JPH0343082A - Production of trans-4-cyanocyclohexanecarboxylic acid and enzyme therefor - Google Patents

Abstract

PURPOSE:To obtain the subject carboxylic acid useful as a production intermediate for tranexamic acid known as a hemostatic agent, etc., with a simple process in high yield by treating dicyanocyclohexane with a specific microorganism or its treated product. CONSTITUTION:The objective trans-4-cyanocyclohexanecarboxylic acid of formula II is produced from trans-1, 4-dicyanocyclohexane by the action of a microbial strain belonging to genus Corynebacterium or its treated product. The process is performed preferably e.g. by culturing Corynebacterium Spc-5 strain (FERM 8931) in conventional medium at pH5-10 and 25-32 deg.C for 1-8 days.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel method for producing trans-4-cyanocyclohexanecarboxylic acid.

The purpose of the present invention is to industrially produce trans-4-cyanocyclohexanecarboxylic acid, which is an intermediate for the production of tranexamic acid, which is extremely effective as a medicinal agent such as a hemostatic agent, and cetraxate hydrochloride, which is highly effective as an anti-ulcer agent. , to produce it in high yield.

(Prior Art) As a conventional method for producing trans-4-cyanocyclohexanecarboxylic acid, for example, a method in which hexaterephthalic acid is brought into contact with ammonia gas (Japanese Patent Publication No. 47-197-
23535), a method of contacting 1,4-dicyanocyclohexane with ammonia gas (JP-A-48-34144)
)It has been known. In addition, as a method using microorganisms,
The present inventors have obtained trans-4-cyanocyclohexanecarboxylic acid by allowing microorganisms to act on trans-1,4-dicyanocyclohexane.

(Japanese Patent Publication No. 63-10752, Japanese Patent Application Publication No. 63-12291,
JP-A-63-129988, JP-A-63-294793
) (Problem to be Solved by the Invention) The above chemical synthesis method requires high temperature and high pressure reaction conditions. In addition, it is difficult to produce while maintaining the steric configuration, and many by-products are produced, making the isolation and purification process of the target product extremely complicated.

In addition, in the conventional method using microorganisms, the raw material is cis, trans-1,4-dicyanocyclohexane from which the cis isomer has been removed through a purification process, that is, highly purified trans-1,4-dicyanocyclohexane. was. The present invention provides trans-4-
The objective is to produce cyanocyclohexanecarboxylic acid.

(Means for Solving the Problems) As a result of intensive studies to solve the above problems, the present inventors have found that it is possible to select and maintain the steric configuration at room temperature and normal pressure, and that We have discovered a new production method that utilizes the efficient biochemical action of microorganisms. That is, it is a method for producing trans-4-cyanocyclohexanecarboxylic acid represented by the following formula () from cis, trans-1,4-dicyanocyclohexane represented by the following formula (1) CN by the action of microorganisms. .

The present inventors have demonstrated that Corynebacterium bacteria are compound (1).
) to Compound (II), the trans form was preferentially converted to Compound (If), and the conversion activity of the cis form was found to be extremely low. Furthermore, the present invention was completed by extracting enzymes involved in the conversion of compound (1) to compound (II), ie, nitrile hydratase and amidase, from Corynebacterium bacteria.

Next, a method of implementing the present invention will be explained.

(1) Bacterial strain used The microorganism used in the present invention belongs to the genus Corynebacterium, and specific bacterial strains belonging to these genus include, for example, Corynebacterium sp. No. 8931). The C5 strain has been deposited at the Microbial Technology Research Institute, and its mycological properties have been published in Japanese Patent Application Publication No. 1989-1999.
3-129988.

(2) Culture method The microorganisms used in the present invention can be cultured according to known methods. The medium to be used can be one known as a nutritional source for microorganisms, including blackstrap molasses, carbon sources such as glucose, glycerin, ethanol, and sucrose, nitrogen sources such as ammonium sulfate or urea, and ammonium chloride, meat extract, and yeast. Extract, malt extract, organic nutritional sources such as peptone, phosphoric acid, magnesium, potassium,
Inorganic nutritional sources such as iron and cobalt, and vitamins can be used in appropriate combinations. In addition, cyano compounds such as propionitrile, isobutyronitrile, and crotononitrile, isobutyramide, n
-An amide compound such as butyramide, or further, divalent iron ions may be added in appropriate amounts. Medium pH
should be selected in the range of 5 to 1O, and the culture temperature should be selected in the range of 18 to 38
℃, preferably 25 to 32℃. Culture days are 1-8
It may be cultured within a range of days until the activity is maximized.

(3) Enzyme From the bacterial cells of Corynebacterium sp. C5 strain, preferentially convert the trans form of the compound of formula (1) into the formula (If).
The cis-converting compound, which has a low conversion activity, was extracted and isolated. As a result, it was revealed that the conversion of the compound of formula (1) to the compound of formula (II) in strain C5 occurred by a combination of nitrile hydratase and amidase.

Furthermore, these two enzymes were recognized as novel nitrile hydratase and ashortase due to their enzymatic action and substrate specificity. The physical and chemical properties of nitrile hydratase and amidase are explained as follows.

(A) Nitrile hydratase ■Enzyme action It acts on the nitrile group of a nitrile compound and acts as a catalyst for the reaction of hydration converting the nitrile group into an amide group.

■Substrate specificity We investigated the different substrate types of nitrile hydratase.

The results are shown in Table 1.

Table 1 From Table 1, this enzyme can contain saturated aliphatic nitriles such as n-butyronitrile and n-valeronitrile, unsaturated aliphatic nitriles such as acrylonitrile and methacrylonitrile, aromatic nitriles such as benzonitrile, 3-cyanopyridine, etc. It has been found that the present invention acts on heterocyclic nitriles, as well as dinitrile compounds such as malononitrile and trans-1,4-dicyanocyclohexane.

In terms of compounds with a fast nitrile group hydration rate, n
-Aliphatic nitrile compounds having 4 to 5 carbon atoms, such as butyronitrile, n-valeronitrile, iso-valeronitrile, and the like.

Furthermore, 1,4-dicyanocyclohexane is characterized in that the hydration rate of the trans isomer is significantly higher than that of the cis isomer.

■Molecular weight liquid chromatograph [Column;TSK-gelG30
The molecular weight was determined to be 61,400 by 0-3W (0,75×60C11). Further, as a result of 5DS-PAGE electrophoresis, there was a single band, and the molecular weight of the subunit was 2pH6.900.

(2) Optimum pH The results of the examination of the optimum pt- are shown in FIG. 1, and the optimum pH was 8.0 to 8.5.

■Dissolve temperature-stable nitrile hydratase in 10mM potassium phosphate buffer (pH 7.5 (contains 3mM Na salt of isovalerate)) and measure the residual activity after standing at 10-50°C for 10 minutes. As a result, 1
No decrease in activity was observed between 0 and 40°C, and residual activity was 40% at 45°C.

■Stabilizer Nitrile hydratase is 10mM containing various stabilizers.
Dialysis was performed in potassium phosphate buffer (pH 7.5) for 3 days. The residual activity after dialysis (activity before dialysis is taken as 100%) is shown in Table 2.

table Isovaleric acid was the best stabilizer.

■After dissolving the metal and the inhibitor '438 nitrile hydratase in 100mM phosphate buffer (pH 7,5), add 1 portion of each of the following substances.
The enzyme activity was measured by adding mM each, and the effect on nitrile hydratase activity involved in trans-4-cyanocyclohexanecarboxylic acid amide administration was examined. The results are shown in Table 3.

Table 3 Nitrile hydratase was strongly inhibited by Hg++ and phenylhydrazine.

■Presence of PQQ (pyrroloquinoline quinone) The presence of PQQ (pyrroloquinoline quinone) in nitrile hydratase can be determined by the following method! Approved.

15μ of nitrile hydratase in 6N hydrochloric acid at 120°C.
After being treated for 48 hours and hydrolyzed, it was concentrated and neutralized with an aqueous solution of caustic soda. After drying the treated solution, it was redissolved in 10% methanol solution, and G-10 (1,OX
120cm) column, and the passing liquid 2. 1j! l! Collected. Examinations (i) to (iv) were conducted using this solution, and the presence of PQQ was confirmed.

(i) The presence of PQQ was confirmed by reactivating the membrane-bound D-glucose dehydrogenase apoenzyme.

(ii) 252 nm and 360 nm characteristic of PQQ,
Absorption was observed.

(ii) Fluorescence spectrum (IIITAcIII 650
-10S type detector) Excitation peak: 375 rv+, emission peak: 460 ns+, which coincided with the PQQ standard.

(iv) Acetobacter, a PQQ demanding strain
The presence of PQQ was confirmed by a bioassay method using aceti.

(B) Amidase ■ Enzyme action Acts on the amide group of an amide compound and acts as a catalyst for the reaction of hydrolyzing the amide group into a carboxyl group and ammonia.

■Substrate specificity The substrate specificity of amidase was investigated. Table 4 shows the results.
Shown below.

Table 4 From Table 4, this enzyme contains saturated aliphatic amides such as propionamide and n-valeramide, cyclic saturated aliphatic amides such as trans-4-cyanocyclohexanecarboxylic acid agodo, and unsaturated aliphatic amides such as methacrylamide. Amides, aromatic acids such as benzadamide, and nicotinamide, etc.? ! [It can be seen that it acts on i-cyclic amide compounds. In addition, the hydrolysis rate of trans-4-cyanocyclohexanecarboxylic acid amide is particularly high;
The activity of the 4-cyanocyclohexanecarboxylic acid agodo is low.

■Optimal pH The results of the optimum pH study are shown in Figure 2, and the optimum pH is 6.
5 to 10.0.

■Optimal temperature The results of the optimal temperature study are shown in Figure 3, and the optimal temperature is approximately 5.
It is 0°C.

■ Effects of metals and inhibitors Amidase was buffered in 100mM phosphate buffer (PH7.5).
), the following substances were added at 1mM each, the enzyme activity was measured, and the effect on the amidase activity involved in trans-4-cyanocyclohexanecarboxylic acid produced $C was investigated. The results are shown in Table 5.

Table 5 It was slightly inhibited by Hg"4 and l-CM B.

(4) Reaction method In the present invention, the reaction method for converting the compound of formula (1) to the compound of formula (II) is specifically a method of culturing the microorganism in the presence of the compound of formula (1). , microbial culture, and bacterial cells collected therefrom or treated bacterial cells (
For example, crushed bacterial cells, organic solvent-treated bacterial cells, or enzymes separated and extracted from bacterial cells by a known method) and the formula (
There are two methods of contacting with the compound of 1).

Alternatively, the bacterial cells, treated bacterial cells, or enzymes extracted from the bacterial cells may be immobilized by a known method such as celite, calcium alginate, carrageenan, etc., and then reacted with the compound of formula (1).

As the reaction medium, an aqueous medium such as water, a buffer solution, or a mixture of water and an organic solvent such as methanol, chloroform, or methylene chloride can be used.
1) The compound of formula (I) is added as a powder or oil, or dissolved in a suitable solvent.The concentration of the compound of formula (I) is about 0.01 to 60% by weight, preferably 1.0%.
~40% by weight.

When using bacterial cells in the reaction, the concentration of bacterial cells is usually 0.1
~8! The reaction temperature is 4 to 65°C, preferably 15 to 55°C, and the reaction pH is 4 to 11, preferably 6.5 to 9.0. The reaction time is usually 0.5 to 100%.
All you have to do is choose an appropriate time within the time range, and the consumed expression (
The compound 1) may be added continuously or intermittently so that the concentration in the reaction solution is maintained within the above range.

The weight ratio of the cis form and the trans form in the compound of formula (1) used as a raw material is 0.1:9.9 to 9°570.5, but if it is enriched in the trans form, it will be produced. formula(
The purity of the trans isomer of the compound n) is higher.

(5) Minute purification The produced compound of formula (II) can be easily purified to a high purity by a known method such as solvent extraction or crystallization after removing insoluble substances such as bacterial cells from the reaction completed solution. Crystals can be obtained. Furthermore, unreacted cis-1,4-dicyanocyclohexane can also be easily recovered and subjected to isomerization to be subjected to the enzymatic reaction again.

(6) Production of cis,trans-1,4-dicyanocyclohexane [compound (■)] Compound (1) is a mixture of dimethyl-1,4-cyanocyclohexane carboxylate and a compound that can be a source of ammonia, such as ammonia, urea. etc. 200~350”
C, preferably by heating at 230 to 300°C. In addition, to increase the reaction rate of this step, mineral acids such as hydrochloric acid, phosphoric acid, and sulfuric acid, oxides such as alkoxide, phosphorus pentoxide, and tin oxide, or organic acids such as acetic acid, propionic acid, and benzoic acid are used. may be used as a catalyst. Moreover, regardless of whether the configuration of the dimethyl-1,4-cyclohexanecarboxylate used is trans, cis, or a trans/cis mixture, the resulting compound (1) gives a trans/cis mixture. In the present invention, the compound (1) thus produced can be used as it is as a raw material for an enzyme reaction without being isolated and purified.

(Effects of the Invention) By utilizing the present invention, trans-4-cyanocyclohexanecarboxylic acid can be produced at a high concentration under reaction conditions of room temperature and normal pressure. Furthermore, since the trans isomer can be produced from a raw material containing a mixture of cis and trans isomers that does not require purification, it is economically extremely useful.

(Examples) Next, the present invention will be described with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples in any way.

Example 1 Dimethyl-1,4-cyclohexanecarboxylate 1
00g (0.5 mol) and 1.0g of tin oxide were charged into a reactor, and 0.0g of ammonia gas was charged at 260-280°C.
The reaction was carried out for 10 hours while introducing at a flow rate of 25 mol/Hr. After the reaction was completed, 2.000 d of methanol was added, and substances insoluble in methanol were collected by filtration, and the filtrate was concentrated and cooled to give cis.

70 g of trans-1,4-dicyanocyclohexane (purity 97.5%) was obtained. This product contained 42% cis isomer and 58% trans isomer.

Example 2 Glycerol 0.5g, polypeptone 0.3g, potassium phosphate 0.08g, dipotassium phosphate 0.12g,
Sodium chloride 0.1g, isobutyronitrile 0.05
g, magnesium sulfate heptahydrate 0.02g, iron sulfate heptahydrate 0.004g, biotin 100μg, thiamine hydrochloride 1
Corynebacterium sp. strain C5, which had been pre-cultured in the same medium, was inoculated into 100 d of a sterilized medium containing 00 μg and adjusted to pH 7.0, and cultured at 30° C. for 2 days.

After the culture is completed, the bacterial cells are collected by centrifugation, and 0.
After suspending 4 g DCW (dry weight) in an Erlenmeyer flask containing 100 ral of 0.05 M potassium phosphate buffer y-(pH 7,5), cis, trans-1,4- prepared in Example 1 was suspended. Dicyanocyclohexane (cis:
20 g of trans=42:58) was added and reacted at 30° C. with shaking.

The reaction was terminated after 50 hours. As a result of high performance liquid chromatography analysis of the reaction solution, bacterial cells were removed by centrifugation from the reaction solution containing 10.6 g of trans-4-cyanocyclohexanecarboxylic acid and 0.45 g of cis-4-cyanocyclohexanecarboxylic acid. 8.4 g of crystals of trans-4-cyanocyclohexanecarboxylic acid were obtained by operations such as removal, extraction, concentration, and crystallization.

This product showed a single peak in high performance liquid chromatography analysis.

Melting point 150-152°C IR (KBr) 2230cm-' Cv = CH) Elemental analysis CH Calculated value 62.75 $ 7.19 X Actual value
61.5 $ 7.35% High performance liquid chromatography analysis was conducted as follows. Analyzer: Waters 6000A pump, Tosoh T-8 differential refractometer, column nipper pack C18, solvent: water/acetonitrile = 77/23 (volume ratio) → -PICB-7 (
Waters Inc.) 1.5% by volume.

Comparative example Glucose 5.0g, polypeptone 3.0g, phosphoric acid-
Potassium 0.8g, dipotassium phosphate 1°2g1 sodium chloride 1.0g, isobutyronitrile 0.5g, magnesium sulfate heptahydrate 0.2g, iron sulfate heptahydrate 0.04g
, contains 1 mg of biotin, 1 μg of thiamine hydrochloride, and has a pH of 7.
Candida giamondi (Candida
ida guilliermondii) IFo
0454 was inoculated and cultured at 32°C for 5 days, and then the bacterial cells were collected by centrifugation.

4. After suspending OgDCW (dry weight) in an Erlenmeyer flask containing 0.05 M potassium phosphate buffer (pH 7,5), cis, trans-1,4-dicyanocyclohexane prepared in Example 1 was added. 2 g of (cis:trans=42:5B) was added and reacted at 30°C with shaking. The reaction was terminated after 80 hours, and high performance liquid chromatography analysis of the reaction solution revealed that 0.12 g of trans-4-cyanocyclohexanecarboxylic acid and 0.26 g of cis-4-cyanocyclohexanecarboxylic acid were produced.

Example 3 Isolation and purification of nitrile hydratase of Corynebacterium sp. 05 strain After culturing Corynebacterium sp. C5 strain in the same medium as in Example 2, 8. OOOxg.

Bacterial cells were obtained by centrifuging for 20 minutes.

After the bacterial cells were washed, they were crushed while cooling using a Dynor x-mill. The disrupted solution was centrifuged, and the resulting supernatant was used as a cell-free extract.

The cell-free extract was added to DEAE-cellulose [suspended in 10 mM phosphate buffer C pH 1,5] and stirred for 30 minutes. DEAE - 0.4M after cellulose removal
Resuspend in 100mM buffer containing NaCl and incubate for 30
Mix slowly for a minute. Ammonium sulfate was added to the filtered filtrate to bring the degree of saturation to 60%, the pH was adjusted to 7.5, and the mixture was stirred for 1 hour. Then, centrifugation was performed to collect the precipitated protein group. After dissolving the recovered protein in a small amount of 10 mM phosphate buffer, it was placed in a cellophane tube and left in the same buffer at 4°C for 18 hours, and ammonium sulfate in the aqueous protein solution was removed by dialysis.

The protein group aqueous solution after the dialysis operation is DEAE-Sep
Chromatographed on hacel (5X 25 Cm). The t8 syneresis was made using 100mM phosphate buffer (NaC
1 concentration O~0.3M) was used. A portion of each chromatography eluate fraction was obtained, trans-1°4-dicyanocyclohexane was added thereto, and the presence or absence of hydration activity of the nitrile group was examined by liquid chromatography analysis. Fractions showing activity were collected, ammonium sulfate was added to precipitate proteins, and then collected by centrifugation. After dialysis, ammonium sulfate was added to achieve 15% saturation, and P
henyl-3epharose CL-4B
Chromatographic separation was performed using a column (2X16C11). Fractions with activity were collected and recovered in the same manner as above, followed by dialysis treatment. From this step onwards, 30mM invaleric acid was added as an enzyme stabilizer.

After the dialysis solution was again subjected to DEA E-5 ephacel column chromatography and active fractions were collected, Ph
Purification of the nitrile hydratase was completed by performing enyl-5epharose CL-4B column chromatographic separation. Through the above series of operations, the specific activity increased approximately 130 times. In addition, the molecular weight was measured using a liquid chromatograph [column; TSK-get G 3000].
-3W (0,75X 60cm) ) by 61
It was determined to be 400°. Furthermore, as a result of 50S-PAGE electrophoresis, a single band was observed, and the molecular weight of the subunit was 2.
The pH was 6.900. Furthermore, as a result of measuring the visible and ultraviolet spectra, there was a broad peak with maximum absorption at 710 ns, and a Schilder peak at 410 ns+.

Example 4 Isolation and purification of amodase of Corynebacterium sp. strain C5 A cell-free extract was obtained in the same manner as in Example 3.

The cell-free extract was added to DEAE-cellulose (suspended in OmM phosphate buffer (pH 7,5)) and stirred for 30 minutes. After removing the DEAE-cellulose,
Resuspend in 100mM buffer containing M NaCl2 and mix gently for 30 minutes.

Add ammonium sulfate to the filtered filtrate to bring the saturation level to 60.
%, the pH was adjusted to 7.5 and stirred for 1 hour.

Next, centrifugation was performed and the precipitated protein group was collected. After dissolving the recovered protein in a small amount of 10 mM phosphate buffer, put it into a cellophane tube and incubate for 4 minutes.
The mixture was allowed to stand for 18 hours in the same buffer as above, and the ammonium sulfate in the aqueous protein solution was removed by dialysis.

The protein group aqueous solution after the dialysis operation is DEAE-Sep
Chromatographed on hacel (5X 25 cn+). The eluent was 100mM phosphate buffer (NaC
7! A concentration of 0 to 0.4 M) was used.

A portion of each chromatographic elution fraction was obtained, and a fraction with amidase activity using trans-4-cyanocyclohexanecarboxylic acid amide as a substrate was collected.

After adding ammonium sulfate to the active fraction to precipitate proteins, 10 mM potassium phosphate buffer (pH 7,
5) and dialysis was performed. After the dialysis was completed, it was passed through a hydroxyapatite (5×20C11) column. The enzyme was not adsorbed and was washed out with 10mM potassium potassium phosphate buffer. The active fractions were collected, 30% saturated ammonium sulfate was added, and the precipitate was removed by centrifugation. The supernatant is Ph
Chromatography was performed on an enyl-5epharose CL-4B column (1,5×2 Bcm, packed with 10 mM buffer containing 30% saturated ammonium sulfate). Elution was performed by gradient of ammonium sulfate concentration from 30% to 0% in 10mM phosphate buffer. The active fractions were collected and precipitated with 60% saturated ammonium sulfate. The precipitate was 0.2M NaC.
Dissolved in 10mM potassium phosphate buffer containing i,
Dialysis was performed. Dialysis finished fluid is Ce1lu1ofine
It was separated by GC-700m column (2×83 cm) and 45% saturated ammonium sulfate was added to the active fraction.

The resulting precipitate was removed by centrifugation, the supernatant was precipitated by adding 55% saturated ammonium sulfate, the precipitate was dissolved in 10 mM phosphate buffer containing 20% glycerol, and dialyzed. After dialysis, D E A
E-5ephaccl column (1,75X21.5c
Chromatographic separation was performed by m). Elution was performed by increasing the NaCi concentration in the buffer from O to 0.3M. The active fraction is again Phenyl-3epharo
se CL 4 B column, active fractions were collected, precipitated with ammonium sulfate, and then dialyzed. After dialysis, DEA E-5epha
Fractionation using a cel column was performed twice, and the product could be purified to a specific activity approximately 220 times that of the cell-free extract.

Example 5 After dissolving nitrile hydratase 2■ prepared in Example 3 and amidase 15■ prepared in Example 4 in LO ml of 0.1M potassium phosphate buffer (pH 7,5), cis, trans-1,4 2 g of -dicyanocyclohexane (cis:trans=42:58) was added, and the reaction was carried out at 30°C for 8 hours.

High performance liquid chromatography analysis of the reaction completed solution revealed that trans-4-cyanocyclohexanecarboxylic acid was 1.28%.
g, cis-4-cyanocyclohexanecarboxylic acid 0.0
6g was produced.

[Brief explanation of drawings]

Figure 1 is a graph showing the results of determining the optimum pH of nitrile hydratase, Figure 2 is a graph showing the results of determining the optimum pH of amidase, and Figure 3 is the result of determining the optimum temperature of amidase. This is a graph showing. Figure 1: pH of reaction solution: 0-m-0; Potassium phosphorus: 1-1-0; Tris-HCl buffer: 1-11: Glycine-NaOH buffer; Figure 2: pH e-+; Quen Acid-NcLOH buffer Δ-1Δ
Tris-maleic acid buffer -m-;
MOPS buffer - 1; Tris-ML acid buffer 0-10; glycine-NaOH buffer Figure 3 (°C)

Claims (3)

[Claims] (1) Corynebacterium
Production of trans-4-cyanocyclohexanecarboxylic acid, characterized in that trans-4-cyanocyclohexanecarboxylic acid is obtained from cis,trans-1,4-dicyanocyclohexane by the action of a microorganism belonging to the genus Ium) or a preparation thereof. Method. (2) A nitrile hydratase prepared from Corynebacterium bacteria and having the following physicochemical properties. [1] Enzyme action Acts on the nitrile group of a nitrile compound and acts as a catalyst for the reaction that hydrates and converts the nitrile group into an amide group. [2] Substrate specificity Saturated aliphatic nitriles such as n-butyronitrile and n-valeronitrile, unsaturated aliphatic nitriles such as acronitrile and methacronitrile, aromatic nitriles such as benzonitrile, and complex compounds such as 3-cyanopyridine Acts on dinitrile compounds such as cyclic nitrile, malononitrile, trans-1,4-dicyanocyclohexane. In particular, the hydration rate of n-valeronitrile and iso-valeronitrile is high. Furthermore, in 1,4-dicyanocyclohexane, the hydration rate of the trans isomer is significantly higher than that of the cis isomer. [3] Molecular weight The molecular weight is 61,400 (liquid chromatography method), and the molecular weight of the subunit is 26,900. (SDS-PAGE) [4] Optimal pH The hydration effect of the nitrile group is optimal at pH 8.0 to 8.5. [5] Temperature stability Hydration activity does not decrease even when treated at pH 7.5 at 10 to 40°C for 10 minutes. [6] Stabilized by adding the stabilizer isovaleric acid. [7] Inhibited by inhibitors such as Hg^+^+ and phenylhydrazine. [8] Contains PQQ (pyrroloquinoline quinone) in the molecule. (3) An amidase prepared from Corynebacterium bacteria and having the following physicochemical properties. [1] Enzyme action Acts on the amide group of an amide compound and shows a function as a catalyst for the reaction of hydrolyzing the amide group into a carboxyl group and ammonia. [2] Substrate specificity Saturated aliphatic amides such as propionamide and n-valeramide, cyclic saturated aliphatic amides such as trans-4-cyanocyclohexanecarboxylic acid amide, unsaturated aliphatic amides such as methacrylamide, aromatics such as benzamide, etc. amide, and heterocyclic amide compounds such as nicotinamide. Further, in particular, the hydrolysis rate of trans-4-cyanocyclohexanecarboxylic acid amide is high, whereas the activity of cis-4-cyanocyclohexanecarboxylic acid amide is low. [3] Optimal pH The hydrolysis effect of the amide group is optimal at pH 6.5 to 10.0. [4] Optimal temperature The optimal temperature is about 50°C. [5] Inhibited by inhibitors such as Hg^+^+ and ¥p¥-CMB.