and recovering the produced carboxylic acid.
[5] The organic solvent is at least selected from the group consisting of water-soluble alcohols, water-soluble ketones, amides, dimethyl sulfoxide, esters, hydrocarbons optionally substituted with halogen, higher alcohols and ethers. The production method according to [4], which is one type.
[6] The production method according to [4] or [5], wherein the nitrile compound is arylacetonitrile.
[7] The production method according to any one of [4] to [6], wherein the content of the organic solvent is 90% (V / V (resistance of organic solvent / total volume of solution)) or less.

  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 enzyme of the present invention is preferably a purified enzyme. Such a purified enzyme exhibits extremely high organic solvent resistance and nitrile hydrolysis activity, has excellent storage stability, and suppresses the generation of side reactions, which is advantageous for industrial use.

[Embodiment of the Invention]
The present invention provides a nitrilase derived from Arthrobacter sp. F-73 strain and having specific physicochemical properties. The nitrilase of the present invention exhibits excellent organic solvent resistance.

Arthrobacter sp. F-73 strain The “Arrobacter sp. F-73 strain” containing the nitrilase of the present invention is a microbial strain isolated from the soil collected from the campus of Gifu University. Has been deposited as "FERM P-20349" at the Institute for Patent Biological Deposits. This strain can be obtained from the trustee based on this accession number.

  Alternatively, the isolation of the strain of the genus Arthrobacter is aimed, for example, by first culturing a test sample containing the microorganism of the genus Arthrobacter in a nitrile-containing medium and measuring the carboxylic acid accumulated in the culture. The presence of microorganisms having nitrilase activity can be identified. The test sample can be a material collected from soil, rivers, or lakes. A method for isolating and identifying microorganisms belonging to the genus Arthrobacter can be referred to, for example, “Bergey's Manual of Determinative Bacteriology, 9th Edition” (Edited by John G. Holt, Williams & Wilkins). .

  The microorganism is cultured in a general medium used for culturing bacteria. The medium for culturing the microorganism 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 a 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.

Nitrilase The nitrilase of the present invention is a nitrilase derived from Arthrobacter sp. F-73 strain and having the following physicochemical properties.
[1] Action:
Acts on the nitrile group of the nitrile compound, oxidizing the nitrile group to a carboxyl group,
[2] Substrate specificity:
Acts on 2-thiophene acetonitrile but does not act on acetonitrile or benzonitrile,
[3] Optimum pH:
The oxidation activity of nitrilase is optimal at pH 6.0 to 8.0 (reaction temperature 30 ° C.),
[4] Optimum temperature:
The nitrile group oxidation action shows maximum activity at 40-45 ° C.,
[5] pH stability:
pH 5.0-11.0 is the stable region, and [6] molecular weight:
The molecular weight by gel filtration is about 530 kDa,
It is separated into subunits having a molecular weight of about 44 kDa by SDS-polyacrylamide gel electrophoresis.
[7] Organic solvent resistance:
An aqueous solution containing 20% (V / V) (volume of organic solvent / total volume of solution) of any organic solvent selected from the group consisting of methanol, ethanol, acetone, DMSO and 2-propanol at 60 ° C. Shows 50% or more residual activity when treated for minutes.

  In the present invention, “nitrilase” is an enzyme that hydrolyzes a nitrile compound to convert it into a carboxylic acid. The nitrilase having specific physicochemical properties of the present invention is resistant 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 the nitrilase of the present invention, it is possible to industrially produce the corresponding carboxylic acid from a wide range of substrate compounds such as a nitrile that is hardly soluble in water from a water-soluble nitrile. The nitrilase of the present invention is derived from Arthrobacter sp. F-73 strain.

The “nitrilase activity” in the present invention can be confirmed as follows. The enzyme activity was measured at 20 ° C. in 50 mM potassium phosphate buffer (pH 7.0), 1 mM dithiothreitol (hereinafter abbreviated as “DTT”) in a reaction solution (2 ml) with an appropriate amount of enzyme solution (10 ml). The reaction is started by adding the substrate (standard reaction conditions when the substrate is 2-thiopheneacetonitrile). The reaction is stopped by adding 0.2 ml of 3N HCl. The reaction time is 60 minutes.
The reaction solution is centrifuged to obtain a supernatant. The substrate is quantitatively analyzed by HPLC. HPLC analysis was performed using Wakosil-II C18TS (4.6 × 150 mm) and 10 mM KH ₂ PO ₄ / H ₃ PO ₄ (pH 2.8) / CH ₃ CN (13 when the substrate was 2-thiopheneacetonitrile) as a developing solvent. / 7 (V / V)) (flow rate 1 ml / min), and detection at 235 nm.
The amount of enzyme that catalyzes the production of 1 micromole (1 μmol) of 2-thiopheneacetic acid per minute under standard reaction conditions is defined as 1 unit.
Protein quantification can be performed, for example, by the Bradford method (Bradford, M. (1976) Anal. Biochem. 72, 248-254.) Using a protein quantification kit manufactured by Bio-Rad.
In the examination of substrate specificity, the generated ammonia is colorimetrically determined by the indophenol method (Fawcett, JK & Scott, JE (1960) J. Clin. Pathol. 13, 156-159.). Can do.

  “Organic solvent resistance” in the present invention means that enzyme activity is maintained after treatment with an organic solvent. For example, when the nitrilase of the present invention is treated with 20% (V / V) acetone at 20 ° C. for 60 minutes, the enzyme activity does not substantially decrease. In the present invention, “the enzyme activity does not substantially decrease” means a case where the residual activity of the enzyme is 10% or more as compared with a case where the treatment is performed under the same conditions without an organic solvent.

The nitrilase of the present invention includes acetone, methanol, ethanol, 1-propanol, 2-propanol, DMSO, DMF, n-heptanol, n-octanol, n-hexane, n-octane, n-hexadecane, and methyl t-butyrate. It has resistance to organic solvents such as diisopropyl alcohol and ethyl acetate.
The nitrilase of the present invention is 20% (V / V) of these organic solvents with respect to any organic solvent selected from the group consisting of methanol, ethanol, acetone, DMSO and 2-propanol. (The volume of the organic solvent / the total volume of the solution) Treated with an aqueous solution at 20 ° C. for 60 minutes, the residual activity is 50% or more, preferably 50% -100%, more preferably 65% -100%.

  The nitrilase used in the present invention is preferably a purified enzyme. In the present invention, “purified enzyme” means an enzyme that has been electrophoretically purified to almost a single band, and does not include a mere crudely purified enzyme that has not been subjected to fractionation or chromatography. Such a purified enzyme is industrially advantageous because it is superior in storage stability and suppresses the formation of by-products as compared with the crude enzyme.

The nitrilase derived from the Arthrobacter sp. F-73 strain of the present invention is presumed to be a 12-mer of subunits, and the amount of enzyme obtained was very small. Difficult to obtain. For this reason, it is necessary to combine various purification means for stable acquisition of the enzyme.
For example, the microorganism producing the enzyme is first sufficiently grown and then the cells are collected and crushed in a suitable buffer solution to obtain a cell-free extract. A reducing agent such as 2-mercaptoethanol or a protease inhibitor such as phenylmethanesulfonyl fluoride (PMFS) can be added to the buffer.

  In the preparation of the enzyme from Arthrobacter sp. F-73, in the preparation of the cell-free extract, dithiothreitol was present as a reducing agent, and the temperature of the solution containing the enzyme was 5 ° C. or lower. Preferably there is. Under such conditions, an enzyme that is a 12-mer can be stably obtained.

  It is necessary to purify nitrilase from the obtained cell-free extract by combining fractionation based on protein solubility and various types of chromatography.

  As a fractionation method based on protein solubility, for example, precipitation with an organic solvent such as acetone or dimethyl sulfoxide, salting out with ammonium sulfate, or the like can be used. On the other hand, for chromatography, cation exchange, anion exchange, gel filtration, hydrophobic chromatography, and many affinity chromatography using chelates, dyes, antibodies and the like are known. More specifically, for example, hydrophobic chromatography using phenyl-Toyopearl, anion exchange chromatography using DEAE-Sepharose, hydrophobic chromatography using butyl-Toyopearl, affinity chromatography using Blue-Sepharose, Through gel filtration using Superdex 200 or the like, the nitrilase of the present invention can be stably purified to almost a single band by electrophoresis.

For example, the enzyme purification method preferably includes the following steps.
(1) A step of fractionating the cell-free extract with ammonium sulfate,
(2) a step of performing anion exchange chromatography, and (3) a step of performing hydrophobic chromatography.

In this case, the step of fractionating the cell-free extract with ammonium sulfate is preferably performed under the condition that dithiothreitol is present as a reducing agent and the pH of the solution containing the enzyme is 7.0 ± 0.3.
The step of performing anion exchange chromatography is preferably carried out by adjusting the concentration so that dithiothreitol is present as a reducing agent and the enzyme concentration in the buffer solution eluted from the column does not fall below 100 μg / ml.
The step of performing hydrophobic chromatography is preferably carried out by adjusting the concentration so that dithiothreitol is present as a reducing agent and the enzyme concentration in the buffer solution eluted from the column does not fall below 100 μg / ml.
Under such conditions, an enzyme that is a 12-mer can be stably obtained.

  The enzyme having nitrilase activity of the present invention can be a substantially pure protein. In the present invention, a substantially pure protein means that it is substantially free from other biological molecules. More specifically, a substantially pure protein means a purity of usually 75% or more, or 80% or more, preferably 85% or more, more preferably 95% or more, and even more preferably 99% or more by dry weight. Have Methods for determining protein purity are known. Specifically, the purity of the protein can be known by various column chromatography or electrophoresis analysis such as SDS-PAGE.

Method for Producing Carboxylic Acid Using Nitrilase The present invention provides a method for producing carboxylic acid from nitrile using the nitrilase.

  In the method for producing a carboxylic acid of the present invention, a nitrilase derived from the above-mentioned microorganism or having the same physicochemical properties is used, but it is preferably a purified nitrilase enzyme. The 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 nitrilase of the present invention.

  In the production method of the present invention, the starting nitrile is preferably arylacetonitrile, cyanohydrins, saturated nitrile, unsaturated nitrile, more preferably arylacetonitrile.

In the present invention, “arylacetonitrile” means a compound in which an acetonitrile group is bonded to a C _6-10 aryl ring or a 5- to 10-membered heteroaryl ring. The C _6-10 aryl ring refers to an aromatic hydrocarbon ring having 6 to 10 carbon atoms, and specific examples include benzene, toluene, naphthalene and the like. A 5- to 10-membered heteroaryl ring has 5 to 10, preferably 5 or 6, atoms constituting the ring, and 1 to 2 (preferably 1) heteroatoms in the atoms constituting the ring. An aromatic ring containing (a sulfur atom, an oxygen atom or a nitrogen atom, preferably a nitrogen atom) is meant, and specific examples include a pyridine ring and a thiophene ring.

  As the aryl acetonitrile, for example, preferably 2-thiopheneacetonitrile, 3-thiopheneacetonitrile, 3-pyridineacetonitrile, 2-pyridineacetonitrile, 2-tolylacetonitrile, 3-tolylacetonitrile, 4-tolylacetonitrile, benzylcyanide, naphthylacetonitrile Etc. Of these, 2-thiopheneacetonitrile, 3-thiopheneacetonitrile, 3-pyridineacetonitrile, 2-pyridineacetonitrile, 4-tolylacetonitrile and the like are more preferable.

  Examples of cyanovidrins include mandelonitrile, 2-chloromandelonitrile, 2-hydroxy-4-methylthiobutyronitrile and the like.

  Examples of the saturated nitrile include propyronitrile, butyronitrile, valeronitrile, 2-fluoronitrile, 2-hydroxy-4-methylthiobutyronitrile, 3-cyano-2-methylpropanol, and the like.

  Examples of the unsaturated nitrile include acrylonitrile.

  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 nitrilase of the present invention has nitrilase activity, and this nitrilase 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.
Examples of the water-soluble alcohols include methanol, ethanol, 1-propanol, 2-propanol, n-heptanol, n-octanol, 1,3-propanediol, and ethylene glycol. Among these, methanol, ethanol, Propanol is preferred, and methanol and ethanol are more preferred.
Examples of the water-soluble ketones include acetone. Examples of the amides include dimethylformamide.

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.
Examples of the esters include ethyl acetate, butyl acetate, and methyl t-butyrate.
Examples of the hydrocarbons that may be substituted with halogen include n-hexane, n-octane, n-hexadecane, cyclohexane, and chloroform.
Examples of higher alcohols include 1-octanol.
Examples of ethers include diisopropyl ether, 1,4-dioxane, and tetrahydrofuran.

  The organic solvent may be appropriately added within a range not impairing the hydrolysis reaction of the present invention. Usually, it is preferably 90% (V / V) (volume of the organic solvent / total volume of the solution) with respect to the aqueous reaction solution. In the following, an amount of 80% (V / V) or less, more preferably 60% (V / V) or less can be added. The lower limit of the addition amount is preferably 1%, more preferably about 5%.

  For example, in the case of acetone, methanol, ethanol, dimethyl sulfoxide, hexane, etc., 10 to 50% (V / V) or less, preferably 20 to 40% (V / V) can be added. In the case of dimethylformamide, tetrahydrofuran, 1,3-propanediol and the like, 5 to 30% (V / V) or less, preferably 10 to 20% or less can be added. In the case of ethyl acetate, an amount of 5 to 90% (V / V) or less, preferably 10 to 80% 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.

  Although the density | concentration of the substrate compound in a reaction liquid is not restrict | limited in particular, For example, it is 0.1-10 weight% normally, Preferably it can be 0.2-5.0 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.

  With respect to this substrate concentration, the nitrilase of the present invention can efficiently advance the enzyme reaction by setting the amount of the enzyme activity to, for example, 1 mU / mL to 100 U / mL, preferably 100 mU / mL or more. The enzyme can be brought into contact with the substrate by dissolving or dispersing in the reaction solution. Alternatively, an enzyme 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 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 enzyme of the present invention come into contact with each other 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.

[Example 1]
culture:
The pre-culture (4 ml) of Arthrobacter sp. F-73 strain was cultured with shaking at 28 ° C. for 24 hours using a nutrient medium containing peptone and yeast extract. The main culture is a medium comprising 0.4% (w / v) sodium L-glutamate, 0.15% yeast extract, 0.1% monopotassium phosphate, 0.02% magnesium sulfate (7 hydrate) ( 40 ml, pH 7.0) was placed in a 500 ml shoulder shake flask and sterilized. To this, a preculture (4 ml) was inoculated, and at the same time, 0.3% (V / V) isovaleronitrile was added as an inducer for nitrilase production and cultured at 28 ° C. for 72 hours with shaking. In this way, cells containing highly active nitrilase were prepared.

Preparation of cell-free extract:
The strain was collected by centrifugation at 15,000 rpm for 20 minutes at 5 ° C. The cell body obtained from the culture solution (2 L) (5.2 g dry cell amount) was suspended in 0.85% (w / v) NaCl and centrifuged twice, and the cell body Was thoroughly washed. The cells were suspended at 50 ° C. or less in 50 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol (DTT). A cell extract was prepared by performing a crushing operation at 100 W for 20 minutes using an ultrasonic generator (Model 201 Kubota Seisakusho) at 5 ° C. or lower. This was centrifuged at 13,000 rpm for 20 minutes to remove the cells, and a cell-free extract was obtained.

Enzyme purification:
In purifying the enzyme of the present invention, various purification means were combined in order to purify the enzyme stably and with high purity.
(1) Ammonium sulfate fraction Ammonium sulfate was added to the cell-free extract so as to be 40% saturated. At this time, 10% (w / v) ammonia solution was added to maintain pH 7.0. After stirring for 4 hours, centrifugation (15,000 rpm, 20 minutes) was performed, and the precipitate fraction was suspended in 50 mM potassium phosphate buffer (pH 7.0) (Buffer A) containing 1 mM DTT. The suspension was dialyzed thoroughly with Buffer A to remove ammonium sulfate. After dialysis, the mixture was centrifuged to remove the precipitate and obtain a supernatant.

(2) DEAE-Sephacel column chromatography (anion exchange chromatography)
The enzyme solution was placed on a DEAE-Sephacel column (Φ30 × 250 mm) that had been sufficiently equilibrated with Buffer A, and the same buffer was poured to wash. Thereafter, it was washed with Buffer A containing 0.25 M KCl, but the activity was not eluted. Next, when Buffer A containing 0.35 M KCl was passed, the target nitrilase activity was eluted. To this fraction, ammonium sulfate was added to 20% saturation. During this time, the amount of the solution was adjusted so that the enzyme concentration in the buffer solution eluted from the column was 109 μg / ml.

(3) Butyl-Toyopearl column chromatography (hydrophobic chromatography)
The active fraction was placed on a Butyl-Toyopearl column (Φ20 × 200 mm) sufficiently equilibrated with Buffer A containing 20% saturated ammonium sulfate, and washed thoroughly with the same buffer. Next, after washing with Buffer A containing 10% saturated ammonium sulfate, the desired nitrilase activity was eluted with Buffer A containing 5% saturated ammonium sulfate. This active fraction was collected and dialyzed thoroughly against Buffer A containing 15% saturated ammonium sulfate. During this time, the amount of the solution was adjusted so that the enzyme concentration in the buffer solution eluted from the column was 376 μg / ml.

(4) Phenyl-Sepharose column chromatography The active fraction was placed on a Phenyl-Sepharose column (Φ20 × 10 mm) sufficiently equilibrated with Buffer A containing 15% saturated ammonium sulfate, and washed with the same Buffer. After washing with Buffer A, the target nitrilase activity was eluted by flowing Buffer A containing 10% (V / V) ethylene glycol. The active fraction was collected and dialyzed thoroughly with Buffer A.

Table 1 shows the results of purifying the nitrilase of Arthrobacter sp. F-73. The enzyme purified in this way showed a single band by SDS-PAGE, confirming that it was highly purified.
The final purified sample of the nitrilase of the present Arthrobacter sp. F-73 strain was 4.6 mg of total protein, a specific activity of 61.1 units / mg under standard reaction conditions, and a purification yield of 13.8%.

Properties of the enzyme obtained:
(1) Molecular weight, subunit molecular weight From SDS gel electrophoresis, the subunit of this nitrilase was calculated to be about 44 kDa, and the molecular weight was calculated to be about 530 kDa from gel filtration chromatography, which was considered to be a 12-mer of subunits of the same size. .

(2) Optimum pH, temperature, and heat stability The enzyme reaction was carried out at 30 ° C for 15 minutes to examine the optimum pH of the reaction, which was pH 6.0-8.0, pH 5.0 or lower, pH 9.0. As a result, the activity decreased rapidly. The enzyme solution was kept for 30 minutes under various pH conditions, and then reacted at pH 7.0 for 10 minutes to examine pH stability. As a result, it was stable in the range of pH 5.0 to 11.0.
The enzyme was kept at various temperatures for 15 minutes or 30 minutes and then reacted at 30 ° C. for 10 minutes. The enzyme was stable at 40-45 ° C., but was inactivated when it exceeded 50 ° C. Moreover, when the optimal temperature of reaction made to react for 10 minutes at 20-60 degreeC was examined, the maximum activity was shown at 40-45 degreeC.

[Example 2]
Substrate specificity:
In Examples, the enzyme activity was measured at 20 ° C. by adding 10 mM of the substrate shown in Table 2 to a reaction solution (2 ml) containing 50 mM potassium phosphate buffer (pH 7.0), 1 mM DTT and an appropriate amount of enzyme solution. To start the reaction. The reaction was stopped by adding 0.2 ml of 3N HCl. The reaction time was 60 minutes. The reaction solution was centrifuged to obtain a supernatant.

  The organic acid produced by the reaction was quantitatively analyzed by high performance liquid chromatography or gas chromatography.

HPLC analysis was carried out using a Wakosil-II 5C18TS (4.6 × 150 mm) in a system of 10 mM, KH ₂ PO ₄ / H ₃ PO ₄ (pH 2.8) / CH ₃ CN, in a phosphate buffer and acetonitrile solvent. The quantitative ratio was varied depending on the substrate.

  For example, mandelic acid is detected at 17/3 (v / v) at 235 nm, 2-chloromandelic acid is detected at 13/7 (v / v) at 220 nm, 2-, 3-thiopheneacetic acid is detected at 65:35 (v / V) detected at 235 nm, 3-tolylacetonitrile, benzyl cyanide, naphthoacetonitrile detected at 230 nm at 1: 1 (v / v), 2-tolylacetonitrile, 2-, 3-pyridineacetonitrile 1 (v / v) at 230 nm, 2-hydroxy-4-methylthiobutyronitrile and 2-hydroxy-4-methylthiobutyric acid at 9/1 (v / v) at 210 nm, 2-furancarboxylic acid is 9/1 (v / v) was detected at 230 nm, and acrylic acid was detected at 4/1 (v / v) at 210 nm.

  GC analysis was performed using a gas column (Shimadzu model GC-14A) and a glass column packed with Thermo 3000 (5% on Chromosorb W) 80/100 mesh, and the temperature of the injector, detector, and column was set to 230 respectively. , 230, and 150 ° C.

  Table 2 shows the results of substrate specificity (relative activity) of this nitrilase for various substrates. This enzyme was shown as a relative activity with 2-thiopheneacetonitrile as the best substrate and its activity (43 μmol / min / mg protein) as 100. Ammonia and the corresponding carboxylic acid were produced using arylacetonitriles such as 3-thiopheneacetonitrile, 3-pyridineacetonitrile, 2-pyridineacetonitrile, 4-tolylacetonitrile, 3-tolylacetonitrile, benzylcyanide as good substrates.

  In addition, it also acted on α-cyanohydrins such as mandelonitrile, 2-chloromandelonitrile, 2-hydroxy-4-methylthiobutyronitrile. It also acted on acrylonitrile and n-valeronitrile, but benzonitrile, p-cyanophenol, o-cyanophenol, o, m or p-tolunitrile, 3-cyanopyridine, cyanopyrazine, 2-cyanothiophene, 3-indoleacetonitrile It did not act on methacrylonitrile, isovaleronitrile, isobutyronitrile, crotonnitrile, acetonitrile, isobutyronitrile and the like. Therefore, this nitrilase was judged to fall within the category of allylacetonitrilase. The Km value for 2-thiopheneacetonitrile showed a small value of 15 μM or less, and because the value was too small, an accurate Km value could not be measured.

Protein quantification was performed by the Bradford method (Bradford, M. (1976) Anal. Biochem. 72, 248-254.) Using a protein quantification kit manufactured by Bio-Rad.
In the examination of substrate specificity, the produced ammonia was colorimetrically determined by the indophenol method (Fawcett, JK & Scott, JE (1960) J. Clin. Pathol. 13, 156-159.).

[Example 3]
Inhibitor effect:
When the nitrilase activity was measured by adding various inhibitors, it was greatly inhibited by addition of SH inhibitors such as 1 mM Cu ²⁺ , Hg ²⁺ , Ag ⁺ , 0.1 mM p-chloromercurybenzoic acid (p-CMB). Received. These inhibitions were restored by adding 5 mM DTT.

[Example 4]
Effect of organic solvent addition:
The activity was examined under the condition of organic solvent addition. The results are shown in Table 3. Although the resistance of the purified enzyme to the organic solvent was reduced as compared with the fungus body, it exhibited solvent resistance and activity under the conditions of addition of the water-soluble organic solvent shown in Table 3.

  The mixture of n-butanol and water (1: 9, V / V) and the mixture of toluene and water (1: 9, V / V) were strongly inhibited and almost completely deactivated. However, n-heptanol, n-octanol, n-hexane, n-octane, n-hexadecane, methyl t-butyrate, diisopropyl ether and water mixture (1: 1, V / V) are completely inhibited. There wasn't. The mixed liquids of ethyl acetate and water (9: 1, 8: 2) showed activity of 75% and 38%, respectively, compared to the case of no addition.

  The fact that such purified nitrilase has excellent organic solvent resistance is a very surprising property first discovered in the present invention. For example, cell bodies have barriers due to cell membranes, and cell-free crude enzymes have certain organic solvent resistance to cell and cell-free reactions (crude enzymes) due to the presence of impurities and impurities that promote stabilization. Is possible. However, whether or not the purified nitrilase has excellent organic solvent resistance is unpredictable and was confirmed for the first time after isolation and purification.

[Example 5]
Production of various carboxylic acids using purified nitrilase in aqueous solution in the presence of organic solvent:
Test containing 0.1 mg of the purified enzyme obtained in Example 1 and 2 ml of reaction solution (containing 20 mM of the substrate listed in Table 4, potassium phosphate buffer (pH 7.0) 100 mM methanol 5% (v / v)) After reacting in a tube at 25 ° C. for 2 hours, an additional 20 mM of substrate was further added, and further reacted at 25 ° C. for 2 hours. After a total of 4 hours of reaction, the produced carboxylic acid was quantified. The results are shown in Table 4. 2-thiopheneacetonitrile, 3-thiopheneacetonitrile, 3-pyridineacetonitrile, 2-pyridineacetonitrile, 4-tolylacetonitrile, 3-tolylacetonitrile, benzylcyanide, (R, S) -mandelonitrile, 2- In hydroxy-4-methylthiobutyronitrile, the corresponding carboxylic acid was obtained in high yield. The results are shown in Table 4.

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

Claims (4)

A purified nitrilase derived from Arthrobacter sp. F-73 strain represented by accession number FERM P-20349 and having the physicochemical properties [1] to [7] below in an aqueous solution containing an organic solvent is converted into a nitrile compound. A method for producing a carboxylic acid, comprising a step of contacting the carboxylic acid with the product and recovering the produced carboxylic acid,
The nitrile compound is 2-thiopheneacetonitrile, 3-thiopheneacetonitrile, 3-pyridineacetonitrile, 2-pyridineacetonitrile, 4-tolylacetonitrile, 3-tolylacetonitrile, benzylcyanide, mandelonitrile, and 2-hydroxy-4- Selected from the group consisting of methylthiobutyronitrile,
The organic solvent consists of methanol, ethanol, propanol, acetone, DMSO, DMF, n-heptanol, n-octanol, n-hexane, n-octane, n-hexadecane, methyl t-butyrate, diisopropyl ether, and ethyl acetate. A method selected from the group;
[1] Action:
Acts on the nitrile group of the nitrile compound, oxidizing the nitrile group to a carboxyl group,
[2] Substrate specificity:
Acts on 2-thiophene acetonitrile but does not act on acetonitrile or benzonitrile,
[3] Optimum pH:
The oxidation activity of nitrilase is optimal at pH 6.0 to 8.0 (reaction temperature 30 ° C.),
[4] Optimum temperature:
The nitrile group oxidation action shows maximum activity at 40-45 ° C.,
[5] pH stability:
pH 5.0-11.0 is the stable region, and [6] Molecular weight:
The molecular weight by gel filtration is about 530 kDa,
It is separated into subunits having a molecular weight of about 44 kDa by SDS-polyacrylamide gel electrophoresis.
[7] Organic solvent resistance:
Treatment with an aqueous solution containing 20% (V / V) of any organic solvent selected from the group consisting of methanol, ethanol, acetone, DMSO and 2-propanol at 20 ° C. for 60 minutes gives a residual activity of 50% or more. Show.   The production method according to claim 1, wherein the nitrile compound is 2-hydroxy-4-methylthiobutyronitrile.   The production method according to claim 1 or 2, wherein the content of the organic solvent is 90% (V / V (resistance of organic solvent / total volume of solution)) or less. The production method according to claim 1, wherein the purified nitrilase is obtained through a purification process including the following steps;
(1) A step of preparing a cell-free extract derived from Arthrobacter sp. F-73 strain under the condition that dithiothreitol is present as a reducing agent and the temperature of the solution containing the enzyme is 5 ° C. or lower;
(2) a step of subjecting the cell-free extract to ammonium sulfate fractionation under the condition that dithiothreitol is present as a reducing agent and the pH of the solution containing the enzyme is 7.0 ± 0.3;
(3) performing anion exchange chromatography under the condition that dithiothreitol is present as a reducing agent and the enzyme concentration is not less than 100 μg / ml; and (4) dithiothreitol is present as a reducing agent, A step of performing hydrophobic chromatography under conditions where the concentration does not fall below 100 μg / ml.