JP2023141245A - Method for producing 3-hydroxycarboxylic acid compound or salt thereof - Google Patents

Abstract

To provide a method for efficiently producing a 3-hydroxycarboxylic acid compound through the hydrolysis of a 3-hydroxyamide compound or a salt thereof.SOLUTION: A method for producing a 3-hydroxycarboxylic acid compound or a salt thereof includes reacting a 3-hydroxyamide compound with, in an aqueous medium, at least one selected from the group consisting of bacterial cells and their processed products containing amidase derived from bacteria belonging to Pseudonocardia or Acidophillum.SELECTED DRAWING: None

Description

The present disclosure relates to a method for producing a 3-hydroxycarboxylic acid compound or a salt thereof obtained by hydrolyzing a 3-hydroxyamide compound.

3-Hydroxycarboxylic acid compounds are useful compounds as raw materials for so-called fine chemicals such as medicines and agricultural chemicals. Furthermore, 4-halo-3-hydroxybutanoic acid can be used as a raw material for carnitine, which is useful as a substance related to fatty acid metabolism.
As one of the main methods for producing 3-hydroxycarboxylic acid compounds, a hydrolysis method using a 3-hydroxynitrile compound as a raw material is often used. Various methods are known. Alternatively, in recent years, a method has been known in which a 3-hydroxynitrile compound is hydrolyzed using a nitrilase-containing microbial cell and/or a treated product of the microbial cell as a catalyst.

In recent years, methods using catalysts derived from microorganisms have been attracting industrial attention because of their high conversion and selectivity of 3-hydroxyamide compounds.
As a method for hydrolyzing 3-hydroxynitrile to 3-hydroxycarboxylic acid, for example, Patent Document 1 discloses that an enzyme catalyst having nitrile hydratase activity and amidase activity is used to first convert aliphatic nitrile into amide by nitrile hydratase. A method of converting the amide into the corresponding carboxylic acid by amidase was proposed, and useful microorganisms include Acidovorax sp., Comamonas sp., Dietzia sp., Scytalidium sp. ), Rhodococcus, and Pseudomonas are disclosed.
As an enzyme having the activity of hydrolyzing carnitinamide to produce carnitine, for example, Patent Document 2 discloses an amidase belonging to the genus Pseudomonas.
As a method for producing 4-halo-3-hydroxybutyric acid, for example, Patent Document 3 describes a method in which a hydrolase acts on 4-halo-3-hydroxybutyronitrile to hydrate it to 4-halo-3-hydroxybutyronitrile. A method has been proposed for degrading microorganisms belonging to the genera Arthrobacter, Caseobacter, Nocardia, Psuedonocardia, and Rhodococcus as useful microorganisms. A nitrile hydrolase is disclosed.

Special Publication No. 2004-535754 Japanese Unexamined Patent Publication No. 63-56294 Japanese Patent Application Publication No. 4-360689

Patent Documents 1 to 3 mentioned above disclose a method for obtaining a 3-hydroxycarboxylic acid compound or a salt thereof using an enzyme derived from a microorganism. A method for producing a 3-hydroxycarboxylic acid compound or a salt thereof from a 3-hydroxyamide compound using an amidase derived from at least one bacterium selected from the genus Acidophilium and genus Acidophilium is not disclosed.
In addition, the method of producing 3-hydroxycarboxylic acid compounds by hydrolyzing 3-hydroxynitrile compounds using an alkali catalyst or metal catalyst can be used to treat waste generated during the reaction process and to remove generated by-products. There are concerns that this will be difficult.
Under these circumstances, 3-hydroxycarboxylic acid compounds can be more efficiently converted into 3-hydroxycarboxylic acid compounds in the hydrolysis reaction of 3-hydroxyamide compounds using bacterial cells containing amidase and/or a treated product of the bacterial cells as catalysts derived from microorganisms. or the production of its salt.

An object of an embodiment of the present disclosure is to provide a method for efficiently producing a 3-hydroxycarboxylic acid compound or a salt thereof obtained by hydrolyzing a 3-hydroxyamide compound.

The present inventor conducted extensive research in order to solve the above problems. As a result, the 3-hydroxycarboxylic acid compound or its salt can be efficiently processed by allowing the 3-hydroxyamide compound to interact with amidase derived from at least one of the bacteria selected from the genus Pseudonocardia and the genus Acidophilium. They have discovered that it can be produced in a straightforward manner, leading to the present invention.

That is, the present disclosure includes the following aspects.
<1> At least one species selected from the group consisting of bacterial cells and bacterial cell-treated products containing amidase derived from at least one bacteria selected from the genus Pseudonocardia and the genus Acidophilium, and 3-hydroxyamide. A method for producing a 3-hydroxycarboxylic acid compound or a salt thereof, the method comprising reacting the compound with the compound in an aqueous medium.
<2> The 3-hydroxyamide compound is reacted with at least one member selected from the group consisting of nitrile hydratase-containing microbial cells and bacterial cell-treated products, and the 3-hydroxynitrile compound in an aqueous medium. The method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to <1>, further comprising obtaining the 3-hydroxycarboxylic acid compound or a salt thereof.
<3> The 3-hydroxycarboxylic acid compound or its salt according to <2>, which is a method for producing a 3-hydroxycarboxylic acid compound or a salt thereof from a 3-hydroxynitrile compound in the presence of the nitrile hydratase and the amidase. Method of manufacturing salt.
<4> The method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to <2> or <3>, wherein the nitrile hydratase is a nitrile hydratase derived from a bacterium of the genus Pseudonocardia.

According to one embodiment of the present disclosure, it is possible to provide a method for efficiently producing a 3-hydroxycarboxylic acid compound or a salt thereof obtained by hydrolyzing a 3-hydroxyamide compound.

Below, the content of the present disclosure will be explained in detail. Although the description of the constituent elements described below may be made based on typical embodiments of the present disclosure, the present disclosure is not limited to such embodiments.
The explanation of a nucleotide sequence in the present disclosure may be made by focusing on the sequence on one strand even when the nucleic acid strand holding the nucleotide sequence forms a double strand. For the other strand of the duplex, the description of such sequences should translate to its complementary sequence.
In this specification, "~" indicating a numerical range is used to include the numerical values written before and after it as the lower limit and upper limit.
In the numerical ranges described step by step in this disclosure, the upper limit or lower limit described in one numerical range may be replaced with the upper limit or lower limit of another numerical range described step by step. . Furthermore, in the numerical ranges described in this disclosure, the upper limit or lower limit of the numerical range may be replaced with the values shown in the Examples.
Furthermore, in the description of groups (atomic groups) in this specification, descriptions that do not indicate substituted or unsubstituted include those having no substituent as well as those having a substituent. For example, the term "alkyl group" includes not only an alkyl group having no substituent (unsubstituted alkyl group) but also an alkyl group having a substituent (substituted alkyl group).
In the present disclosure, when there are multiple substances corresponding to each component in the composition, unless otherwise specified, the amount of each component in the composition means the total amount of the multiple substances present in the composition. do.
Furthermore, the term "process" in this specification refers not only to an independent process, but also to the term "process" when the intended purpose of the process is achieved, even if the process cannot be clearly distinguished from other processes. included. Furthermore, in the present disclosure, "mass%", "weight%", and "Wt%" have the same meaning, and "mass parts" and "weight parts" have the same meaning.
Furthermore, in the present disclosure, a combination of two or more preferred embodiments is a more preferred embodiment.

(Method for producing 3-hydroxycarboxylic acid compound or its salt)
The method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the first embodiment of the present disclosure provides a method for producing a 3-hydroxycarboxylic acid compound or a salt thereof using a bacterial cell containing an amidase derived from at least one bacterium selected from the genus Pseudonocardia and the genus Acidophilium. and a 3-hydroxyamide compound in an aqueous medium.
The method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the second embodiment of the present disclosure includes at least one species selected from the group consisting of nitrile hydratase-containing microbial cells and bacterial cell-treated products, and 3-hydroxynitrile. a 3-hydroxyamide compound obtained by reacting the compound in an aqueous medium, and a bacterial cell containing an amidase derived from at least one bacterium selected from the above-mentioned Pseudonocardia genus and Acidophilium genus; This is a method for producing a 3-hydroxycarboxylic acid compound or a salt thereof by reacting the 3-hydroxycarboxylic acid compound or a salt thereof with at least one member selected from the group consisting of treated bacterial cells in an aqueous medium.

[First embodiment]
The method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure provides bacterial cells and treated bacterial cells containing amidase derived from at least one bacterium selected from the genus Pseudonocardia and the genus Acidophilium. The method includes causing at least one member selected from the group consisting of the following to react with a 3-hydroxyamide compound in an aqueous medium.
The method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure provides bacterial cells and treated bacterial cells containing amidase derived from at least one bacterium selected from the genus Pseudonocardia and the genus Acidophilium. A 3-hydroxycarboxylic acid compound or a salt thereof can be efficiently produced by reacting a 3-hydroxyamide compound with at least one selected from the group consisting of:

The method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure provides bacterial cells and treated bacterial cells containing amidase derived from at least one bacterium selected from the genus Pseudonocardia and the genus Acidophilium. The method may further include a step other than reacting the 3-hydroxyamide compound with at least one member selected from the group consisting of the following in an aqueous medium.
The details of the method for producing the 3-hydroxycarboxylic acid compound or salt thereof of the present disclosure will be described below.

<Bacterial cells and processed bacterial cells containing amidase derived from specific bacteria>
In the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure, an amidase derived from at least one bacterium selected from the genus Pseudonocardia and the genus Acidophilium (hereinafter referred to as "an amidase derived from a specific bacterium") is used. 3-hydroxycarboxylic acid compound of 3-hydroxyamide compound or its salt. Used as a hydrolysis catalyst.
Examples of amidases derived from bacteria of the genus Pseudonocardia include amidases derived from Pseudonocardia thermophila JCM3095 and the like.
Furthermore, examples of amidases derived from bacteria of the genus Acidophilium include those derived from Acidphilium cryptum and the like.
Amidases derived from specific bacteria may be used alone or in combination of two or more.
Furthermore, in the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure, an amidase derived from a specific bacterium and an amidase not derived from a specific bacterium may be used in combination; It is preferable that the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof does not use an amidase that is not derived from a specific bacterium.

Among amidases derived from specific bacteria, it is easy to express amidases in hosts that grow at room temperature, such as Escherichia coli, so the amidase must be derived from organisms that do not belong to hyperthermophiles or hyperthermophiles. is more preferred, especially amidase derived from Pseudonocardia thermophila JCM3095.

[Mutant amidase]
As the amidase derived from a specific bacterium, for example, an amidase derived from Pseudonocardia thermophila JCM3095 or an amidase derived from Acidphilium cryptum in which part of the amino acid sequence is substituted with another amino acid can also be used.

As the amidase in one embodiment of the present disclosure, a transformant that has been transformed to produce amidase can also be used.
The method for obtaining this transformant is to clone the amidase-encoding gene registered in the gene database GenBank into an expression vector, thereby obtaining the amidase-encoding DNA and the DNA that hybridizes to this DNA. can.

There are no particular restrictions on the vector used for cloning, and vectors constructed for genetic recombination from phages or plasmids that can autonomously propagate within a host microorganism are suitable.

Examples of phage include Lambda gt10, Lambda gt11, etc. when Escherichia coli is used as a host bacterium.
In addition, when using Escherichia coli as a host bacterium, examples of plasmids include pBTrp2, pBTac1, pBTac2 (all manufactured by Boehringer Mannheim), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX- 1 (manufactured by PROMEGA), PQE -8 (manufactured by Qiagen), PQE -30 (Qiagen), PBLUESCRIPTII SK +, PBLUESCRIPTII SK ( -) (manufactured by STRATAGENE), PET -3 (NOVAGEN) Made by company), PUC18 ( (manufactured by Takara Bio Co., Ltd.), pSTV28 (manufactured by Takara Bio Co., Ltd.), pSTV29 (manufactured by Takara Bio Co., Ltd.), pUC118 (manufactured by Takara Bio Co., Ltd.), and the like.
Note that when an enzyme derived from a fungus different from the host is introduced and expressed by genetic recombination or the like, it is more preferable to modify the codon usage to suit the host.

The promoter is not particularly limited as long as it can express amidase in the host cell. Examples include promoters derived from Eschenchia coli, phage, etc., such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PH promoter, and PSE promoter. Furthermore, artificially designed and modified promoters such as the tac promoter and lacT7 promoter can also be used. Furthermore, Np promoter (Japanese Patent Publication No. 8-24586) can also be used for expression in Bacillus bacteria.

The ribosome binding sequence is not particularly limited as long as it can express amidase in the host cell; ) is preferably used.

In order to efficiently perform transcription and translation, we express a protein that is a fusion of a protein with the N-terminus or a portion thereof deleted of the protein having the protein activity and the N-terminal portion of the protein encoded by the expression vector. It's okay.

The medium for culturing the transformant is not particularly limited as long as it is a medium in which the transformant can proliferate. As the medium, a liquid medium containing a carbon source, a nitrogen source, other nutrients, etc. is usually used.
Examples of carbon sources for the medium include sugars such as glucose, sucrose, and starch, alcohols such as sorbitol, methanol, ethanol, and glycerol, organic acids and their salts such as fumaric acid, citric acid, and acetic acid, and carbonized paraffin. Examples include hydrogens and mixtures thereof.
Nitrogen sources for the medium include, for example, ammonium salts of inorganic acids such as ammonium sulfate and ammonium nitrate, ammonium salts of organic acids such as ammonium fumarate, organic or inorganic nitrogen-containing compounds such as meat extract, yeast extract, urea, and amino acids; , mixtures thereof, and the like.
In addition, nutrients used in normal culture, such as inorganic salts such as magnesium chloride and ferric chloride, trace metal salts, and vitamins, may be added to the medium as appropriate. Furthermore, if necessary, factors that promote the growth of transformants, buffer substances that are effective in maintaining the pH of the medium, and the like may be added to the medium.

The transformant can be cultured under conditions suitable for growth, for example, at a medium pH of 2 to pH 12, preferably pH 4 to pH 10, and a temperature of 5°C to 50°C, more preferably 20°C to 50°C.
The transformant can be cultured under either anaerobic or aerobic conditions, depending on the type of transformant, but it is more preferable to culture the transformant under aerobic conditions.

The culture time of the transformant is, for example, about 1 hour to 240 hours, preferably about 5 hours to 120 hours, and more preferably about 12 hours to 72 hours.

The cultured transformant may be used for the reaction as it is, or may be used for the reaction after being subjected to various treatments. Examples of this treatment include crushing the transformant, freeze-drying, and extraction.
As the above-mentioned extraction treatment, conventional methods such as an ultrasonic method, a freeze-thaw method, a lysozyme method, etc. can be used. By performing such treatment, amidase can be extracted from the transformant. The obtained amidase may be used unpurified or after being purified.
To purify amidase, for example, a transformant separated from a culture medium by centrifugation or the like is washed with water or the like, suspended in a buffer solution whose pH is in the stable range of the enzyme, and incubated at a low temperature (3°C to 18°C). The transformant is disrupted by treatment with a French press or ultrasound at a temperature of 3°C to 10°C (preferably 3°C to 10°C). Then, fragments of the transformant are separated and removed by centrifugation or the like, and the resulting extract of the transformant is fractionated with ammonium sulfate in a conventional manner and dialyzed to obtain a crude extract of amidase. Highly pure amidase can be obtained by subjecting this crude extract of amidase to column chromatography using, for example, Sephadex G-200.
Furthermore, a transformant in which the permeability of the cell membrane is changed by using a surfactant or an organic solvent such as toluene can also be used.
Alternatively, the transformant or amidase may be immobilized and used by a conventional method such as a polyacrylamide gel method.

<3-hydroxyamide compound>
Examples of the 3-hydroxyamide compound used in the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure include the below-mentioned 3-hydroxycarboxylic acid compound or a salt thereof obtained by the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure. Examples include 3-hydroxyamide compounds corresponding to 3-hydroxycarboxylic acid compounds. Specifically, the 3-hydroxyamide compound is a 3-hydroxyamide compound in which the amide group in the 3-hydroxyamide compound is formed into a carboxy group through a hydrolysis reaction using amidases derived from Pseudonocardia and Acidophilium. There are no particular limitations as long as it is an amide compound.

The 3-hydroxyamide compound may have a group (ie, a substituent) other than the amide group and the hydroxy group.
Examples of the above-mentioned substituents include halogen atoms, hydroxy groups, amino groups, alkylthio groups, and alkoxy groups.
Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

The 3-hydroxyamide compound is preferably a 3-hydroxyamide compound having 3 to 20 carbon atoms, more preferably a 3-hydroxyamide compound having 3 to 15 carbon atoms, and still more preferably a 3-hydroxyamide compound having 3 to 10 carbon atoms. -Hydroxyamide compounds.
When counting the number of carbon atoms in a 3-hydroxyamide compound, the amide group is also counted as the number of carbon atoms; for example, the number of carbon atoms in 3-hydroxypropionamide ((HO)CH ₂ CH ₂ CONH ₂ ) is 3.
From the viewpoint of efficiently producing a 3-hydroxycarboxylic acid compound or a salt thereof, the 3-hydroxyamide compound is preferably a substituted or unsubstituted saturated aliphatic 3-hydroxyamide compound, and has 3 to 20 carbon atoms. More preferably, it is a substituted or unsubstituted saturated aliphatic 3-hydroxyamide compound.

Specifically, the 3-hydroxyamide compound includes, for example, 3-hydroxypropionamide,
3-hydroxybutyramide, 3-hydroxy-3-methylbutyramide,
3-Hydroxypentanamide, 3-hydroxy-3-methylpentanamide, 3-ethyl-3-hydroxypentanamide, 3-hydroxy-3,4-dimethylpentanamide, 2-propyl-3-hydroxypentanamide, 3- Hydroxy-3,4,4-trimethylpentanamide, 3-ethyl-3-hydroxy-4-methylpentanamide, 3-hydroxy-3-isopropyl-4-methylpentanamide, 3-ethyl-3-hydroxy-4, 4-dimethylpentanamide, 3-hydroxy-4,4-dimethyl-3-isopropylpentanamide, 3-tert-butyl-3-hydroxy-4,4-dimethylpentanamide,
3-hydroxyhexanamide, 3-hydroxy-3-methylhexanamide, 3-hydroxy-3,4-dimethylhexanamide, 3-hydroxy-3,5-dimethylhexanamide, 3-ethyl-3-hydroxyhexanamide, 3-Hydroxy-3-propylhexanamide, 3-hydroxy-3-isopropylhexanamide, 3-ethyl-3-hydroxy-4-methylhexanamide, 3-ethyl-3-hydroxy-5-methylhexanamide, 3- tert-butyl-3-hydroxyhexanamide, 3-hydroxy-4-methyl-3-propylhexanamide, 3-hydroxy-5-methyl-3-propylhexanamide, 3-hydroxy-4-methyl-3-isopropylhexane Amide, 3-hydroxy-5-methyl-3-isopropylhexanamide, 3-isobutyl-3-hydroxy-4-methylhexanamide, 3-isobutyl-3-hydroxy-5-methylhexanamide, 3-sec-butyl- 3-hydroxy-4-methylhexanamide, 3-tert-butyl-3-hydroxy-4-methylhexanamide,
3-Hydroxyheptanamide, 3-hydroxy-3-methylheptanamide, 3-ethyl-3-hydroxyheptanamide, 3-hydroxy-3-propylheptanamide, 3-hydroxy-3-isopropylheptanamide, 3-butyl- 3-hydroxyheptanamide, 3-isobutyl-3-hydroxyheptanamide, 3-sec-butyl-3-hydroxyheptanamide, 3-tert-butyl-3-hydroxyheptanamide,
3-hydroxyoctanamide,
unsubstituted saturated aliphatic 3-hydroxyamide compounds such as 3-hydroxynonanamide;
4-fluoro-3-hydroxybutyramide, 4-chloro-3-hydroxybutyramide, 4-bromo-3-hydroxybutyramide, 4-iodo-3-hydroxybutyramide, 4-chloro-3-hydroxy-3- saturated aliphatic 3-hydroxyamide compounds substituted with halogen atoms such as methylbutyramide and 4-chloro-3-hydroxy-5-methylhexanamide;
3,4-dihydroxybutyramide, 3,4-dihydroxy-2-methylbutyramide, 3,4-dihydroxy-3-methylbutyramide,
3,5-dihydroxypentanamide, 3,5-dihydroxy-3-methylpentanamide,
Saturated aliphatic 3-hydroxyamide compounds substituted with hydroxy groups such as 3,5-dihydroxyhexanamide, 3,5-dihydroxyheptanamide, 3,5-dihydroxyoctanamide, 3,5-dihydroxydecanamide,
3-hydroxy-4-methoxybutyramide, 3-hydroxy-4-ethoxybutyramide, 3-hydroxy-4-propyloxybutyramide 3-hydroxy-5-methoxypentanamide, 3-hydroxy-5-methoxy-3- saturated aliphatic 3-hydroxyamide compounds substituted with alkoxy groups such as methylpentanamide, 3-hydroxy-5-ethoxypentanamide and 3-hydroxy-5-methoxyhexanamide;
Examples include saturated aliphatic 3-hydroxyamide compounds substituted with alkylthio groups such as 3-hydroxy-2-methyl-2-methylthiopropionamide and 3-hydroxy-4-methylthiobutylamide.

<3-hydroxycarboxylic acid compound>
The 3-hydroxycarboxylic acid compound is not particularly limited as long as it can be obtained from the above-mentioned 3-hydroxyamide compound.
Groups other than the carboxy group and hydroxy group (ie, substituents) possessed by the 3-hydroxycarboxylic acid compound have the same meanings as the substituents possessed by the above-mentioned 3-hydroxyamide compound, and preferred embodiments thereof are also the same.
The 3-hydroxycarboxylic acid compound preferably has 3 to 20 carbon atoms, more preferably 3 to 15 carbon atoms, and even more preferably 3 to 10 carbon atoms.
The 3-hydroxycarboxylic acid compound is preferably a substituted or unsubstituted saturated aliphatic 3-hydroxycarboxylic acid compound, and a substituted or unsubstituted saturated aliphatic 3-hydroxycarboxylic acid compound having 3 to 20 carbon atoms. It is more preferable that there be.

Specifically, the 3-hydroxycarboxylic acid compound includes, for example, 3-hydroxypropionic acid,
3-hydroxybutanoic acid, 3-hydroxy-3-methylbutanoic acid,
3-Hydroxypentanoic acid, 3-hydroxy-3-methylpentanoic acid, 3-ethyl-3-hydroxypentanoic acid, 3-hydroxy-3,4-dimethylpentanoic acid, 2-propyl-3-hydroxypentanoic acid, 3- Hydroxy-3,4,4-trimethylpentanoic acid, 3-ethyl-3-hydroxy-4-methylpentanoic acid, 3-hydroxy-3-isopropyl-4-methylpentanoic acid, 3-ethyl-3-hydroxy-4, 4-dimethylpentanoic acid, 3-hydroxy-4,4-dimethyl-3-isopropylpentanoic acid, 3-tert-butyl-3-hydroxy-4,4-dimethylpentanoic acid,
3-hydroxyhexanoic acid, 3-hydroxy-3-methylhexanoic acid, 3-hydroxy-3,4-dimethylhexanoic acid, 3-hydroxy-3,5-dimethylhexanoic acid, 3-ethyl-3-hydroxyhexanoic acid, 3-Hydroxy-3-propylhexanoic acid, 3-hydroxy-3-isopropylhexanoic acid, 3-ethyl-3-hydroxy-4-methylhexanoic acid, 3-ethyl-3-hydroxy-5-methylhexanoic acid, 3- tert-butyl-3-hydroxyhexanoic acid, 3-hydroxy-4-methyl-3-propylhexanoic acid, 3-hydroxy-5-methyl-3-propylhexanoic acid, 3-hydroxy-4-methyl-3-isopropylhexane Acid, 3-hydroxy-5-methyl-3-isopropylhexanoic acid, 3-isobutyl-3-hydroxy-4-methylhexanoic acid, 3-isobutyl-3-hydroxy-5-methylhexanoic acid, 3-sec-butyl- 3-hydroxy-4-methylhexanoic acid, 3-tert-butyl-3-hydroxy-4-methylhexanoic acid,
3-hydroxyheptanoic acid, 3-hydroxy-3-methylheptanoic acid, 3-ethyl-3-hydroxyheptanoic acid, 3-hydroxy-3-propylheptanoic acid, 3-hydroxy-3-isopropylheptanoic acid, 3-butyl- 3-hydroxyheptanoic acid, 3-isobutyl-3-hydroxyheptanoic acid, 3-sec-butyl-3-hydroxyheptanoic acid, 3-tert-butyl-3-hydroxyheptanoic acid,
3-hydroxyoctanoic acid,
unsubstituted saturated aliphatic 3-hydroxycarboxylic acid compounds such as 3-hydroxynonanoic acid,
4-fluoro-3-hydroxybutanoic acid, 4-chloro-3-hydroxybutanoic acid, 4-bromo-3-hydroxybutanoic acid, 4-iodo-3-hydroxybutanoic acid, 4-chloro-3-hydroxy-3- saturated aliphatic 3-hydroxycarboxylic acid compounds substituted with halogen atoms such as methylbutanoic acid and 4-chloro-3-hydroxy-5-methylhexanoic acid;
3,4-dihydroxybutanoic acid, 3,4-dihydroxy-2-methylbutanoic acid, 3,4-dihydroxy-3-methylbutanoic acid,
3,5-dihydroxypentanoic acid, 3,5-dihydroxy-3-methylpentanoic acid,
saturated aliphatic 3-hydroxycarboxylic acid compounds substituted with hydroxy groups such as 3,5-dihydroxyhexanoic acid, 3,5-dihydroxyheptanoic acid, 3,5-dihydroxyoctanoic acid, and 3,5-dihydroxydecanoic acid;
3-hydroxy-4-methoxybutanoic acid, 3-hydroxy-4-ethoxybutanoic acid, 3-hydroxy-4-propyloxybutanoic acid,
Substituted with alkoxy groups such as 3-hydroxy-5-methoxypentanoic acid, 3-hydroxy-5-methoxy-3-methylpentanoic acid, 3-hydroxy-5-ethoxypentanoic acid, 3-hydroxy-5-methoxyhexanoic acid saturated aliphatic 3-hydroxycarboxylic acid compound,
Examples include saturated aliphatic 3-hydroxycarboxylic acid compounds substituted with alkylthio groups such as 3-hydroxy-2-methyl-2-methylthiopropionic acid and 3-hydroxy-4-methylthiobutanoic acid.

Although known synthesis methods may be used to synthesize the 3-hydroxyamide compound, from the viewpoint of efficiently obtaining the desired product, the 3-hydroxyamide compound is synthesized from the 3-hydroxynitrile compound using a microbial catalyst. Preferably, it is a method of synthesizing a 3-hydroxyamide compound from a 3-hydroxynitrile compound using at least one selected from the group consisting of a bacterial cell containing nitrile hydratase and a treated bacterial cell. It is more preferable that there be.

From the viewpoint of efficiently obtaining the desired product, the 3-hydroxyamide compound used in the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure is prepared from a bacterial cell containing nitrile hydratase and a bacterial cell-treated product. It is preferable that the compound be obtained by reacting at least one member selected from the group consisting of a 3-hydroxynitrile compound and a 3-hydroxynitrile compound in an aqueous medium.
The details of the bacterial cells and the bacterial cell-treated product containing nitrile hydratase used for the synthesis of the 3-hydroxyamide compound will be described later.

<Water (raw water)>
The raw water used in the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure is not particularly limited, and purified water such as pure water, distilled water, ion-exchanged water, etc. can be used.
In this specification, "pure water" means water whose electrical conductivity is 100 μS/m or less.

<Aqueous medium>
The aqueous medium used in the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure is not particularly limited, and usually water is used, but a mixture of water and a water-soluble organic solvent can also be used. . The water used as the aqueous medium is not particularly limited, and purified water such as pure water, distilled water, ion exchange water, etc. can be used. A part of the water used as the aqueous medium can also be used as raw material water in the reaction.
Examples of water-soluble organic solvents that can be mixed with water include alcoholic solvents such as methanol, ethanol, 1-propanol, 2-propanol, 2-methoxyethanol, 2-ethoxyethanol, ethylene glycol, and propylene glycol;
Ether solvents such as dimethyl ether, diethyl ether, diisopropyl ether, dioxane, ethylene glycol dimethyl ether,
Ketone solvents such as acetone, methyl ethyl ketone, methyl isobutyl ketone,
Examples include aprotic polar solvents such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, 1-methyl-2-pyrrolidinone, and 1,3-dimethyl-2-imidazolidinone.
When a mixture of water and a water-soluble organic solvent is used, the mixing ratio of water and a water-soluble organic solvent is determined by the amidase catalyst (i.e., a bacterial cell containing amidase derived from a specific bacterium and/or a processed product of the bacterial cell). Although there is no particular limitation as long as the amidase activity of can be maintained satisfactorily, the ratio of water to water-soluble organic solvent is preferably 100:100 to 100:1 by volume, more preferably 100:50 to 100. :1, more preferably 100:30 to 100:1.

<pH adjuster>
In the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure, a pH adjuster can also be used during the hydrolysis reaction of the 3-hydroxyamide compound. The pH adjuster is used to adjust the pH of the reaction mixture to a suitable range for maintaining good activity of the amidase catalyst.
In the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure, a pH adjuster may be used as necessary.
The pH adjuster adjusts the pH of the mixture of the amidase-containing bacterial cells and/or their processed product (amidase catalyst) and the 3-hydroxyamide compound to a suitable range for maintaining the activity of the amidase catalyst. Can be adjusted.

When the pH suitable for the hydrolysis reaction of the 3-hydroxyamide compound is lower than 7, an acid can be used as the pH adjuster.
As the acid used as the pH adjuster, either an inorganic acid or an organic acid can be used. Examples of inorganic acids include hydrohalic acids such as hydrogen chloride, hydrogen bromide, and hydrogen iodide, hypochlorous acid, chlorous acid, chloric acid, perchloric acid, hypobromous acid, bromous acid, and bromine. Examples include halogenated oxoacids such as perbromic acid, hypoiodic acid, iodic acid, iodic acid, and periodic acid, sulfuric acid, nitric acid, phosphoric acid, and boric acid. Examples of organic acids include carboxylic acids such as formic acid, acetic acid, propionic acid, acrylic acid, methacrylic acid, crotonic acid, oxalic acid, malonic acid, fumaric acid, maleic acid, citric acid, lactic acid, benzoic acid, methanesulfonic acid, Examples include sulfonic acids such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
The acid used as a pH adjuster can be used in any state of gas, solid, or liquid, but in consideration of ease of supply to the reaction tank, it is preferable to use one in liquid state. Moreover, it is more preferable to use the acid in a gaseous or solid state as an aqueous solution.
Considering the controllability of pH adjustment in the reaction tank, it is more preferable to use the acid in a liquid state as an aqueous solution. When the acid is used as an aqueous solution, the concentration of the acid is not particularly limited, but from the viewpoint of easy pH control, it is preferably 0.1 wt% or more and 99 wt% or less, more preferably 1 wt% or more and 90 wt% or less, and even more preferably is 1 wt% or more and 50 wt% or less.

When the pH suitable for the hydrolysis reaction of the 3-hydroxyamide compound is higher than 7, a base can be used as a pH adjuster.
As the base used as a pH adjuster, either an inorganic base or an organic base can be used. Examples of inorganic bases include alkali metal hydroxides such as lithium hydroxide, sodium hydroxide, and potassium hydroxide, alkaline earth metal hydroxides such as magnesium hydroxide and calcium hydroxide, lithium carbonate, and sodium carbonate. , alkali metal carbonates such as potassium carbonate, alkali metal hydrogen carbonates such as lithium hydrogen carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, and ammonia. Examples of the organic base include trimethylamine, triethylamine, aniline, and pyridine.
The base used as a pH adjuster can be used in any state of gas, solid, or liquid, but in consideration of ease of supply to the reaction tank, it is preferable to use a base in liquid state. The base in gas or solid state is preferably used as an aqueous solution. Considering the controllability of pH adjustment in the reaction tank, it is preferable to use the base in a liquid state as an aqueous solution. The concentration of the base when used as an aqueous solution is not particularly limited, but from the viewpoint of easy pH control, it is preferably 0.1 wt% or more and 99 wt% or less, more preferably 1 wt% or more and 90 wt% or less, and even more preferably 1 wt%. The content is 50 wt% or less.

A method of reacting a 3-hydroxyamide compound with at least one member selected from the group consisting of bacterial cells and bacterial cell-treated products (amidase catalysts) containing amidase derived from specific bacteria (hydrolysis). There are no particular restrictions on the reaction method (reaction method), but examples include methods (1) to (3).
(1) A method in which the amidase catalyst, reaction raw materials (3-hydroxyamide compound and raw water), aqueous medium, and pH adjuster are charged into a reaction tank in their entirety at once and then the reaction is carried out (batch reaction).
(2) After charging the amidase catalyst, reaction raw materials (3-hydroxyamide compound and raw water), aqueous medium, and part of the pH adjuster into the reaction tank, the remaining amidase catalyst, reaction raw materials ( 3-Hydroxyamide compound and raw material water), an aqueous medium, and a pH adjuster are supplied to carry out the reaction (semi-batch reaction).
(3) Continuous or intermittent supply of amidase catalyst, reaction raw materials (3-hydroxyamide compound and raw water), aqueous medium and pH adjuster, and reaction solution (amidase catalyst, unreacted raw materials, aqueous medium, pH adjustment) A method in which the reaction is carried out continuously without taking out the entire amount of the reaction liquid in the reaction tank (continuous reaction).

In the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure, the amidase catalyst is preferably used in a suspended bed; for example, a suspension of the amidase catalyst is prepared, and the suspension is reacted. Just supply it to the tank.

The reaction tank temperature, which is the temperature of the liquid in the reaction tank, is usually set at 0°C to 50°C, preferably 10°C to 40°C, although it depends on the heat resistance of the amidase catalyst. It is preferable that the reaction tank temperature is within the above range because the activity of the amidase catalyst can be maintained well.

The pressure inside the reaction tank is generally normal pressure, but it can also be carried out under increased pressure in order to increase the solubility of the 3-hydroxyamide compound. The pressurizing conditions are preferably, for example, 0.01 MPa or more and 1 MPa or less.

The pH in the reaction tank is not particularly limited as long as it is within a suitable range for maintaining good activity of the amidase catalyst, but is preferably in the range of pH 5 to pH 10. It is preferable that the pH is within the above range because the activity of the amidase catalyst can be maintained well.

[Second embodiment]
The method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure includes at least one member selected from the group consisting of nitrile hydratase-containing microbial cells and bacterial cell-treated products and a 3-hydroxynitrile compound in an aqueous medium. The 3-hydroxyamide compound obtained by the reaction in an aqueous medium and at least one species selected from the group consisting of bacterial cells and bacterial cell-treated products containing amidase derived from the above-mentioned specific bacteria. Preferably, the method is one for producing a 3-hydroxycarboxylic acid compound or a salt thereof.
Further, a method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure is a method for producing a 3-hydroxycarboxylic acid compound or a salt thereof from a 3-hydroxynitrile compound in the presence of the nitrile hydratase and amidase. There may be.
Specifically, at least one species selected from the group consisting of bacterial cells and bacterial cell-treated products containing nitrile hydratase, and at least one species selected from the group consisting of bacterial cells and bacterial cell-treated products containing the above-mentioned amidase. A method of producing a 3-hydroxycarboxylic acid compound or a salt thereof by mixing the 3-hydroxycarboxylic acid compound or a salt thereof can be mentioned.

<Nitrile hydratase-containing bacterial cells and/or treated bacterial cells>
In the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure, at least one species selected from the group consisting of nitrile hydratase-containing microbial cells and bacterial cell-treated products (hereinafter, these are simply referred to as "nitrile hydratase"). (also referred to as "catalyst") may be used as a catalyst for hydration of a 3-hydroxynitrile compound to a 3-hydroxyamide compound.

In this specification, nitrile hydratase refers to an enzyme that acts on the cyano group of a nitrile compound to hydrate the cyano group and convert it into an amide group.
The nitrile hydratase used in the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure only needs to be able to efficiently hydrate a 3-hydroxynitrile compound, and can be derived from microorganisms, plants, molds, animals, insects, etc. It may also be a nitrile hydratase.

Examples of microorganisms that produce nitrile hydratase include the genus Corynebacterium, the genus Nocardia, the genus Pseudomonas, the genus Bacillus, the genus Bacteridium, and the genus Brevi. Brevibacterium, Micrococcus, Rhodococcus, Aeromonas, Citrobacter, Agrobacterium, Erwinia, Enterobacter ( Examples include microorganisms belonging to the genus Enterobacter, Streptomyces, Rhizobium, Pseudonocardia, and the like.
The nitrile hydratase used in the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure is preferably a nitrile hydratase derived from the genus Pseudomonas or the genus Pseudonocardia, more preferably a nitrile hydratase derived from the genus Pseudonocardia. preferable.

A bacterial cell containing nitrile hydratase derived from the genus Pseudonocardia is one that produces nitrile hydratase derived from the genus Pseudonocardia and retains nitrile hydratase activity in an aqueous solution containing a nitrile compound and an amide compound. There is no particular limitation as long as it is a bacterial cell that exists.
One type of bacterial cells containing nitrile hydratase derived from the genus Pseudonocardia may be used, or two or more types may be used in combination.

In addition, transformants in which the nitrile hydratase gene cloned from these bacterial cells is highly expressed in any host, and one or more constituent amino acids of the nitrile hydratase using recombinant DNA technology Transformants that express mutant nitrile hydratase that has further improved resistance to amide compounds, resistance to nitrile compounds, and temperature resistance by substitution, deletion, deletion, or insertion of amino acids are This is preferable because productivity is improved.
Note that the arbitrary host mentioned here includes Escherichia coli as a representative example, as shown in the Examples below, but is not particularly limited to Escherichia coli, and includes Bacillus species such as Bacillus subtilis. Other strains such as fungi, yeasts and actinomycetes are also included. As an example of such a strain, MT-10822 [this strain was acquired on February 7, 1996 at the Institute of Biotechnology and Industrial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture (currently Kisarazu, Chiba Prefecture). Ichikazusa Kamatari 2-5-8 National Institute of Technology and Evaluation (Biotechnology Center, Patent Organism Depositary Center) under the accession number FERMBP-5785 under the Budapest Treaty on the International Recognition of the Deposit of Microbial Organisms for the Purposes of Patent Procedures It has been deposited. ].

The vector used for cloning the nitrile hydratase derived from the genus Pseudonocardia contains the gene encoding the nitrile hydratase derived from the genus Pseudonocardia. It can be obtained by linking the encoding genes.
Vectors are not particularly limited, and include commercially available expression plasmids such as pET-21a(+), pKK223-3, pUC19, pBluescriptKS(+), and pBR322, and nitrile vectors derived from Pseudonocardia. By incorporating a gene encoding hydratase, an expression plasmid for the nitrile hydratase can be constructed. In addition, the host organism used for transformation may be one that is stable, capable of self-replication, and capable of expressing the characteristics of the foreign DNA. For example, Escherichia coli is a good example, but it is limited to Escherichia coli. By introducing it into Bacillus subtilis, yeast, etc., a transformant having the ability to produce nitrile hydratase can be obtained.

The above-mentioned nitrile hydratase-producing microbial cells derived from the genus Pseudonocardia may be appropriately cultured and grown to produce nitrile hydratase by a known method.
The medium used for culturing the bacterial cells that produce nitrile hydratase derived from the genus Pseudonocardia may be either a synthetic medium or a natural medium as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic salts, and other nutrients. is also available.
For example, after inoculating the bacterial cells into a normal liquid medium such as LB medium or M9 medium, the appropriate culture temperature (generally 20°C to 50°C, but 50°C or higher in the case of thermophilic bacteria) ) can be prepared by culturing in Cultivation can be carried out in a liquid medium containing the above-mentioned culture components using conventional culture methods such as shaking culture, aerated agitation culture, continuous culture, and fed-batch culture.
The culture temperature of the transformant is preferably 15°C to 37°C.
Culture conditions are not particularly limited and may be appropriately selected depending on the type of culture and culture method, as long as the strain can grow and produce nitrile hydratase.

The above-mentioned nitrile hydratase-producing microorganisms derived from the genus Pseudonocardia may be subjected to various treatments, such as collection by centrifugation or crushing to produce crushed microorganisms, in order to react with nitrile compounds. A treated bacterial cell product that produces nitrile hydratase derived from the genus Pseudonocardia often refers to bacterial cells that have been subjected to some kind of treatment.
The bacterial cell treatment product includes nitrile hydratase that has been extracted by crushing the bacterial cells that produce nitrile hydratase and removing insoluble matter from the crushed bacterial cell material. The nitrile hydratase in the state removed is sometimes referred to as free nitrile hydratase. Furthermore, free nitrile hydratase does not contain insoluble matter derived from bacterial cells and/or crushed bacterial cells.
The form of the microbial cells to be disrupted is not particularly limited as long as it contains microbial cells that produce nitrile hydratase derived from the genus Pseudonocardia. Examples include bacterial collections separated or collected by washing, and those obtained by washing these bacterial collections with physiological saline or the like.
As a method for removing nitrile hydratase from a microbial cell that produces nitrile hydratase, a known method can be used, for example, the method described in Japanese Patent No. 5430659 can be used.

<Nitrile hydratase derived from Pseudonocardia>
The nitrile hydratase derived from the genus Pseudonocardia may be a wild type nitrile hydratase derived from the genus Pseudonocardia (hereinafter sometimes simply referred to as "wild type nitrile hydratase"), or a mutant type nitrile hydratase. It may be a nitrile hydratase derived from the genus Pseudonocardia (hereinafter also simply referred to as "mutant nitrile hydratase").
Mutant nitrile hydratase is a nitrile hydratase in which one or more of the amino acids constituting the nitrile hydratase is substituted, deleted, deleted, or inserted with other amino acids, and is a nitrile hydratase that is a nitrile hydratase that has been substituted, deleted, deleted, or inserted with another amino acid, and is a nitrile hydratase that is produced by the recombinant DNA described below It can be produced using technology.
In order to improve the productivity of the 3-hydroxycarboxylic acid compound or its salt, the nitrile hydratase derived from the genus Pseudonocardia must be a mutant nitrile hydratase derived from the genus Pseudonocardia (mutant nitrile hydratase). is preferred.

[Mutant nitrile hydratase]
In order to improve the productivity of 3-hydroxycarboxylic acid compounds or salts thereof, the mutant nitrile hydratase includes an α subunit having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1; In the nitrile hydratase derived from Pseudonocardia thermophila having a β subunit having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, the following amino acid substitution positions (1L) to (37L) It is preferable that the mutant nitrile hydratase is substituted with another amino acid residue at at least one position selected from the group consisting of.

The amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence of the α subunit of nitrile hydratase derived from wild-type Pseudonocardia thermophila.
The amino acid sequence represented by SEQ ID NO: 2 is the amino acid sequence of the β subunit of nitrile hydratase derived from wild-type Pseudonocardia thermophila.
The α subunit having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 is the α subunit possessed by the nitrile hydratase derived from wild type Pseudonocardia thermophila (wild type α subunit ) means that the α subunit has an amino acid sequence that is 90% or more identical to the amino acid sequence of the wild type α subunit.
Similarly, the β subunit having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 is the β subunit possessed by the nitrile hydratase derived from wild type Pseudonocardia thermophila (wild type β This means that the β subunit has an amino acid sequence that is 90% or more identical to the amino acid sequence of the wild type β subunit.

In addition, the basic structural unit of mutant nitrile hydratase is a dimer in which an α subunit and a β subunit associate, and the dimers further associate to form a tetramer. The cysteine residue, which is the 111th amino acid residue from the N-terminus of the α subunit, is converted to cysteine sulfinic acid (Cys-SOOH), and the cysteine residue, which is the 113th amino acid residue from the N-terminus, is converted to cysteine sulfenic acid (Cys-SOOH). -SOH) are each subjected to post-translational modification, and the polypeptide chain of the α subunit and cobalt atom are bonded to each other via this modified amino acid residue to form an active center.

The mutant nitrile hydratase is preferably a nitrile hydratase derived from Pseudonocardia thermophila from the viewpoint of improving the productivity of the 3-hydroxycarboxylic acid compound or its salt.
In the present disclosure, nitrile hydratase derived from Pseudonocardia thermophila refers to wild-type Pseudonocardia thermophila having an α subunit having the amino acid sequence of SEQ ID NO: 1 and a β subunit having the amino acid sequence of SEQ ID NO: 2. Not only the nitrile hydratase derived from thermophila but also the nitrile hydratase derived from wild type Pseudonocardia thermophila, those skilled in the art would recognize that it is a modified sequence of nitrile hydratase derived from wild type Pseudonocardia thermophila. The concept also includes modified nitrile hydratases that have been modified to a discernible extent.
Modified nitrile hydratases included in the concept of nitrile hydratase derived from Pseudonocardia thermophila include (i) one or more amino acid residues compared to the wild-type nitrile hydratase derived from Pseudonocardia thermophila; (ii) deletion of one or more amino acid residues other than amino acid residue substitutions (1A) to (37A), (iii) insertion of an amino acid residue, (iv) α-sub Addition of an amino acid residue to one or both of the N-terminus and C-terminus of the amino acid sequence of the unit; (v) Addition of an amino acid residue to one or both of the N-terminus and C-terminus of the amino acid sequence of the β subunit Also included are modified nitrile hydratases with one or more modifications selected from the group consisting of.
Note that, as will be described in detail below, when introducing amino acid residue substitutions included in the mutant nitrile hydratase, the mutant nitrile hydratase may be produced using a modified nitrile hydratase.

-Modified nitrile hydratase-
In the α subunit of the modified nitrile hydratase, the number of substituted amino acid residues is, for example, 1 to 20, or 1 to 15, or 1 to 10, or 1 to 8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 1. The number of substituted amino acid residues may be zero.
In the β subunit of the modified nitrile hydratase, the number of substituted amino acid residues is, for example, 1 to 20, or 1 to 15, or 1 to 10, or 1 to 8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 1. The number of substituted amino acid residues may be zero.

In the α subunit of the modified nitrile hydratase, the number of amino acid residues deleted is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, Or it is 1. The number of deleted amino acid residues may be zero.
In the β subunit of the modified nitrile hydratase, the number of amino acid residues deleted is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, Or it is 1. The number of deleted amino acid residues may be zero.

In the alpha subunit of the modified nitrile hydratase, the number of inserted amino acid residues is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, or It is 1. The number of inserted amino acid residues may be zero.
In the β subunit of the modified nitrile hydratase, the number of inserted amino acid residues is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, or It is 1. The number of inserted amino acid residues may be zero.

In the α subunit of the modified nitrile hydratase, the total number of amino acid residues substituted, deleted, or inserted is, for example, 1 to 20, or 1 to 14, or 1 to 8, or 1 to 4, or 1 to 2, or 1. In cases where there is terminal addition, the total number of substituted, deleted or inserted amino acid residues may be zero.
In the β subunit of the modified nitrile hydratase, the total number of substituted, deleted, or inserted amino acid residues is, for example, 1 to 20, or 1 to 14, or 1 to 8, or 1 to 4, or 1 to 2, or 1. In cases where there is terminal addition, the total number of substituted, deleted or inserted amino acid residues may be zero.

In the α subunit of the modified nitrile hydratase, the number of terminally added amino acid residues is, for example, 1 to 60, or 1 to 40, or 1 to 20, or 1 to 10 per terminal. , or 1 to 5, or 1 to 3, or 1. The number of added amino acid residues may be zero.
In the β subunit of the modified nitrile hydratase, the number of terminally added amino acid residues is, for example, 1 to 60, or 1 to 40, or 1 to 20, or 1 to 10 per terminal. , or 1 to 5, or 1 to 3, or 1. The number of added amino acid residues may be zero.
The terminal additional amino acid residue may be, for example, a secretory signal sequence. Terminal additional amino acid residues may be present only at the N-terminus, only at the C-terminus, or at both the N-terminus and the C-terminus.

The similarity between the modified nitrile hydratase and the wild-type Pseudonocardia thermophila-derived nitrile hydratase can also be expressed by sequence identity.
The amino acid sequence of the α-subunit of the modified nitrile hydratase is, for example, 70% or more different from the amino acid sequence of the α-subunit of the wild-type Pseudonocardia thermophila-derived nitrile hydratase represented by SEQ ID NO: 1. have sequence identity, or have 80% or more sequence identity, or have 85% or more sequence identity, or have 90% or more sequence identity, or have 95% or more sequence identity; have a sequence identity of 96% or more, or have a sequence identity of 97% or more, or have a sequence identity of 98% or more, or have a sequence identity of 99% or more. have

The amino acid sequence of the β subunit of the modified nitrile hydratase is, for example, 70% or more different from the amino acid sequence of the β subunit of the wild-type Pseudonocardia thermophila-derived nitrile hydratase represented by SEQ ID NO: 2. have sequence identity, or have 80% or more sequence identity, or have 85% or more sequence identity, or have 90% or more sequence identity, or have 95% or more sequence identity; have a sequence identity of 96% or more, or have a sequence identity of 97% or more, or have a sequence identity of 98% or more, or have a sequence identity of 99% or more. have

Note that alignment of sequences can be performed with ClustalW (1.83) using initial parameters (including gap open penalty: 10 and gap extension penalty: 0.05).

When the amino acid sequence of the modified nitrile hydratase is aligned with the amino acid sequence of the wild-type nitrile hydratase derived from Pseudonocardia thermophila, depending on the manner of modification of the modified nitrile hydratase, corresponding amino acid residues may differ from each other in the alignment. Even so, the distance from the N-terminus of the subunit defined above may vary.
In the present disclosure, in such a case, for a modified nitrile hydratase, the X-th amino acid residue from the N-terminus of a subunit refers to the Refers to the amino acid residue that corresponds in alignment to the X-th amino acid residue from the end.
For example, in the present disclosure, "the 40th amino acid residue from the N-terminus of the α subunit", "the 40th Asp residue from the N-terminus of the α subunit", or "the 40th amino acid residue from the N-terminus of the α subunit" The "Asp residue" may be located at a position other than the 40th position from the N-terminus of the α subunit on the amino acid sequence of the modified nitrile hydratase.
For example, as a result of insertion of an amino acid residue, the 41st amino acid residue (not necessarily Asp) from the N-terminus of the α subunit of the modified nitrile hydratase becomes a nitrile derived from wild-type Pseudonocardia thermophila. It may correspond to the Asp residue, which is the 40th amino acid residue from the N-terminus of the α subunit of hydratase. In such a case, "the 40th amino acid residue from the N-terminus of the α subunit", "the 40th Asp residue from the N-terminus of the α subunit", or "the N-terminus of the α subunit" in the description of this disclosure The term "Asp residue which is the 40th amino acid residue from" refers to the 41st amino acid residue from the N-terminus of the α subunit in the modified nitrile hydratase.

In this specification, amino acids, peptides, and proteins are represented using the abbreviations adopted by the IUPAC-IUB Committee on Biochemical Nomenclature (CBN) shown below. Furthermore, unless otherwise specified, the sequences of amino acid residues of peptides and proteins are expressed from the left end to the right end, from the N-terminus to the C-terminus. Furthermore, unless otherwise specified, sequences of amino acid residues of peptides and proteins are expressed such that the N-terminal amino acid residue is the first amino acid residue.
A=Ala=alanine, C=Cys=cysteine,
D=Asp=aspartic acid, E=Glu=glutamic acid,
F=Phe=phenylalanine, G=Gly=glycine,
H=His=histidine, I=Ile=isoleucine,
K=Lys=lysine, L=Leu=leucine,
M=Met=methionine, N=Asn=asparagine,
P=Pro=proline, Q=Gln=glutamine,
R=Arg=arginine, S=Ser=serine,
T=Thr=threonine, V=Val=valine,
W=Trp=tryptophan, Y=Tyr=tyrosine

-Amino acid substitution positions (1L) to (37L)-
The mutant nitrile hydratase is substituted with another amino acid residue at at least one position selected from the group consisting of amino acid substitution positions (1L) to (37L) (hereinafter also referred to as "specific amino acid substitution position"). Preferably, it is a mutant nitrile hydratase.
In the mutant nitrile hydratase, either the α subunit or the β subunit may be substituted with another amino acid residue at a specific amino acid substitution position, or both the α subunit and the β subunit may be substituted with another amino acid residue at a specific amino acid substitution position. Other amino acid residues may be substituted at specific amino acid substitution positions.

(1L) In the α subunit, the 13th position from the N-terminus of SEQ ID NO: 1,
(2L) In the α subunit, the 27th position from the N-terminus of SEQ ID NO: 1,
(3L) In the α subunit, the 36th position from the N-terminus of SEQ ID NO: 1,
(4L) In the α subunit, the 40th position from the N-terminus of SEQ ID NO: 1,
(5L) In the α subunit, the 43rd position from the N-terminus of SEQ ID NO: 1,
(6L) In the α subunit, the 71st position from the N-terminus of SEQ ID NO: 1,
(7L) In the α subunit, the 92nd position from the N-terminus of SEQ ID NO: 1,
(8L) In the α subunit, the 94th position from the N-terminus of SEQ ID NO: 1,
(9L) In the α subunit, the 148th position from the N-terminus of SEQ ID NO: 1,
(10L) In the α subunit, the 197th position from the N-terminus of SEQ ID NO: 1,
(11L) In the α subunit, the 204th position from the N-terminus of SEQ ID NO: 1,
(12L) In the β subunit, the fourth position from the N-terminus of SEQ ID NO: 2,
(13L) In the β subunit, the 10th position from the N-terminus of SEQ ID NO: 2,
(14L) In the β subunit, the 24th position from the N-terminus of SEQ ID NO: 2,
(15L) In the β subunit, the 32nd position from the N-terminus of SEQ ID NO: 2,
(16L) In the β subunit, the 37th position from the N-terminus of SEQ ID NO: 2,
(17L) In the β subunit, the 41st position from the N-terminus of SEQ ID NO: 2,
(18L) In the β subunit, the 46th position from the N-terminus of SEQ ID NO: 2,
(19L) In the β subunit, the 48th position from the N-terminus of SEQ ID NO: 2,
(20L) In the β subunit, the 51st position from the N-terminus of SEQ ID NO: 2,
(21L) In the β subunit, the 72nd position from the N-terminus of SEQ ID NO: 2,
(22L) In the β subunit, the 79th position from the N-terminus of SEQ ID NO: 2,
(23L) In the β subunit, the 96th position from the N-terminus of SEQ ID NO: 2,
(24L) In the β subunit, the 107th position from the N-terminus of SEQ ID NO: 2,
(25L) In the β subunit, the 110th position from the N-terminus of SEQ ID NO: 2,
(26L) In the β subunit, the 118th position from the N-terminus of SEQ ID NO: 2,
(27L) In the β subunit, the 127th position from the N-terminus of SEQ ID NO: 2,
(28L) In the β subunit, the 146th position from the N-terminus of SEQ ID NO: 2,
(29L) In the β subunit, the 160th position from the N-terminus of SEQ ID NO: 2,
(30L) In the β subunit, the 186th position from the N-terminus of SEQ ID NO: 2,
(31L) In the β subunit, the 205th position from the N-terminus of SEQ ID NO: 2,
(32L) In the β subunit, the 206th position from the N-terminus of SEQ ID NO: 2,
(33L) In the β subunit, the 215th position from the N-terminus of SEQ ID NO: 2,
(34L) In the β subunit, the 217th position from the N-terminus of SEQ ID NO: 2,
(35L) In the β subunit, the 226th position from the N-terminus of SEQ ID NO: 2,
(36L) In the β subunit, the 230th position from the N-terminus of SEQ ID NO: 2,
(37L) 231st from the N-terminus of SEQ ID NO: 2 in the β subunit.

-Other amino acid substitution positions-
In addition to the specific amino acid substitution positions described above, the mutant nitrile hydratase may be substituted with other amino acids at positions other than the specific amino acid substitution positions (hereinafter referred to as "other substitution positions").
Other substitution positions are not particularly limited, but include, for example, the 6th, 19th, 48th, 126th, and 188th positions from the N-terminus of SEQ ID NO: 1 in the α subunit.
In addition, other substitution positions include, for example, the 8th, 33rd, 40th, 47th, 61st, 108th, 112th, 168th, 176th from the N-terminal substitution of SEQ ID NO: 2 in the β subunit, Examples include the 200th, 212th, 218th, etc.
From the viewpoint of efficiently producing a 3-hydroxycarboxylic acid compound or a salt thereof, the mutant nitrile hydratase is preferably substituted with other amino acids at the specific amino acid substitution position and at other substitution positions.
Other positions include positions 6, 19, 48, and 126 from the N-terminus of SEQ ID NO: 1 in the α subunit, and positions 108, 176, and 212 from the N-terminus of SEQ ID NO: 2 in the β subunit. More preferably, at least one position selected from the group consisting of .

In the mutant nitrile hydratase, the number substituted with other amino acid residues at the specific amino acid substitution position and other substitution positions is not particularly limited, and may include 1 to 20, 1 to 15, 1 to 10, 1 to 8, It may be 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1.
From the viewpoint of efficiently producing a 3-hydroxycarboxylic acid compound or a salt thereof, the number substituted with other amino acid residues is preferably 1 to 20, more preferably 2 to 15, More preferably, it is 3 to 12.

-Amino acid residue substitutions (1A) to (37A)-
In the mutant nitrile hydratase, from the viewpoint of efficiently obtaining a 3-hydroxycarboxylic acid compound or a salt thereof, the above substitution (i.e., substitution at a specific amino acid substitution position) consists of the following (1A) to (37A). It is preferable that at least one substitution selected from the group (hereinafter referred to as "specific amino acid residue substitution") is included.

(1A) In the α subunit, substitution of the amino acid residue Ile corresponding to the 13th position from the N-terminus of SEQ ID NO: 1 with Leu,
(2A) In the α subunit, substitution of the amino acid residue Met corresponding to the 27th position from the N-terminus of SEQ ID NO: 1 with He;
(3A) In the α subunit, substitution of the amino acid residue Thr corresponding to the 36th position from the N-terminus of SEQ ID NO: 1 with a Met group,
(4A) In the α subunit, substitution of the amino acid residue Asp corresponding to the 40th position from the N-terminus of SEQ ID NO: 1 with Asn;
(5A) In the α subunit, substitution of amino acid residue Ala to Val corresponding to the 43rd position from the N-terminus of SEQ ID NO: 1,
(6A) In the α subunit, substitution of the amino acid residue Arg corresponding to the 71st position from the N-terminus of SEQ ID NO: 1 with a His group,
(7A) In the α subunit, substitution of the amino acid residue Asp corresponding to the 92nd position from the N-terminus of SEQ ID NO: 1 with Glu,
(8A) In the α subunit, substitution of the amino acid residue Met corresponding to the 94th position from the N-terminus of SEQ ID NO: 1 with He;
(9A) In the α subunit, substitution of the amino acid residue Gly corresponding to the 148th position from the N-terminus of SEQ ID NO: 1 with Asp;
(10A) In the α subunit, substitution of amino acid residue Gly to Cys corresponding to the 197th position from the N-terminus of SEQ ID NO: 1;
(11A) In the α subunit, substitution of the amino acid residue Val corresponding to the 204th position from the N-terminus of SEQ ID NO: 1 with Arg;
(12A) In the β subunit, substitution of the amino acid residue Val corresponding to the fourth position from the N-terminus of SEQ ID NO: 2 with Met;
(13A) In the β subunit, substitution of the amino acid residue Thr corresponding to the 10th position from the N-terminus of SEQ ID NO: 2 with Asp;
(14A) In the β subunit, substitution of amino acid residue Val corresponding to the 24th position from the N-terminus of SEQ ID NO: 2 with He;
(15A) In the β subunit, substitution of the amino acid residue Val corresponding to the 32nd position from the N-terminus of SEQ ID NO: 2 with Gly;
(16A) In the β subunit, substitution of the amino acid residue Phe corresponding to the 37th position from the N-terminus of SEQ ID NO: 2 with Leu,
(17A) In the β subunit, substitution of amino acid residue Phe corresponding to the 41st position from the N-terminus of SEQ ID NO: 2 with He;
(18A) In the β subunit, substitution of the amino acid residue Met corresponding to the 46th position from the N-terminus of SEQ ID NO: 2 with Lys;
(19A) In the β subunit, substitution of amino acid residue Leu to Val corresponding to the 48th position from the N-terminus of SEQ ID NO: 2,
(20A) In the β subunit, substitution of amino acid residue Phe corresponding to the 51st position from the N-terminus of SEQ ID NO: 2 with Ala;
(21A) In the β subunit, substitution of amino acid residue Trp corresponding to the 72nd position from the N-terminus of SEQ ID NO: 2 with Phe,
(22A) In the β subunit, substitution of the amino acid residue His corresponding to the 79th position from the N-terminus of SEQ ID NO: 2 with Asn;
(23A) In the β subunit, substitution of the amino acid residue Gln corresponding to the 96th position from the N-terminus of SEQ ID NO: 2 with Arg;
(24A) In the β subunit, substitution of the amino acid residue Pro corresponding to the 107th position from the N-terminus of SEQ ID NO: 2 with Met;
(25A) In the β subunit, substitution of the amino acid residue Glu corresponding to the 110th position from the N-terminus of SEQ ID NO: 2 with Asn;
(26A) In the β subunit, substitution of amino acid residue Phe corresponding to the 118th position from the N-terminus of SEQ ID NO: 2 with Ala;
(27A) In the β subunit, substitution of amino acid residue Leu to Ala corresponding to the 127th position from the N-terminus of SEQ ID NO: 2,
(28A) In the β subunit, substitution of the amino acid residue Arg corresponding to the 146th position from the N-terminus of SEQ ID NO: 2 with Gly;
(29A) In the β subunit, substitution of the amino acid residue Arg corresponding to the 160th position from the N-terminus of SEQ ID NO: 2 with Leu,
(30A) In the β subunit, substitution of the amino acid residue Leu corresponding to the 186th position from the N-terminus of SEQ ID NO: 2 with Glu,
(31A) In the β subunit, substitution of the amino acid residue Gly corresponding to the 205th position from the N-terminus of SEQ ID NO: 2 with Val;
(32A) In the β subunit, substitution of the amino acid residue Pro corresponding to the 206th position from the N-terminus of SEQ ID NO: 2 with Leu or Gln;
(33A) In the β subunit, substitution of the amino acid residue Tyr corresponding to the 215th position from the N-terminus of SEQ ID NO: 2 with Asn;
(34A) In the β subunit, substitution of the amino acid residue Asp corresponding to the 217th position from the N-terminus of SEQ ID NO: 2 with Gly;
(35A) In the β subunit, substitution of the amino acid residue Val corresponding to the 226th position from the N-terminus of SEQ ID NO: 2 with He;
(36A) In the β subunit, substitution of the amino acid residue Ala corresponding to the 230th position from the N-terminus of SEQ ID NO: 2 with Glu,
(37A) Substitution of amino acid residue Ala to Val corresponding to the 231st position from the N-terminus of SEQ ID NO: 2 in the β subunit.

In addition, from the viewpoint of efficiently obtaining a 3-hydroxycarboxylic acid compound or a salt thereof, the mutant nitrile hydratase has the following group consisting of (1B) to (18B) (hereinafter referred to as , also referred to as "amino acid residue substitution group B").
(1B) In the α subunit, substitution of the amino acid Leu corresponding to the 6th position from the N-terminus of SEQ ID NO: 1 with Thr or Ala;
(2B) In the α subunit, substitution of the amino acid Ala to Val corresponding to the 19th position from the N-terminus of SEQ ID NO: 1,
(3B) In the α subunit, substitution of the amino acid Thr corresponding to the 36th position from the N-terminus of SEQ ID NO: 1 with Trp, Ser, Gly, or Ala;
(4B) In the α subunit, substitution of amino acid Asn corresponding to position 48 from the N-terminus of SEQ ID NO: 1 to Gln;
(5B) In the α subunit, substitution of amino acid Phe corresponding to the 126th position from the N-terminus of SEQ ID NO: 1 with Tyr;
(6B) In the α subunit, the amino acid Thr to Gly corresponds to the 188th position from the N-terminus of SEQ ID NO: 1. (7B) The amino acid Gly to Ala corresponds to the 8th position from the N-terminus of SEQ ID NO: 2 in the β subunit. replacement,
(8B) Substitution of amino acid Ala to Val or Met corresponding to the 33rd position from the N-terminal substitution of SEQ ID NO: 2 in the β subunit,
(9B) Substitution of amino acid Thr to He, Val or Leu corresponding to the 40th position from the N-terminal substitution of SEQ ID NO: 2 in the β subunit,
(10B) Substitution of amino acid Ala to Val, Gly, Trp, Ser, Leu or Thr corresponding to the 61st position from the N-terminal substitution of SEQ ID NO: 2 in the β subunit,
(11B) Substitution of amino acid Glu to Asp or Arg corresponding to the 108th position from the N-terminal substitution of SEQ ID NO: 2 in the β subunit,
(12B) Substitution of the amino acid Lys to Val or He corresponding to the 112th position from the N-terminal substitution of SEQ ID NO: 2 in the β subunit (13B) Amino acid corresponding to the 150th position from the N-terminal substitution of SEQ ID NO: 2 in the β subunit Replacement of Ala with Asn or Ser from residue (14B) Substitution of amino acid Thr with Glu corresponding to position 168 from N-terminal substitution of SEQ ID NO: 2 in β subunit (15B) N-terminus of SEQ ID NO: 2 in β subunit Substitution of amino acid Tyr to Ala, Thr, Met or Cys corresponding to the 176th position from the substitution,
(16B) Substitution of amino acid Ala to Glu corresponding to the 200th position from the N-terminal substitution of SEQ ID NO: 2 in the β subunit,
(17B) Substitution of amino acid Ser to Tyr corresponding to the 212th position from the N-terminal substitution of SEQ ID NO: 2 in the β subunit,
(18B) Substitution of amino acid Cys to Met or Ser corresponding to the 218th position from the N-terminal substitution of SEQ ID NO: 2 in the β subunit.

In addition, the mutant nitrile hydratase may include specific amino acid residue substitutions, (1B) to ( 18B) substitutions (hereinafter referred to as "other amino acid residue substitutions") may be included.

In the mutant nitrile hydratase, the total number of specific amino acid residue substitutions and the above (1B) to (18B) substitutions is not particularly limited, and may be, for example, 1 to 20, or 1. It's okay.
From the viewpoint of efficiently obtaining a 3-hydroxycarboxylic acid compound or a salt thereof, it is preferably 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5 , 1-4, 1-3, 1-2, or 1.

-Method for introducing amino acid residue substitution-
In the mutant nitrile hydratase, as a method for introducing a specific amino acid residue substitution into a specific amino acid substitution position, a publicly known introduction method can be used.
For example, specific amino acid residue substitutions may be introduced into wild-type nitrile hydratase derived from Pseudonocardia thermophila, or modified nitrile hydratase derived from wild-type Pseudonocardia thermophila. Specific amino acid residue substitutions may be introduced into hydratase (hereinafter also referred to as "modified nitrile hydratase").

-Example of amino acid sequence of wild type nitrile hydratase-
In the nitrile hydratase derived from wild-type Pseudonocardia thermophila, the Met residue located at the beginning of SEQ ID NO: 1 is the first amino acid residue from the N-terminus of the amino acid sequence of the α subunit, for example, α The 40th amino acid residue from the N-terminus of the amino acid sequence of the subunit is an Asp residue, and the 43rd amino acid residue from the N-terminus of the α subunit amino acid sequence is an Ala residue.
In addition, in the nitrile hydratase derived from wild-type Pseudonocardia thermophila, the Met residue located at the beginning of SEQ ID NO: 2 is the first amino acid residue from the N-terminus of the amino acid sequence of the β subunit, and β The 205th amino acid residue from the N-terminus of the amino acid sequence of the subunit is a Gly residue, and the 206th amino acid residue from the N-terminus of the amino acid sequence of the β subunit is a Pro residue. The 215th amino acid residue from the N-terminus of the sequence is a Tyr residue.

It can be used when replacing at least one of the specific amino acid substitution positions (1L) to (37L) with another amino acid residue (preferably amino acid residue substitutions (1A) to (37A)). The modified nitrile hydratase sequence can be selected from nitrile hydratase sequences registered in GenBank provided by the National Center for Biotechnology Information (NCBI) or nitrile hydratase sequences described in known literature. can.

It can be used when replacing at least one of the specific amino acid substitution positions (1L) to (37L) with another amino acid residue (preferably amino acid residue substitutions (1A) to (37A)). The modified nitrile hydratase may be a modified nitrile hydratase containing at least one amino acid residue substitution selected from the group consisting of (1B) to (18B).

<First embodiment of nitrile hydratase>
The first embodiment of the mutant nitrile hydratase includes (4L), (5L), and (31L) to (33L) from the viewpoint of efficiently obtaining a 3-hydroxycarboxylic acid compound or a salt thereof. Preferably, at least one position selected from the group consisting of is substituted with another amino acid residue, and at least one position selected from the group consisting of (4A), (5A), and (31A) to (33A) It is preferable to include substitutions of one amino acid residue, and more preferably substitutions of amino acid residues (4A), (5A), (31A) to (33A), and (3B).

In the first embodiment, for example, one or more of the amino acid residue substitutions (4A), (5A), (31A) to (33A) are added to the above wild-type Pseudonocardia thermophila-derived nitrile hydratase or It can be introduced into a modified nitrile hydratase to obtain a mutant nitrile hydratase.

The modified nitrile hydratase that can be used when introducing at least one amino acid residue substitution selected from the group consisting of (4A), (5A), and (31A) to (33A) is α 36th amino acid residue from the N-terminus of the subunit (amino acid residue corresponding to the 36th Thr residue from the N-terminus in the amino acid sequence of the α subunit of nitrile hydratase derived from wild-type Pseudonocardia thermophila) group) may be substituted with a Trp residue (hereinafter also referred to as amino acid residue substitution (3Bf)).
From the viewpoint of efficiently obtaining a 3-hydroxycarboxylic acid compound or a salt thereof, the mutant nitrile hydratase contains at least one compound selected from the group consisting of (4A), (5A), and (31A) to (33A). It is preferable to include one amino acid residue substitution and an amino acid residue substitution (3Bf).

When the mutant nitrile hydratase contains amino acid residue substitutions (4A), (5A), and (31A) to (33A), the amino acid residue substitutions (4A), (5A), and (31A) The modified nitrile hydratase that can be used as a target for introducing one or more of ~(33A) has the above amino acid residues when compared with the amino acid sequence of the nitrile hydratase derived from wild-type Pseudonocardia thermophila. It may contain one or more amino acid residue substitutions selected from substitution group B, may have amino acid residue substitutions (3Bf), (1A), (2A), (6A) ) to (30A) and (34A) to (37A). Two or more of the above amino acid residue substitutions may be included in combination. Many examples of such combinations are also described in International Publication No. 2010/055666. The modified nitrile hydratase should also, of course, have nitrile hydratase activity.

For example, if it contains a combination of three,
A combination of Thr36Ser and Asp92Glu in the α subunit and Ala33Val in the β subunit,
A combination of Met94Ile in the α subunit and Ala61Gly and Ala150Asn in the β subunit,
A combination of Val4Met, Tyr176Ala and Asp217Val in the β subunit, a combination of Ala33Met, His79Asn and Tyr176Thr in the β subunit,
A combination of Thr40Val, Cys218Met and Val226Ile in the β subunit,
Examples include:
In this specification, for example, "Thr36Ser in the α subunit" refers to the 36th amino acid residue from the N-terminus of the α subunit (α subunit of nitrile hydratase derived from wild-type Pseudonocardia thermophila). This means that the amino acid residue (which corresponds in alignment to the 36th Thr residue from the N-terminus in the amino acid sequence) is replaced with a Ser residue.

Also, for example, if it contains a combination of 8,
A combination of Ile13Leu, Ala19Val, Arg71His and Phe126Tyr in the α subunit and Phe37Leu, Gln96Arg, Glu108Asp and Ala200Glu in the β subunit (International Publication No. 2010/055666: Transformant No. 59),
A combination of Leu6Thr, Met27Ile, Thr36Met and Phe126Tyr in the α subunit and Thr10Asp, Pro107Met, Phe118Val and Ala200Glu in the β subunit (International Publication No. 2010/055666: Transformant number 68),
A combination of Leu6Thr, Thr36Met and Phe126Tyr in the α subunit and Thr10Asp, Phe118Val, Ala200Glu, Pro206Leu and Ala230Glu in the β subunit (International Publication No. 2010/055666: Transformant number 92),
A combination of Leu6Thr, Ala19Val and Phe126Tyr in the α subunit and Leu48Val, His79Asn, Glu108Arg, Ser212Tyr and Ala230Glu in the β subunit (International Publication No. 2010/055666: Transformant number 85),
A combination of Thr36Met, Gly148Asp and Val204Arg in the α subunit and Phe41Ile, Phe51Val, Glu108Asp, Pro206Leu and Ala230Glu in the β subunit (International Publication No. 2010/055666: Transformant number 93),
is given as an example.

In addition, in the first embodiment, when the mutant nitrile hydratase includes amino acid residue substitutions (4A), (5A), and (31A) to (33A), the amino acid residue substitution (4A), The amino acid sequence of the modified nitrile hydratase that can introduce at least one amino acid residue substitution selected from the group consisting of (5A) and (31A) to (33A) is a nitrile hydratase derived from wild type Pseudonocardia thermophila. (1A), (2A), (6A) to (30A), and (35A) to (37A), as well as amino acid residue substitution group B and amino acid residue substitution (3Bf ) may include amino acid residue substitutions other than those in
In the first embodiment, the introduction of at least one amino acid residue substitution selected from the group consisting of (4A), (5A), and (31A) to (33A) is carried out in such a group of amino acid residue substitutions. In addition to one or more amino acid residue substitutions and/or amino acid residue substitutions (3Bf) in B, or without substitutions to amino acid residue substitution group B or amino acid residue substitutions (3Bf), A 3-hydroxycarboxylic acid compound or a salt thereof can also be efficiently produced for a modified nitrile hydratase containing amino acid residue substitutions other than amino acid residue substitution group B and amino acid residue substitution (3Bf).

In the first embodiment, when the mutant nitrile hydratase contains at least one amino acid residue substitution selected from the group consisting of (4A), (5A), (31A) to (33A) and (3Bf), It may also contain a combination of two amino acid residue substitutions shown in Table 1. It goes without saying that mutant nitrile hydratase is not limited to these combinations of amino acid residue substitutions.

When the mutant nitrile hydratase contains at least one amino acid residue substitution selected from the group consisting of (4A), (5A), (31A) to (33A) and (3Bf), the three amino acids shown in Table 2 It may also include combinations of residue substitutions. It goes without saying that mutant nitrile hydratase is not limited to these combinations of amino acid residue substitutions.

When the mutant nitrile hydratase contains at least one amino acid residue substitution selected from the group consisting of (4A), (5A), (31A) to (33A) and (3Bf), the four amino acids shown in Table 3 It may also include combinations of residue substitutions. It goes without saying that mutant nitrile hydratase is not limited to these combinations of amino acid residue substitutions.

When the mutant nitrile hydratase contains at least one amino acid residue substitution selected from the group consisting of (4A), (5A), (31A) to (33A) and (3Bf), the five amino acids shown in Table 4 It may also include combinations of residue substitutions. It goes without saying that mutant nitrile hydratase is not limited to these combinations of amino acid residue substitutions.

When the mutant nitrile hydratase contains at least one amino acid residue substitution selected from the group consisting of (4A), (5A), (31A) to (33A) and (3Bf), all (6 shown in Table 5) combinations of amino acid residue substitutions). It goes without saying that mutant nitrile hydratase is not limited to these combinations of amino acid residue substitutions.

The amino acid sequence of a modified nitrile hydratase capable of introducing at least one amino acid residue substitution selected from the group consisting of (4A), (5A), and (31A) to (33A) is disclosed in WO 2010/055666 It may be the amino acid sequence of the modified nitrile hydratase prepared in No. 1, etc., or a sequence similar thereto. Therefore, in the first embodiment, the mutant nitrile hydratase has, for example, at least one of amino acid residue substitutions (4A), (5A), and (31A) to (33A) in the following nitrile hydratase. It may be one introduced into any of the nitrile hydratase (1) to (46) (hereinafter also referred to as mutant nitrile hydratase B).

The following nitrile hydratases (2) to (46) are nitrile hydratases whose activities were confirmed in the Examples in International Publication No. 2010/055666, and the following nitrile hydratases (1) are wild type nitrile hydratases. It is a nitrile hydratase derived from Pseudonocardia thermophila.
Furthermore, SEQ ID NOS: 16 to 73 below are the SEQ ID NOs listed in the sequence listing in International Publication No. 2018/124247.

(1) A nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 1 and a β subunit having the amino acid sequence of SEQ ID NO: 2,
(2) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 16 and a β subunit having the amino acid sequence of SEQ ID NO: 33;
(3) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 17 and a β subunit having the amino acid sequence of SEQ ID NO: 33;
(4) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 18 and a β subunit having the amino acid sequence of SEQ ID NO: 34;
(5) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 19 and a β subunit having the amino acid sequence of SEQ ID NO: 34;
(6) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 20 and a β subunit having the amino acid sequence of SEQ ID NO: 35;
(7) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 20 and a β subunit having the amino acid sequence of SEQ ID NO: 36;
(8) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 21 and a β subunit having the amino acid sequence of SEQ ID NO: 37;
(9) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 21 and a β subunit having the amino acid sequence of SEQ ID NO: 38;
(10) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 21 and a β subunit having the amino acid sequence of SEQ ID NO: 39;
(11) A nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 21 and a β subunit having the amino acid sequence of SEQ ID NO: 40,
(12) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 18 and a β subunit having the amino acid sequence of SEQ ID NO: 41;
(13) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 18 and a β subunit having the amino acid sequence of SEQ ID NO: 42;
(14) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 21 and a β subunit having the amino acid sequence of SEQ ID NO: 43;
(15) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 22 and a β subunit having the amino acid sequence of SEQ ID NO: 44;
(16) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 23 and a β subunit having the amino acid sequence of SEQ ID NO: 45;
(17) A nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 24 and a β subunit having the amino acid sequence of SEQ ID NO: 46,
(18) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 25 and a β subunit having the amino acid sequence of SEQ ID NO: 47;
(19) A nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 18 and a β subunit having the amino acid sequence of SEQ ID NO: 48,
(20) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 23 and a β subunit having the amino acid sequence of SEQ ID NO: 49;
(21) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 16 and a β subunit having the amino acid sequence of SEQ ID NO: 50;
(22) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 26 and a β subunit having the amino acid sequence of SEQ ID NO: 51;
(23) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 27 and a β subunit having the amino acid sequence of SEQ ID NO: 52;
(24) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 28 and a β subunit having the amino acid sequence of SEQ ID NO: 53;
(25) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 17 and a β subunit having the amino acid sequence of SEQ ID NO: 54;
(26) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 29 and a β subunit having the amino acid sequence of SEQ ID NO: 55;
(27) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 18 and a β subunit having the amino acid sequence of SEQ ID NO: 56;
(28) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 18 and a β subunit having the amino acid sequence of SEQ ID NO: 57;
(29) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 30 and a β subunit having the amino acid sequence of SEQ ID NO: 58;
(30) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 29 and a β subunit having the amino acid sequence of SEQ ID NO: 59;
(31) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 31 and a β subunit having the amino acid sequence of SEQ ID NO: 60;
(32) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 18 and a β subunit having the amino acid sequence of SEQ ID NO: 61;
(33) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 32 and a β subunit having the amino acid sequence of SEQ ID NO: 62;
(34) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 30 and a β subunit having the amino acid sequence of SEQ ID NO: 63;
(35) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 30 and a β subunit having the amino acid sequence of SEQ ID NO: 64;
(36) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 30 and a β subunit having the amino acid sequence of SEQ ID NO: 65;
(37) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 25 and a β subunit having the amino acid sequence of SEQ ID NO: 54;
(38) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 30 and a β subunit having the amino acid sequence of SEQ ID NO: 66;
(39) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 1 and a β subunit having the amino acid sequence of SEQ ID NO: 67;
(40) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 1 and a β subunit having the amino acid sequence of SEQ ID NO: 68;
(41) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 1 and a β subunit having the amino acid sequence of SEQ ID NO: 69;
(42) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 1 and a β subunit having the amino acid sequence of SEQ ID NO: 70;
(43) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 1 and a β subunit having the amino acid sequence of SEQ ID NO: 71;
(44) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 21 and a β subunit having the amino acid sequence of SEQ ID NO: 72;
(45) a nitrile hydratase having an α subunit having the amino acid sequence of SEQ ID NO: 1 and a β subunit having the amino acid sequence of SEQ ID NO: 73;
(46) A nitrile hydratase in which the 36th amino acid residue from the N-terminus of the α subunit in any one of the nitrile hydratases (1) to (45) above is a Trp residue;
(47) α consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence of the α subunit of a specific nitrile hydratase, which is any one of the nitrile hydratases described in (1) to (46) above. consisting of a subunit variant and an amino acid sequence having 70% or more sequence identity with the amino acid sequence of the β subunit of a specific nitrile hydratase, which is any one of the nitrile hydratases (1) to (46). A modified nitrile hydratase having a β subunit variant.

The above nitrile hydratases (2) to (45) are nitrile hydratases (1) (wild type) having an α subunit having the amino acid sequence of SEQ ID NO: 1 and a β subunit having the amino acid sequence of SEQ ID NO: 2. The amino acid residues shown in Tables 6 to 12 below are different when compared to the nitrile hydratase (type nitrile hydratase), and are described in International Publication No. 2010/055666 along with the transformant number.
In the table below, the numbers in the mutation site column represent the position from the N-terminus of the amino acid sequence of the corresponding subunit. Moreover, the transformant number is the number assigned in International Publication No. 2010/055666.
Furthermore, the sequence numbers listed in Tables 6 to 12 are the sequence numbers listed in the sequence listing in International Publication No. 2018/124247.

In the above nitrile hydratase (47), the sequence identity to the α subunit of the specific nitrile hydratase, which is any one of the nitrile hydratases (1) to (46), may be 80% or more, or It may be 85% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more. Similarly, in (34) above, the sequence identity to the β subunit of a specific nitrile hydratase, which is any one of nitrile hydratases (1) to (46), may be 80% or more, Alternatively, it may be 85% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more.
Further, an α subunit variant refers to an α subunit that satisfies the sequence identity defined above but has an amino acid sequence different from that of a specific nitrile hydratase α subunit. A β subunit variant refers to a β subunit that meets the sequence identity defined above, but has an amino acid sequence that differs from the β subunit of a particular nitrile hydratase.

Nitrile hydratases (2) to (46) are also modified nitrile hydratases that have at least one amino acid residue substitution selected from the group consisting of (4A), (5A), and (31A) to (33A). It can be used to introduce amino acid residue substitutions.
In addition, other modified nitrile hydratases having amino acid sequences similar to those of these nitrile hydratases (2) to (33) are also modified by amino acid residue substitutions (4A), (5A), and (31A) to (33A). ) can be used to introduce at least one amino acid residue substitution selected from the group consisting of:
Therefore, matters describing the differences in amino acid sequence between the modified nitrile hydratase and the wild-type nitrile hydratase from Pseudonocardia thermophila (including explanations of the extent and examples of sequence modification) are "Nitrile hydratase derived from wild-type Pseudonocardia thermophila" can be read as any of the above nitrile hydratases (1) to (46) and applied as is. The modified nitrile hydratase should also, of course, have nitrile hydratase activity.

For example, regarding the amino acid residue substitutions and combinations thereof that the mutant nitrile hydratase B may contain, and the 36th amino acid residue from the N-terminus of the α subunit of the nitrile hydratase, the mutant nitrile hydratase B is The combinations a to o of the amino acid residues listed in Table 1, the combinations p to ai of the amino acid residues listed in Table 2, the combinations aj to ax of the amino acid residues listed in Table 3, and the combinations of amino acid residues listed in Table 4. It may contain a combination of amino acid residues selected from the group consisting of combinations ay to bd, and combinations of amino acid residues be listed in Table 5. That is, such a combination of amino acid residue substitutions can be introduced into the amino acid sequences of the above nitrile hydratases (1) to (46).

Further, for example, the nitrile hydratase (47) has the following effect on any of the nitrile hydratases (1) to (46): (i) substitution of one or more amino acid residues with another amino acid residue; , (ii) Deletion of one or more amino acid residues other than those in (a) to (e) above, (iii) Insertion of amino acid residues, (iv) At the N-terminus and C-terminus of the amino acid sequence of the α subunit. (v) addition of an amino acid residue to one or both of the N-terminus and C-terminus of the amino acid sequence of the β subunit; It may also be a modified nitrile hydratase with the following modifications.

In the α subunit of nitrile hydratase (47), the number of substituted amino acid residues when based on the α subunit of any of the nitrile hydratase (1) to (46) above is, for example, 1 ~20, or 1~15, or 1~10, or 1~8, or 1~7, or 1~6, or 1~5, or 1~ 4, or 1 to 3, or 1 to 2, or 1. The number of substituted amino acid residues may be zero.
In the β subunit of nitrile hydratase (47), the number of substituted amino acid residues when based on the β subunit of any of the nitrile hydratase (1) to (46) above is, for example, ~20, or 1~15, or 1~10, or 1~8, or 1~7, or 1~6, or 1~5, or 1~ 4, or 1 to 3, or 1 to 2, or 1. The number of substituted amino acid residues may be zero.

In the α subunit of nitrile hydratase (47), the number of amino acid residues deleted when based on the α subunit of any of the nitrile hydratase (1) to (46) above is, for example, 1-10, or 1-7, or 1-4, or 1-2, or 1. The number of deleted amino acid residues may be zero.
In the β subunit of nitrile hydratase (47), the number of amino acid residues deleted when based on the β subunit of any of the nitrile hydratase (1) to (46) above is, for example, 1-10, or 1-7, or 1-4, or 1-2, or 1. The number of deleted amino acid residues may be zero.

In the alpha subunit of nitrile hydratase (47), the number of inserted amino acid residues is, for example, 1 ~10, or 1 to 7, or 1 to 4, or 1 to 2, or 1. The number of inserted amino acid residues may be zero.
In the β subunit of nitrile hydratase (47), the number of inserted amino acid residues is, for example, 1 when based on the β subunit of any of the nitrile hydratase (1) to (46) above. ~10, or 1 to 7, or 1 to 4, or 1 to 2, or 1. The number of inserted amino acid residues may be zero.

The total number of amino acid residues substituted, deleted, or inserted in the α subunit of nitrile hydratase (47), based on the α subunit of any of the nitrile hydratase (1) to (46) above. is, for example, from 1 to 20, or from 1 to 14, or from 1 to 8, or from 1 to 4, or from 1 to 2, or from 1. In cases where there is terminal addition, the total number of substituted, deleted or inserted amino acid residues may be zero.
In the β subunit of a modified nitrile hydratase obtained by modifying nitrile hydratase (47), substitution, deletion, or insertion based on the β subunit of any of the nitrile hydratase described in (1) to (46) above. The total number of amino acid residues determined is, for example, from 1 to 20, alternatively from 1 to 14, alternatively from 1 to 8, alternatively from 1 to 4, alternatively from 1 to 2, or alternatively 1. In cases where there is terminal addition, the total number of substituted, deleted or inserted amino acid residues may be zero.

In the α subunit of nitrile hydratase (47), the number of terminally added amino acid residues when based on the α subunit of any of the nitrile hydratase (1) to (46) above is, for example, 1 to 60, or 1 to 40, or 1 to 20, or 1 to 10, or 1 to 5, or 1 to 3, or 1 per end. The number of added amino acid residues may be zero.
In the β subunit of nitrile hydratase (47), the number of terminally added amino acid residues when based on the β subunit of any of the nitrile hydratase (1) to (46) above is, for example, 1 to 60, or 1 to 40, or 1 to 20, or 1 to 10, or 1 to 5, or 1 to 3, or 1 per end. The number of added amino acid residues may be zero.

Nitrile hydratase (47) has one or more amino acid residue substitutions selected from the above amino acid residue substitution group B when compared with the amino acid sequence of nitrile hydratase (1) to (46). It may also include. Two or more of the above amino acid residue substitutions may be included in combination.

In the above, the α and/or β subunit variant (47) does not have any amino acid insertions or amino acid residue deletions compared to the amino acid sequence of the α and/or β subunit of a specific nitrile hydratase. Alternatively, it may have only amino acid residue substitutions or terminal additions. Regarding the position of the amino acid residue in the α and/or β subunit variant of (47), as described above, alignment with the designated amino acid residue in the α and/or β subunit of a specific nitrile hydratase is performed. means the corresponding position above.

In a first embodiment, the mutant nitrile hydratase is
A modified α subunit having 70% or more sequence identity to a specific nitrile hydratase of any of the above nitrile hydratases (1) to (46) or to the α subunit of a specific nitrile hydratase. and for a modified nitrile hydratase having a modified β subunit having 70% or more sequence identity to the β subunit of a particular nitrile hydratase, but not the particular nitrile hydratase itself;
It may be one in which at least one of the following amino acid residue substitutions has been introduced (hereinafter also referred to as mutant nitrile hydratase C):
Substitution of the amino acid residue corresponding to the 40th amino acid residue from the N-terminus of the α subunit of a specific nitrile hydratase with Asn,
Substitution of the amino acid residue corresponding to the 43rd amino acid residue from the N-terminus of the α subunit of a specific nitrile hydratase with Val,
Substitution of the amino acid residue corresponding to the 205th amino acid residue from the N-terminus of the β subunit of a specific nitrile hydratase with Val,
Substitution of the amino acid residue corresponding to the 206th amino acid residue from the N-terminus of the β subunit of a specific nitrile hydratase with Gln,
Substitution of the amino acid residue corresponding to the 215th amino acid residue from the N-terminus of the β subunit of a specific nitrile hydratase with Asn.

In the above explanation, when the nitrile hydratase before introduction is a modified nitrile hydratase, "the amino acid residue corresponding to the A-th amino acid residue from the N-terminus of the α subunit of a specific nitrile hydratase" means: When the amino acid sequence of the α subunit of the modified nitrile hydratase is aligned with the amino acid sequence of the α subunit of a specific nitrile hydratase, the refers to the amino acid residues on the amino acid sequence of the α subunit of the modified nitrile hydratase that correspond to the amino acid residues in the α subunit of the modified nitrile hydratase.
Similarly, "the amino acid residue corresponding to the A-th amino acid residue from the N-terminus of the β subunit of a specific nitrile hydratase" refers to the amino acid sequence of the β subunit of a modified nitrile hydratase. The amino acid sequence of the β subunit of the modified nitrile hydratase that corresponds to the A-th amino acid residue from the N-terminus of the amino acid sequence of the β subunit of the specific nitrile hydratase when aligned to the amino acid sequence of the β subunit. Refers to the amino acid residue of
In addition, when the nitrile hydratase before introduction is a specific nitrile hydratase, "the amino acid residue corresponding to the A-th amino acid residue from the N-terminus of the α subunit of the specific nitrile hydratase" means the specific nitrile hydratase. Refers to the A-th amino acid residue from the N-terminus of the α-subunit of a nitrile hydratase, and "the amino acid residue corresponding to the A-th amino acid residue from the N-terminus of the β-subunit of a specific nitrile hydratase" Refers to the A-th amino acid residue from the N-terminus of the β subunit of a specific nitrile hydratase.

In the modified nitrile hydratase, the sequence identity of the α subunit to the α subunit of a specific nitrile hydratase may be 80% or more, or 85% or more, or 90% or more, or 95% or more. % or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more. Similarly, the sequence identity of the β subunit to the β subunit of the particular nitrile hydratase may be 80% or more, or 85% or more, or 90% or more, or 95% or more. It may be good, or it may be 96% or more, or it may be 97% or more, or it may be 98% or more, or it may be 99% or more.
Items describing the amino acid sequence differences between the modified nitrile hydratase and the wild type nitrile hydratase from Pseudonocardia thermophila (including explanations of the extent and examples of sequence modification) are "Nitrile hydratase derived from Pseudonocardia thermophila" can be read as a specific nitrile hydratase and applied as is. In the above, the amino acid sequence of the α and/or β subunit of the modified nitrile hydratase is an insertion of an amino acid or a deletion of an amino acid residue compared to the amino acid sequence of the α and/or β subunit of a specific nitrile hydratase. It may not have any, but may have only amino acid residue substitutions or terminal additions. The modified nitrile hydratase should also, of course, have nitrile hydratase activity.

Regarding the amino acid residue substitutions and combinations thereof that the mutant nitrile hydratase C may contain, and the amino acid residue corresponding to the 36th position from the N-terminus of the α subunit of a specific nitrile hydratase, the mutant nitrile hydratase C is Combinations a to o of amino acid residues listed in Table 1, combinations p to ai of amino acid residues listed in Table 2, combinations aj to ax of amino acid residues listed in Table 3, amino acid residues listed in Table 4 The combinations of amino acid residues may include combinations of amino acid residues selected from the group consisting of combinations ay to bd, and combinations of amino acid residues be listed in Table 5. That is, such a combination of amino acid residue substitutions can be introduced into the amino acid sequence of a specific nitrile hydratase or modified nitrile hydratase.

Furthermore, for example, the modified nitrile hydratase has one or more amino acid residues selected from the above amino acid residue substitution group B when compared with the amino acid sequence of any of the nitrile hydratases (1) to (46). It may include group substitution. Two or more of the above amino acid residue substitutions may be included in combination.

The modified nitrile hydratase described above consists of the α subunit specified above or a polypeptide with an additional sequence added to its N-terminus or C-terminus, or both, and the β subunit specified above or its polypeptide. It may also be a nitrile hydratase having a polypeptide with an additional sequence added to the N-terminus, C-terminus, or both.
In the α subunit of the modified nitrile hydratase, the number of terminally added amino acid residues is, for example, 1 to 60, or 1 to 40, or 1 to 20, or 1 to 10 per terminal. , or 1 to 5, or 1 to 3, or 1. The number of added amino acid residues may be zero.
In the β subunit of the modified nitrile hydratase, the number of terminally added amino acid residues is, for example, 1 to 60, or 1 to 40, or 1 to 20, or 1 to 10 per terminal. , or 1 to 5, or 1 to 3, or 1. The number of added amino acid residues may be zero.

In the first embodiment, when the mutant nitrile hydratase contains at least one amino acid residue substitution selected from the group consisting of (4A), (5A), and (31A) to (33A), the mutant nitrile hydratase The nitrile hydratase may contain only one amino acid residue substitution of (4A), (5A), and (31A) to (33A), or may contain two or more.
For example, in the case of two combinations, a combination of (4A) and (5A), a combination of (4A) and (31A), a combination of (4A) and (32A), a combination of (4A) and (33A), a combination of (5A) ) and (31A), (5A) and (32A), (5A) and (33A), (31A) and (32A), (31A) and (33A), (32A) ) and (33A), and (32A) and (3Bf).

In the case of three combinations, for example, a combination of (4A), (5A) and (31A), a combination of (4A), (5A) and (32A), a combination of (4A), (5A) and (33A), A combination of (4A), (31A) and (32A), a combination of (4A), (31A) and (33A), a combination of (4A), (32A) and (33A), (5A), (31A) and Examples include the combination of (32A), the combination of (5A), (31A) and (33A), the combination of (5A), (32A) and (33A), and the combination of (31A), (32A) and (33A). It will be done.
In the case of four combinations, for example, a combination of (4A), (5A), (31A) and (32A), a combination of (4A), (5A), (31A) and (33A), (4A), (5A) ), (32A) and (33A), (4A), (31A), (32A) and (33A), and (5A), (31A), (32A) and (33A). It will be done.
Of course, five of (4A) to (33A) may be substituted in combination.

As described above, the modified nitrile hydratase to which at least one of the amino acid residue substitutions (4A), (5A), and (31A) to (33A) is introduced is a wild-type pseudonolyte. The nitrile hydratase derived from Cardia thermophila or the above specific sequences (i) to (v) may contain modifications such as amino acid substitutions. Therefore, compared to the wild type nitrile hydratase derived from Pseudonocardia thermophila or the specific sequences (i) to (v) above, the mutant nitrile hydratase also has the above (4A) and (5A). In addition to amino acid residue substitutions in , and (31A) to (33A), amino acid residue substitutions at positions other than the amino acid residue substitutions in (4A), (5A), and (31A) to (33A) above. It doesn't matter if it includes. As described above, even in a mutant nitrile hydratase containing other amino acid residue substitutions, the effects of specific amino acid residue substitutions located at specific positions in the steric structure can be obtained.

<Second embodiment of nitrile hydratase>
As a second embodiment, the mutant nitrile hydratase is (1L), (2L), (7L), (8L), (10L) from the viewpoint of efficiently obtaining a 3-hydroxycarboxylic acid compound or a salt thereof. ), (12L), (14L), (22L) to (25L), (32L), and (35L) to (37L) substituted with another amino acid residue at least one position selected from the group consisting of (1A), (2A), (7A), (8A), (10A), (12A), (14A), (22A) to (25A), (32A), and (35A) It is more preferable that at least one amino acid residue substitution selected from the group consisting of ) to (37A) is included.
In the second embodiment, the mutant nitrile hydratase is (1L), (2L), (7L), (8L), (10L), (12L), (14L), (22L) to (25L), ( 32L) and at least one amino acid residue substitution selected from the group consisting of (35L) to (37L), in addition to these amino acid residue substitutions, an amino acid residue selected from the above-mentioned amino acid residue substitution group B It may further contain at least one amino acid residue.

In addition, in the second embodiment, the mutant nitrile hydratase is α of nitrile hydratase derived from wild-type Pseudonocardia thermophila from the viewpoint of efficiently producing a 3-hydroxycarboxylic acid compound or a salt thereof. It may further contain an amino acid substitution residue in which the corresponding amino acid residue Thr from the N-terminus of the subunit is substituted.

In the second embodiment, examples of amino acid residue substitutions include, for example, the amino acid residue substitutions listed in Tables 13 to 17, but the mutant nitrile hydratase is not limited to the combinations of these amino acid residue substitutions. Needless to say.
Furthermore, the sequence numbers listed in Tables 13 to 17 are the sequence numbers listed in the sequence listing in International Publication No. 2010/055666.

<Third embodiment of nitrile hydratase>
As a third embodiment, the mutant nitrile hydratase is (6L), (9L), (11L), (13L), (15L) from the viewpoint of efficiently obtaining a 3-hydroxycarboxylic acid compound or a salt thereof. ) to (21L), (26L) to (30L) and (34L) is preferably substituted with another amino acid residue.
In addition to the substitution positions in the third embodiment, in the α subunit, from the N-terminus of SEQ ID NO: 1, the 6th, 19th, 38th, 77th, 90th, 102nd, 106th, 126th, Other amino acids at at least one position selected from the group consisting of the 130th, 142nd, 146th, 187th, 194th and 203rd positions, and the 20th and 21st positions from the N-terminus of SEQ ID NO: 2 in the β subunit It may be substituted with a residue.

In the third embodiment, the mutant nitrile hydratase is (6A), (9A), (11A), (13A), It is preferable that at least one amino acid residue substitution selected from the group consisting of (16A), (17A) to (20A), (26A), (27A), (29A) and (30A) is included.

In the third embodiment, the mutant nitrile hydratase is (6A), (9A), (11A), (13A), In addition to at least one amino acid residue substitution selected from the group consisting of (16A), (17A) to (20A), (26A), (27A), (29A) and (30A), an amino acid residue substitution group B, or at least one amino acid residue substitution selected from the group consisting of (1B) to (3B), (5B), (11B), (15B) and (16B). You can stay there.

In a third embodiment, the mutant nitrile hydratase consists of (6A), (9A), (11A), (13A), (15A) to (21A), (26A) to (30A) and (34A). Examples of the amino acid residue substitutions include the amino acid residue substitutions listed in Tables 18 and 19 below, when the mutant nitrile hydratase contains at least one amino acid residue substitution selected from the group. Needless to say, it is not limited to the combination of amino acid residue substitutions.

Mutant nitrile hydratase generally functions by association of two α subunits as defined above and two β subunits as defined above.

A plasmid capable of expressing a large amount of wild-type nitrile hydratase derived from Pseudonocardia thermophila in a transformant and a cell line transformed with the same plasmid are MT-10822 (Accession No. FERM BP-5785, Ibaraki Prefecture). (Deposited with the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba City, Japan since February 7, 1996) can be used.
Modified nitrile hydratases can also be obtained using conventional genetic engineering techniques. In general, it is said that when a protein has a high degree of homology in its amino acid sequence, there is a high probability that the protein will have a similar structure regardless of the species and genus of the strain containing the protein, so amino acid residue substitution (1A) The effect obtained by introducing one or more of ~(37A) is exerted on nitrile hydratases from Pseudonocardia thermophila as defined above in general, including modified nitrile hydratases.

<Method for producing mutant nitrile hydratase>
The method for producing a mutant nitrile hydratase may include culturing a transformant in a medium, and recovering the mutant nitrile hydratase from at least one of the cultured transformant and the medium. preferable.

Mutant nitrile hydratase can be produced by the following method.
For example, an expression vector containing DNA encoding a mutant nitrile hydratase is prepared, an arbitrary host cell is transformed with this expression vector to obtain a transformant or a cell line, and then a transformant or cell line is obtained. can be cultured to produce mutant nitrile hydratase.
As a method for producing an expression vector, for example, the method described in paragraphs 0111 to 0131 of International Publication No. 2018/124247 can be used.

Here, the gene encoding the wild-type nitrile hydratase derived from Pseudonocardia thermophila consists of the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4. The nucleotide sequence shown in SEQ ID NO: 3 corresponds to the amino acid sequence of the wild type nitrile hydratase derived from Pseudonocardia thermophila shown in SEQ ID NO: 1, and the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence of nitrile hydratase from Cardia thermophila corresponds to the amino acid sequence consisting of SEQ ID NO:2.
The DNA encoding the mutant nitrile hydratase contains at least codons corresponding to the amino acid residue substitution positions (1A) to (37A) in the nucleotide sequence shown in SEQ ID NO: 3 or the nucleotide sequence shown in SEQ ID NO: 4. It has the following nucleotide substitutions at one or more of the following positions (total of 5 positions), and also has nucleotide substitutions corresponding to the modified nitrile hydratase at other positions.

As the DNA encoding the mutant nitrile hydratase, for example, the DNA described in paragraphs 0105 to 0110 of International Publication No. 2018/124247 can be used.

Furthermore, in the second embodiment, the base sequences described in paragraphs 0038 to 0046 of International Publication No. 2010/055666 can be used as the DNA encoding the mutant nitrile hydratase.

In the third embodiment, the DNA encoding the mutant nitrile hydratase is, for example, the 36th position from the N-terminus of the α subunit of the nitrile hydratase derived from wild-type Pseudonocardia thermophila. When creating a mutant in which the Tyr residue, which is an amino acid residue, is replaced with Met, use a DNA in which ACG, which is the 106th to 108th nucleotide from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1, is replaced with ATG. do it.
Specific examples of DNA encoding the mutant nitrile hydratase in the third embodiment are listed in Tables 20 and 21 below.
Mutant nitrile hydratase can be produced using the below-mentioned expression vectors encoding the DNAs listed in Tables 20 and 21.
In Tables 20 and 21, the "position from the 5'end" is the nucleotide position counted from the 5' end of the nucleotide sequence shown by SEQ ID NO: 3 or 4. For example, "position from the 5'end" in SEQ ID NO: 1 is "106th to 108th" means the 106th to 108th nucleotides from the 5' end of the nucleotide sequence shown by SEQ ID NO: 1.

[Method for producing transformants]
Transformants can be produced by known methods. For example, a method may be used in which an expression vector containing a nucleotide sequence encoding a mutant nitrile hydratase and, if necessary, other regions described above is constructed, and the expression vector is transformed into a desired host cell. Specifically, Sambrook, J. , etc. al. , "Molecular Cloning A Laboratory Manual, 3rd Edition", Cold Spring Harbor Laboratory Press, (2001), etc., using general methods known in the fields of molecular biology, bioengineering, and genetic engineering. things to do can.

The transformant not only integrates the expression vector into the host cell, but also introduces silent mutations, if necessary, so that codons that are used less frequently in the host cell become codons that are used more frequently. It can also be produced by
Thereby, it may be possible to increase the amount of protein produced from the mutant nitrile hydratase incorporated into the expression vector.

Regarding the introduction of silent mutations, any method involves adjusting the codons of the nitrile hydratase gene on the expression vector and the signal sequence for secreting the nitrile hydratase gene to the outside of the cell in accordance with the codon usage frequency in the host cell. For example, the method of silent mutagenesis, the mutation point, the type of nucleotide to be changed, etc. are not particularly limited.

-Culture method of transformants-
The conditions for culturing the transformant obtained by transformation with the expression vector are the same as the culture conditions for the host cell before transformation, and known conditions can be used.
As the medium, either a synthetic medium or a natural medium can be used as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and other nutrients.
As the culture medium components, known components used for culture media can be used. For example, organic nutritional sources such as meat extract, yeast extract, malt extract, peptone, NZ amines and potatoes; carbon sources such as glucose, maltose, sucrose, starch and organic acids; nitrogen sources such as ammonium sulfate, urea and ammonium chloride; Acid salts, inorganic nutritional sources such as magnesium, potassium, and iron, and vitamins can be used in appropriate combinations.
When culturing a transformant transformed with an expression vector containing a selection marker, for example, if the selection marker is drug resistant, use a medium containing the corresponding drug, and if the selection marker is auxotrophic. If so, a medium that does not contain the corresponding nutrients may be used.
The pH of the medium can be appropriately selected within the range of pH 4 to pH 8.
The transformant can be cultured using conventional culture methods such as shaking culture, aerated agitation culture, continuous culture, and fed-batch culture in a liquid medium containing the transformant.
Culture conditions may be appropriately selected depending on the type of transformant, medium, and culture method, and are not particularly limited as long as the conditions allow the transformant to grow and produce mutant nitrile hydratase.
The culture is carried out aerobically at a temperature of preferably 20°C to 45°C, more preferably 24°C to 37°C.
The culture period may range from 1 day to 7 days until the content of the protein having the desired mutant nitrile hydratase activity is maximized.

- Mutant nitrile hydratase recovery process -
The method for producing a mutant nitrile hydratase preferably includes a step of recovering the mutant nitrile hydratase from at least one of the cultured transformant and the culture medium.

After culturing the transformed transformant, a method commonly used in this field can be used to recover the mutant nitrile hydratase.
When the mutant nitrile hydratase is secreted outside the transformant, a crude enzyme solution containing the mutant nitrile hydratase can be easily obtained by centrifuging, filtering, etc. the culture of the transformant.
In addition, if the mutant nitrile hydratase accumulates in the transformant, the cultured transformant should be collected by centrifugation, etc., then the collected transformant should be suspended in a buffer solution, and this suspension should be A crude enzyme solution containing the mutant nitrile hydratase may be recovered by disrupting the cell membrane of the transformant according to known methods such as lysozyme treatment, freeze-thawing, and ultrasonic disruption.

The crude enzyme solution containing the recovered mutant nitrile hydratase can be further concentrated by ultrafiltration, etc., and can be used as a concentrated mutant nitrile hydratase (concentrated enzyme) by adding a preservative, etc. . Alternatively, after concentrating the mutant nitrile hydratase, the mutant nitrile hydratase may be made into a powder (powdered enzyme) by a spray drying method or the like.
If the crude enzyme solution containing the recovered mutant nitrile hydratase requires separation and purification, for example, salting out with ammonium sulfate, organic solvent precipitation with alcohol, membrane separation using dialysis, ultrafiltration, etc. Alternatively, separation and purification can be carried out by appropriately combining known chromatographic separation methods such as ion exchange chromatography, reversed phase high performance chromatography, affinity chromatography, and gel filtration chromatography.

<3-hydroxynitrile compound>
The 3-hydroxynitrile compound used in the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure is used in the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof from the above-mentioned 3-hydroxyamide compound. There are no particular limitations as long as a 3-hydroxyamide compound can be obtained.
Groups other than the cyano group and the hydroxy group (ie, substituents) that the 3-hydroxynitrile compound has have the same meanings as the substituents that the above-mentioned 3-hydroxyamide compound has, and their preferred embodiments are also the same.

The 3-hydroxynitrile compound preferably has 3 to 20 carbon atoms, more preferably 3 to 15 carbon atoms, and still more preferably 3 to 10 carbon atoms.
The 3-hydroxynitrile compound is preferably a substituted or unsubstituted saturated aliphatic 3-hydroxynitrile compound, and preferably a substituted or unsubstituted saturated aliphatic 3-hydroxynitrile compound having 3 to 20 carbon atoms. More preferred.

Specifically, the 3-hydroxynitrile compound includes 3-hydroxypropionitrile,
3-hydroxybutyronitrile, 3-hydroxy-3-methylbutyronitrile,
3-hydroxypentanenitrile, 3-hydroxy-3-methylpentanenitrile, 3-ethyl-3-hydroxypentanenitrile, 3-hydroxy-3,4-dimethylpentanenitrile, 2-propyl-3-hydroxypentanenitrile, 3- Hydroxy-3,4,4-trimethylpentanenitrile, 3-ethyl-3-hydroxy-4-methylpentanenitrile, 3-hydroxy-3-isopropyl-4-methylpentanenitrile, 3-ethyl-3-hydroxy-4, 4-dimethylpentanenitrile, 3-hydroxy-4,4-dimethyl-3-isopropylpentanenitrile, 3-tert-butyl-3-hydroxy-4,4-dimethylpentanenitrile,
3-hydroxyhexanenitrile, 3-hydroxy-3-methylhexanenitrile, 3-hydroxy-3,4-dimethylhexanenitrile, 3-hydroxy-3,5-dimethylhexanenitrile, 3-ethyl-3-hydroxyhexanenitrile, 3-Hydroxy-3-propylhexanenitrile, 3-hydroxy-3-isopropylhexanenitrile, 3-ethyl-3-hydroxy-4-methylhexanenitrile, 3-ethyl-3-hydroxy-5-methylhexanenitrile, 3- tert-butyl-3-hydroxyhexanenitrile, 3-hydroxy-4-methyl-3-propylhexanenitrile, 3-hydroxy-5-methyl-3-propylhexanenitrile, 3-hydroxy-4-methyl-3-isopropylhexane Nitrile, 3-hydroxy-5-methyl-3-isopropylhexanenitrile, 3-isobutyl-3-hydroxy-4-methylhexanenitrile, 3-isobutyl-3-hydroxy-5-methylhexanenitrile, 3-sec-butyl- 3-hydroxy-4-methylhexanenitrile, 3-tert-butyl-3-hydroxy-4-methylhexanenitrile,
3-Hydroxyheptanenitrile, 3-hydroxy-3-methylheptanenitrile, 3-ethyl-3-hydroxyheptanenitrile, 3-hydroxy-3-propylheptanenitrile, 3-hydroxy-3-isopropylheptanenitrile, 3-butyl- 3-hydroxyheptanenitrile, 3-isobutyl-3-hydroxyheptanenitrile, 3-sec-butyl-3-hydroxyheptanenitrile, 3-tert-butyl-3-hydroxyheptanenitrile,
3-hydroxyoctanenitrile,
unsubstituted saturated aliphatic 3-hydroxynitrile compounds such as 3-hydroxynonanenitrile;

4-fluoro-3-hydroxybutyronitrile, 4-chloro-3-hydroxybutyronitrile, 4-bromo-3-hydroxybutyronitrile, 4-iodo-3-hydroxybutyronitrile, 4-chloro-3- Saturated aliphatic 3-hydroxynitrile compounds substituted with halogen atoms such as hydroxy-3-methylbutyronitrile and 4-chloro-3-hydroxy-5-methylhexanenitrile;
3,4-dihydroxybutyronitrile, 3,4-dihydroxy-2-methylbutyronitrile, 3,4-dihydroxy-3-methylbutyronitrile,
3,5-dihydroxypentanenitrile, 3,5-dihydroxy-3-methylpentanenitrile,
Saturated aliphatic 3-hydroxynitrile compounds substituted with hydroxy groups such as 3,5-dihydroxyhexanenitrile, 3,5-dihydroxyheptanenitrile, 3,5-dihydroxyoctanenitrile, 3,5-dihydroxydecanenitrile,
3-hydroxy-4-methoxybutyronitrile, 3-hydroxy-4-ethoxybutyronitrile, 3-hydroxy-4-propyloxybutyronitrile 3-hydroxy-5-methoxypentanenitrile, 3-hydroxy-5-methoxy -Saturated aliphatic 3-hydroxynitrile compounds substituted with alkoxy groups such as 3-methylpentanenitrile, 3-hydroxy-5-ethoxypentanenitrile, 3-hydroxy-5-methoxyhexanenitrile,
Examples include saturated aliphatic 3-hydroxynitrile compounds substituted with alkylthio groups such as 3-hydroxy-2-methyl-2-methylthiopropionitrile and 3-hydroxy-4-methylthiobutyronitrile.

<Water (raw water)>
The raw water used in the hydration reaction from a 3-hydroxynitrile compound to a 3-hydroxyamide compound is the same as the raw water used in the above-mentioned method for producing a 3-hydroxycarboxylic acid compound or a salt thereof from a 3-hydroxyamide compound. They have the same meaning, and their preferred embodiments are also the same.

<Aqueous medium>
The aqueous medium used in the hydration reaction from a 3-hydroxynitrile compound to a 3-hydroxyamide compound is the same as the aqueous medium used in the above-mentioned method for producing a 3-hydroxycarboxylic acid compound or a salt thereof from a 3-hydroxyamide compound. They have the same meaning, and their preferred embodiments are also the same.

<pH adjuster>
The pH adjuster used in the hydration reaction from a 3-hydroxynitrile compound to a 3-hydroxyamide compound is a pH adjuster used in the above-mentioned method for producing a 3-hydroxycarboxylic acid compound or a salt thereof from a 3-hydroxyamide compound. It has the same meaning as agent, and its preferred embodiments are also the same.

A method of reacting a 3-hydroxynitrile compound with at least one member selected from the group consisting of nitrile hydratase-containing microbial cells and their processed products (nitrile hydratase catalysts) in an aqueous medium (hydration reaction). There are no particular limitations on the method (method 2), but examples include methods (1) to (3).
(1) A method in which the nitrile hydratase catalyst, reaction raw materials (3-hydroxynitrile compound and raw water), aqueous medium, and pH adjuster are charged into a reaction tank in their entirety at once and then the reaction is carried out (batch reaction).
(2) After charging the nitrile hydratase catalyst, reaction raw materials (3-hydroxynitrile compound and raw water), aqueous medium, and part of the pH adjuster into the reaction tank, the remaining nitrile hydratase catalyst is continuously or intermittently charged. , a method of carrying out a reaction by supplying reaction raw materials (3-hydroxynitrile compound and raw water), an aqueous medium and a pH adjuster (semi-batch reaction).
(3) Continuous or intermittent supply of nitrile hydratase catalyst, reaction raw materials (3-hydroxynitrile compound and raw material water), aqueous medium and pH adjuster, and reaction solution (nitrile hydratase catalyst, unreacted raw materials, aqueous A method in which the reaction is carried out continuously without taking out the entire amount of the reaction liquid in the reaction tank (continuous reaction).

In the reaction of converting a 3-hydroxynitrile compound into a 3-hydroxyamide compound (hereinafter also referred to as "hydration reaction"), the hydration reaction is preferably carried out in the presence of a catalyst. The nitrile hydratase catalyst is preferably used in a suspended bed; for example, a suspension of the nitrile hydratase catalyst may be prepared and the suspension may be supplied to a reaction tank.

The reaction tank temperature, which is the temperature of the liquid in the reaction tank, is usually set at 0 to 50°C, preferably 10 to 40°C, although it depends on the heat resistance of the nitrile hydratase catalyst. It is preferable that the reaction tank temperature is within the above range because the activity of the nitrile hydratase catalyst can be maintained well.

The reaction is generally carried out under normal pressure, but can also be carried out under increased pressure in order to increase the solubility of the 3-hydroxynitrile compound.

The pH in the reaction tank is not particularly limited as long as it is within a suitable range for maintaining good activity of the nitrile hydratase catalyst, but is preferably in the range of pH 5 to pH 10. It is preferable that the pH is within the above range because the activity of the nitrile hydratase catalyst can be maintained well.

In the present embodiment, a reaction of converting a 3-hydroxynitrile compound into a 3-hydroxyamide compound (hydration reaction) and a reaction of converting a 3-hydroxyamide compound into a 3-hydroxycarboxylic acid compound or a salt thereof (hereinafter referred to as (sometimes referred to as "hydrolysis reaction") may be carried out separately, the hydration reaction and the hydrolysis reaction may be carried out simultaneously, or the 3-hydroxyamide compound may be A method of performing a hydrolysis reaction after isolation and purification may also be used.

Alternatively, for example, a 3-hydroxycarboxylic acid compound or a salt thereof may be produced in one pot by simultaneously adding a 3-hydroxynitrile compound, nitrile hydratase, and amidase. "One-pot" as used herein means that several reactions are carried out in the same reaction vessel, and specifically, it means that the hydration reaction and the hydrolysis reaction are carried out in the same reaction vessel.

3-hydroxynitrile compounds can be catalyst poisons depending on the type of enzyme. Therefore, using an enzyme that is highly stable against 3-hydroxynitrile compounds is advantageous for industrial production because the reaction yield can be improved and the amount of enzyme used can be reduced.
By using nitrile hydratase and amidase, which are highly stable for 3-hydroxynitrile compounds, hydration and hydrolysis reactions can be carried out simultaneously.
Nitrile hydratase and amidase derived from bacteria of the genus Pseudonocardia as nitrile hydratase and amidase having high stability against 3-hydroxynitrile compounds, and a method for producing 3-hydroxycarboxylic acid compounds and salts thereof according to the present disclosure Examples include amidases derived from specific bacteria used for
Furthermore, in order to avoid the catalyst poison of the 3-hydroxynitrile compound, the hydrolysis reaction may be carried out after the hydration reaction is completed. In order to avoid the catalyst poison of the 3-hydroxynitrile compound, the amidase may be added after the hydration reaction is completely completed, or the 3-hydroxynitrile compound does not inhibit the hydrolysis reaction during the hydration reaction. After reducing the amount to a certain extent, amidase may be added to carry out the hydrolysis reaction.
The amidases used in the hydrolysis reaction include the above-mentioned amidases, and preferred embodiments are also the same.

Next, examples of the present disclosure will be specifically described, but the present disclosure is not limited to these examples.
<1. Construction of amidase-producing bacteria>
(1) Amidase-producing bacteria of Pseudonocardia thermophila JCM3095 The nucleotide sequence of plasmid pPT-B1 described in JP-A-9-275978 was analyzed, and based on the nucleotide sequence of the plasmid, primers of SEQ ID NO: 5 and 6 in the sequence listing were used. designed. PCR was performed using the chromosomal DNA of Pseudonocardia thermophila JCM3095 as a template to amplify the amidase gene. The amplified gene was digested with EcoRI and HindIII. Similarly, pUC18 (manufactured by Takara Bio Co., Ltd.) digested with restriction enzymes was ligated using Ligation High (manufactured by Toyobo Co., Ltd.), and introduced into Competent High DH5α (manufactured by Toyobo Co., Ltd.) to create Pseudonocardia thermophila JCM3095. Amidase-producing bacteria were created.
Hereinafter, the produced bacteria will be referred to as "Pth-amidase producing bacteria."

(2) Amidase-producing strain of Acidphilium cryptum JF-5 NBRC-14242 was purchased from NBRC (Biological Genetic Resources Division, Biotechnology Headquarters, National Institute of Technology and Evaluation). NBRC-14242 was cultured in NBRC medium number 234, and chromosomal DNA was obtained using DNeasy Blood & Tissue Kit (Qiagen Corporation). SEQ ID NO: 7 and 8 as a primer and the chromosomal DNA obtained by culturing NBRC-14242 as a template to amplify the amidase gene. The amplified amidase gene was digested with EcoRI and HindIII. Similarly, pUC18 (manufactured by Takara Bio Co., Ltd.) digested with restriction enzymes was ligated using Ligation High (Toyobo Co., Ltd.), and introduced into Competent High DH5α (manufactured by Toyobo Co., Ltd.) to obtain amidase-producing bacteria. Created.
Hereinafter, the produced bacterium will be referred to as "Acr-amidase producing bacterium."

(3) Polaromonas sp. Amidase-producing bacteria of JS666 Polaromonas sp. from ATCC (American Type Culture Collection). ATCC-BAA-500D-5, which is the chromosomal DNA of JS666, was purchased. Polaromonas sp. registered with NCIBM (National Center for Biotechnology Information). Based on the amidase gene information of JS666 (http://www.ncbi.nlm.nih.gov/nuccore/91785913, Gene ID: 4010994), SEQ ID NOs: 9 and 10 in the sequence list were used as primers, and ATCC-BAA-500D-5 The amidase gene was amplified using this as a template. The amplified amidase gene was digested with EcoRI and HindIII. pUC18 (manufactured by Takara Bio Co., Ltd.) similarly digested with restriction enzymes was ligated using Ligation High (manufactured by Toyobo Co., Ltd.), and introduced into Competent High DH5α (manufactured by Toyobo Co., Ltd.) to produce amidase-producing bacteria. was created.
Hereinafter, the produced bacteria will be referred to as "Psp-amidase producing bacteria."
Note that the Psp-amidase-producing bacteria are bacteria that produce amidase that is not derived from a specific bacterium.

<2. Cultivation of amidase-producing bacteria>
100 mL of LB liquid medium was prepared in a 500 mL baffled Erlenmeyer flask, and sterilized by autoclaving at 121° C. for 20 minutes. Ampicillin was added to this medium to a final concentration of 100 μg/mL, and each of the prepared amidase-producing bacteria was inoculated into Ichiro bacillus ear and cultured at 30°C and 130 rpm (revolution per minute) for about 20 hours. . Only the bacterial cells were separated from the culture solution by centrifugation (5000 G (49,000 m/s ² ) x 15 minutes), and then the bacterial cells were resuspended in 5 mL of physiological saline, and each bacterial cell suspension was Obtained. The bacterial cell suspension was frozen in a freezer, thawed, and used.

<3-1. Construction and cultivation of Pseudonocardia thermophila JCM3095/nitrile hydratase producing bacteria>
Prepare 100 mL of LB liquid medium containing 40 μg/mL ferric sulfate heptahydrate and 10 μg/mL cobalt chloride dihydrate in a 500 mL baffled Erlenmeyer flask, and autoclave at 121° C. for 20 minutes. Sterilized by After adding ampicillin to this medium to a final concentration of 100 μg/mL, deposited microorganism MT-10822 (FERM BP-5785, Patent Organism Deposit, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture) (deposited with the Center on February 7, 1996) was inoculated into the ear of Ippaku and cultured at 37° C. and 130 rpm for about 20 hours. Only the bacterial cells were separated from the culture solution by centrifugation (5000G x 15 minutes), and then the bacterial cells were resuspended in 5 mL of physiological saline to obtain a bacterial cell suspension. The bacterial cell suspension was frozen in a freezer, thawed, and used. This bacterium is referred to as a "Pth-nitrile hydratase producing bacterium."

<3-2. Construction and cultivation of Pseudomonas putida NBRC12668/nitrile hydratase producing bacteria>
1 above. Construction of amidase-producing bacteria (1) Using the chromosomal DNA used in the amidase-producing bacteria of Pseudomonas putida NBRC12668 as a template, PCR was performed using SEQ ID NO: 11 and 12 in the sequence listing as primers to amplify the nitrile hydratase gene. The amplified nitrile hydratase gene was digested with EcoRI and HindIII. pUC18 (manufactured by Takara Bio Co., Ltd.) similarly digested with restriction enzymes was ligated using Ligation High (manufactured by Toyobo Co., Ltd.), and introduced into Competent High DH5α (manufactured by Toyobo Co., Ltd.) to obtain Pseudomonas putida NBRC12668. We created a nitrile hydratase-producing bacterium.
Hereinafter, the produced bacterium will be referred to as "Ppu-nitrile hydratase producing bacterium."
A 100 mL LB liquid medium containing 40 μg/mL ferric sulfate heptahydrate was prepared in a 500 mL baffled Erlenmeyer flask and sterilized by autoclaving at 121° C. for 20 minutes. After adding ampicillin to this medium to a final concentration of 100 μg/mL, the Ppu-nitrile hydratase-producing bacteria prepared above was inoculated into the Ichiro bacterium ear and cultured at 37°C and 130 rpm for about 20 hours. .
The above culture solution was centrifuged (5000G x 15 minutes) to isolate only the bacterial cells, and then the separated bacterial cells were resuspended in 5 mL of physiological saline to obtain a bacterial cell suspension. The bacterial cell suspension was frozen in a freezer, thawed, and used.

[Example 1]
Wet bacterial cells containing Pth-nitrile hydratase producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 0.1 wt%. Using a 0.1M NaOH aqueous solution as a pH adjuster, the pH at 30°C (hereinafter sometimes simply referred to as "pH") was adjusted to 8.0. 10 g of 3-hydroxypropionitrile (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the above suspension and stirred at 30°C to advance the hydration reaction. Ten hours after adding 3-hydroxypropionitrile, analysis was performed under the following HPLC conditions, and the 3-hydroxypropionitrile concentration was below the detection limit (10 mass ppm or less). Although it was confirmed that the conversion rate from 3-hydroxypropionitrile to 3-hydroxypropionamide was 99% or more, no production of 3-hydroxypropionic acid was observed.
Next, 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water at a wet cell concentration of 10 wt% was added and stirred at 30°C to advance the hydrolysis reaction. When analysis was conducted under the following HPLC conditions 10 hours after addition of the suspension, the 3-hydroxypropionamide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 3-hydroxypropionamide to 3-hydroxypropionic acid was 99% or more.

Here, the analysis conditions were as follows.
・HPLC analysis conditions:
High-performance liquid chromatography device: LC-10A system (manufactured by Shimadzu Corporation)
(UV detector wavelength 250nm, column temperature 40℃)
Separation column: SCR-101H (manufactured by Shimadzu Corporation)
Eluent: 7% (by volume)-acetonitrile aqueous solution

[Example 2]
In Example 1, instead of adding 10 g of a suspension of Pth-amidase-producing bacteria diluted with pure water and a wet bacterial cell concentration of 10% by mass, Acr-amidase-producing bacteria were diluted with pure water and a wet bacterial cell concentration of 10% was added. A hydrolysis reaction was carried out in the same manner as in Example 1, except that 10 g of the mass% suspension was added.
When analyzed by HPLC 10 hours after the start of the hydrolysis reaction, the 3-hydroxypropionamide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 3-hydroxypropionamide to 3-hydroxypropionic acid was 99% or more.

[Comparative example 1]
Wet bacterial cells containing Pth-nitrile hydratase-producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. The pH was adjusted to 8.0 using a 0.1M NaOH aqueous solution as a pH adjuster. 10 g of 3-hydroxypropionitrile (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the above suspension and stirred at 30°C to advance the hydration reaction. When analyzed by HPLC after 10 hours, the 3-hydroxypropionitrile concentration was below the detection limit (10 mass ppm or below). The conversion rate of 3-hydroxypropionitrile to 3-hydroxypropionamide was 99% or more, and no production of 3-hydroxypropionic acid was observed.
Next, 10 g of a suspension of Psp-amidase producing bacteria, which is not an amidase derived from a specific bacterium, diluted with pure water and having a wet bacterial cell concentration of 10% by mass was added and stirred at 30°C to initiate a hydrolysis reaction. I let it progress. Analysis by HPLC after 10 hours revealed that the 3-hydroxypropionamide concentration was 26% by mass, and the conversion rate of 3-hydroxypropionamide to 3-hydroxypropionic acid was 74%.

[Example 3]
Wet bacterial cells containing Ppu-nitrile hydratase producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. The pH was adjusted to 8.0 using a 0.1M NaOH aqueous solution as a pH adjuster. 10 g of 3-hydroxypropionitrile (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the above suspension and stirred at 30°C to advance the hydration reaction. When analyzed by HPLC after 10 hours, the 3-hydroxypropionitrile concentration was below the detection limit (10 mass ppm or below). The conversion rate to 3-hydroxypropionamide was 99% or more, and no production of 3-hydroxypropionic acid was observed. Next, 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water at a wet bacterial cell concentration of 10% by mass was added and stirred at 30°C to advance the hydrolysis reaction. HPLC analysis after 10 hours revealed that the 3-hydroxypropionamide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 3-hydroxypropionamide to 3-hydroxypropionic acid was 99% or more.

[Example 4]
In Example 3, instead of adding 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water and having a wet bacterial cell concentration of 10% by mass, Acr-amidase producing bacteria were diluted with pure water and a wet bacterial cell concentration of 10% was added. A hydrolysis reaction was carried out in the same manner as in Example 3, except that 10 g of the mass% suspension was added.
When HPLC analysis was performed 10 hours after the start of the reaction, the 3-hydroxypropionamide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 3-hydroxypropionamide to 3-hydroxypropionic acid was 99% or more.

[Example 5]
Wet bacterial cells containing Pth-nitrile hydratase-producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. The pH was adjusted to 8.0 using a 0.1M NaOH aqueous solution as a pH adjuster. 10 g of 3-hydroxy-3-methylbutyronitrile (manufactured by Merck & Co., Ltd.) was added to the above suspension and stirred at 30°C to advance the hydration reaction. After 10 hours, analysis was performed under the following HPLC conditions, and the 3-hydroxy-3-methylbutyronitrile concentration was below the detection limit (10 mass ppm or below). The conversion rate of 3-hydroxy-3-methylbutyronitrile to 3-hydroxy-3-methylbutyramide was 99% or more, and no formation of 3-hydroxy-3-methylbutanoic acid was observed.
Next, 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water at a wet bacterial cell concentration of 10% by mass was added and stirred at 30°C to advance the hydrolysis reaction. After 10 hours, analysis was performed under the following HPLC conditions, and the 3-hydroxy-3-methylbutyramide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 3-hydroxy-3-methylbutyramide to 3-hydroxy-3-methylbutanoic acid was 99% or more.
Here, the analysis conditions were as follows.
・HPLC analysis conditions:
High-performance liquid chromatography device: LC-10A system (manufactured by Shimadzu Corporation)
(UV detector wavelength 250nm, column temperature 40℃)
Separation column: SCR-101H (manufactured by Shimadzu Corporation)
Eluent: 7% (by volume)-acetonitrile aqueous solution

[Example 6]
In Example 5, instead of adding 10 g of a suspension of Pth-amidase-producing bacteria diluted with pure water at a wet bacterial cell concentration of 10% by mass, Acr-amidase-producing bacteria were diluted with pure water at a wet bacterial cell concentration of 10%. A hydrolysis reaction was carried out in the same manner as in Example 5, except that 10 g of the mass% suspension was added.
When HPLC analysis was performed 10 hours after the start of the hydrolysis reaction, the 3-hydroxy-3-methylbutyramide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 3-hydroxy-3-methylbutyramide to 3-hydroxy-3-methylbutanoic acid was 99% or more.

[Comparative example 2]
Wet bacterial cells containing Pth-nitrile hydratase-producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. The pH was adjusted to 8.0 using a 0.1M NaOH aqueous solution as a pH adjuster. 10 g of 3-hydroxy-3-methylbutyronitrile (manufactured by Merck & Co., Ltd.) was added to the above suspension and stirred at 30°C to advance the hydration reaction. HPLC analysis after 10 hours revealed that the 3-hydroxy-3-methylbutyronitrile concentration was below the detection limit (10 mass ppm or below). The conversion rate of 3-hydroxy-3-methylbutyronitrile to 3-hydroxy-3-methylbutyramide was 99% or more, and no formation of 3-hydroxy-3-methylbutanoic acid was observed.
Next, 10 g of a suspension of Psp-amidase producing bacteria, which is not an amidase derived from a specific bacterium, diluted with pure water and having a wet bacterial cell concentration of 10% by mass was added and stirred at 30°C to initiate a hydrolysis reaction. I let it progress. HPLC analysis after 10 hours revealed that the 3-hydroxy-3-methylbutyramide concentration was 55% by mass, indicating that 3-hydroxy-3-methylbutyramide had been converted to 3-hydroxy-3-methylbutanoic acid. The rate was 45%.

[Example 7]
92.5 g of epichlorohydrin, 424 g of water, and 65.5 g of sodium sulfate were charged into a 1 liter flask equipped with a stirrer, a thermometer, and a condenser, and the reaction temperature was maintained at 50 to 60°C. , 24.6 g of hydrogen cyanide was directly fed into the reaction solution over 1 hour while stirring. Further, stirring was continued for 6 hours at 50 to 60°C, and then cooled in a water bath. The reaction mixture was extracted twice with 300 ml of ethyl acetate. The organic layer was concentrated under reduced pressure, and the resulting oil was purified by distillation under reduced pressure to obtain 62.5 g of 4-chloro-3-hydroxybutyronitrile as a fraction of 138-141°C/2.4 kPa.

Wet bacterial cells containing Pth-nitrile hydratase-producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. The pH was adjusted to 8.0 using a 0.1M NaOH aqueous solution as a pH adjuster. 10 g of 4-chloro-3-hydroxybutyronitrile obtained by the above method was added to the above suspension and stirred at 30°C to advance the hydration reaction. After 10 hours, analysis was conducted under the following HPLC conditions, and the concentration of 4-chloro-3-hydroxybutyronitrile was below the detection limit (10 mass ppm or below). The conversion rate of 4-chloro-3-hydroxybutyronitrile to 4-chloro-3-hydroxybutyramide was 99% or more, and no production of 4-chloro-3-hydroxybutanoic acid was observed.
Next, 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water at a wet bacterial cell concentration of 10% by mass was added and stirred at 30°C to advance the hydrolysis reaction. After 10 hours, analysis was conducted under the following HPLC conditions, and the 4-chloro-3-hydroxybutyramide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 4-chloro-3-hydroxybutyramide to 4-chloro-3-hydroxybutanoic acid was 99% or more.
Here, the analysis conditions were as follows.
・HPLC analysis conditions:
High-performance liquid chromatography device: LC-10A system (manufactured by Shimadzu Corporation)
(UV detector wavelength 250nm, column temperature 40℃)
Separation column: SCR-101H (manufactured by Shimadzu Corporation)
Eluent: 7% (by volume)-acetonitrile aqueous solution

[Example 8]
In Example 7, instead of adding 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water and having a wet bacterial cell concentration of 10% by mass, Acr-amidase producing bacteria were diluted with pure water and a wet bacterial cell concentration of 10% was added. A hydrolysis reaction was carried out in the same manner as in Example 7, except that 10 g of the mass% suspension was added.
When HPLC analysis was performed 10 hours after the start of the hydrolysis reaction, the 4-chloro-3-hydroxybutyramide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 4-chloro-3-hydroxybutyramide to 4-chloro-3-hydroxybutanoic acid was 99% or more.

[Example 9]
Wet bacterial cells containing Ppu-nitrile hydratase producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. The pH was adjusted to 8.0 using a 0.1M NaOH aqueous solution as a pH adjuster. 10 g of 4-chloro-3-hydroxybutyronitrile obtained by the above method was added to the above suspension and stirred at 30°C to advance the hydration reaction. After 10 hours, analysis was conducted under the following HPLC conditions, and the concentration of 4-chloro-3-hydroxybutyronitrile was below the detection limit (10 mass ppm or below). The conversion rate of 4-chloro-3-hydroxybutyronitrile to 4-chloro-3-hydroxybutyramide was 99% or more, and no formation of 4-chloro-3-hydroxybutanoic acid was observed.
Next, 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water at a wet bacterial cell concentration of 10% by mass was added and stirred at 30°C to advance the hydrolysis reaction. After 10 hours, analysis was conducted under the following HPLC conditions, and the 4-chloro-3-hydroxybutyramide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 4-chloro-3-hydroxybutyramide to 4-chloro-3-hydroxybutanoic acid was 99% or more.

[Example 10]
In Example 9, instead of adding 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water and having a wet bacterial cell concentration of 10% by mass, Acr-amidase producing bacteria were diluted with pure water and a wet bacterial cell concentration of 10% was added. A hydrolysis reaction was carried out in the same manner as in Example 9, except that 10 g of the mass% suspension was added.
When HPLC analysis was performed 10 hours after the start of the hydrolysis reaction, the 4-chloro-3-hydroxybutyramide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 4-chloro-3-hydroxybutyramide to 4-chloro-3-hydroxybutanoic acid was 99% or more.

[Example 11]
Wet bacterial cells containing Pth-nitrile hydratase-producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. To this, 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water with a wet bacterial cell concentration of 10% by mass was added, and the pH was adjusted to 8.0 using a 0.1 M NaOH aqueous solution as a pH adjuster. It was adjusted to 10 g of 3-hydroxypropionitrile (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the above suspension, and the reaction was allowed to proceed by stirring at 30°C. When analyzed by HPLC after 20 hours, the 3-hydroxypropionitrile concentration and 3-hydroxypropionamide concentration were below the detection limit (10 mass ppm or less). The conversion rate of 3-hydroxypropionitrile to 3-hydroxypropionic acid was 99% or more.

[Example 12]
Wet bacterial cells containing Pth-nitrile hydratase-producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. To this, 10 g of a suspension of Acr-amidase producing bacteria diluted with pure water with a wet bacterial cell concentration of 10% by mass was added, and the pH was adjusted to 8.0 using a 0.1 M NaOH aqueous solution as a pH adjuster. It was adjusted to 10 g of 3-hydroxypropionitrile (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the above suspension, and the reaction was allowed to proceed by stirring at 30°C. When analyzed by HPLC after 20 hours, the 3-hydroxypropionitrile concentration and 3-hydroxypropionamide concentration were below the detection limit (10 mass ppm or less). The conversion rate of 3-hydroxypropionitrile to 3-hydroxypropionic acid was 99% or more.

[Example 13]
Wet bacterial cells containing Pth-nitrile hydratase-producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. To this, 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water with a wet bacterial cell concentration of 10% by mass was added, and the pH was adjusted to 8.0 using a 0.1 M NaOH aqueous solution as a pH adjuster. It was adjusted to 10 g of 4-chloro-3-hydroxybutyronitrile was added to the above suspension, and the reaction was allowed to proceed by stirring at 30°C. When analyzed by HPLC after 20 hours, the 4-chloro-3-hydroxybutyronitrile concentration and 4-chloro-3-hydroxybutyramide concentration were below the detection limit (10 mass ppm or less). The conversion rate of 4-chloro-3-hydroxybutyronitrile to 4-chloro-3-hydroxybutanoic acid was 99% or more.

[Example 14]
Wet bacterial cells containing Pth-nitrile hydratase-producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. To this, 10 g of a suspension of Acr-amidase producing bacteria diluted with pure water with a wet bacterial cell concentration of 10% by mass was added, and the pH was adjusted to 8.0 using a 0.1 M NaOH aqueous solution as a pH adjuster. It was adjusted to 10 g of 4-chloro-3-hydroxybutyronitrile was added to the above suspension, and the reaction was allowed to proceed by stirring at 30°C. When analyzed by HPLC after 20 hours, the 4-chloro-3-hydroxybutyronitrile concentration and 4-chloro-3-hydroxybutyramide concentration were below the detection limit (10 mass ppm or less). The conversion rate of 4-chloro-3-hydroxybutyronitrile to 4-chloro-3-hydroxybutanoic acid was 99% or more.

[Comparative example 3]
Wet bacterial cells containing Pth-nitrile hydratase-producing bacteria were diluted with pure water to obtain 100 g of a suspension with a wet bacterial cell concentration of 1.0% by mass. To this, 10 g of a suspension of Psp-amidase producing bacteria diluted with pure water with a wet bacterial cell concentration of 10% by mass was added, and the pH was adjusted to 8.0 using a 0.1 M NaOH aqueous solution as a pH adjuster. 10 g of 4-chloro-3-hydroxybutyronitrile adjusted to 4-chloro-3-hydroxybutyronitrile was added thereto, and the reaction was allowed to proceed by stirring at 30°C. When analyzed by HPLC after 20 hours, the concentration of 4-chloro-3-hydroxybutyronitrile was below the detection limit (10 mass ppm or below). The 4-chloro-3-hydroxybutyramide concentration was 29% by mass, and the conversion rate from 4-chloro-3-hydroxybutyronitrile to 4-chloro-3-hydroxybutanoic acid was 71%.

[Example 15]
<Preparation 1 of microbial cells containing mutant nitrile hydratase>
According to the method described in Example 73 of International Publication No. 2004/56990, No. 113 clone cells were obtained and cultured by the method described in Example 1 of the same document to obtain wet cells containing mutant nitrile hydratase.
The mutation sites and the types of substituted amino acid residues are as shown in Tables 23 to 25 below.
The wet bacterial cells containing the mutant nitrile hydratase obtained by the above method were diluted with pure water to obtain 100 g of a suspension having a wet bacterial cell concentration of 1.0 wt%. The pH was adjusted to 8.0 using a 0.1M NaOH aqueous solution as a pH adjuster. 10 g of 4-chloro-3-hydroxybutyronitrile was added to the above suspension, and the mixture was stirred at 30°C to advance the hydration reaction. After 10 hours, analysis was conducted under the following HPLC conditions, and the concentration of 4-chloro-3-hydroxybutyronitrile was below the detection limit (10 mass ppm or below). The conversion rate of 4-chloro-3-hydroxybutyronitrile to 4-chloro-3-hydroxybutyramide was 99% or more, and no formation of 4-chloro-3-hydroxybutanoic acid was observed.
Next, 10 g of a suspension of Pth-amidase producing bacteria diluted with pure water at a wet bacterial cell concentration of 10% by mass was added and stirred at 30°C to advance the hydrolysis reaction. After 10 hours, analysis was conducted under the following HPLC conditions, and the 4-chloro-3-hydroxybutyramide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 4-chloro-3-hydroxybutyramide to 4-chloro-3-hydroxybutanoic acid was 99% or more.

[Example 16]
In Example 15, instead of adding 10 g of a suspension of Pth-amidase-producing bacteria diluted with pure water to a wet bacterial cell concentration of 10% by mass, Acr-amidase-producing bacteria were diluted with pure water to a wet bacterial cell concentration of 10%. A hydrolysis reaction was carried out in the same manner as in Example 15, except that 10 g of the mass% suspension was added.
When HPLC analysis was performed 10 hours after the start of the hydrolysis reaction, the 4-chloro-3-hydroxybutyramide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 4-chloro-3-hydroxybutyramide to 4-chloro-3-hydroxybutanoic acid was 99% or more.

[Examples 17 to 42]
Wet microbial cells containing mutant nitrile hydratase and their free nitrile hydratase were prepared according to Preparation of Microbial Cells Containing Mutant Nitrile Hydratase 2 to 27 below.
The mutation sites and types of substituted amino acid residues of the mutant nitrile hydratase used in microbial cell preparations 2 to 27 are shown in Tables 22 to 24 below.

<Preparation of microbial cells containing mutant nitrile hydratase 2>
According to the method described in Example 74 of International Publication No. 2004/56990, No. 114 clone bacterial cells were obtained and cultured by the method described in Example 1 of the same document to obtain wet bacterial cells containing mutant nitrile hydratase.

<Preparation of microbial cells containing mutant nitrile hydratase 3>
According to the method described in Example 75 of International Publication No. 2004/56990, No. 115 clone cells were obtained and cultured by the method described in Example 1 of the same document to obtain wet cells containing mutant nitrile hydratase.

<Preparation of microbial cells containing mutant nitrile hydratase 4>
According to the method described in Example 76 of International Publication No. 2004/56990, No. 116 clone cells were obtained and cultured by the method described in Example 1 of the same document to obtain wet cells containing mutant nitrile hydratase.

<Preparation of microbial cells containing mutant nitrile hydratase 5>
According to the method described in Example 77 of International Publication No. 2004/56990, No. 117 clone cells were obtained and cultured by the method described in Example 1 of the same document to obtain wet cells containing mutant nitrile hydratase.

<Preparation of microbial cells containing mutant nitrile hydratase 6>
According to the method described in Example 78 of International Publication No. 2004/56990, No. 118 cloned bacterial cells were obtained and cultured by the method described in Example 1 of the same document to obtain wet bacterial cells containing mutant nitrile hydratase.

<Preparation 7 of microbial cells containing mutant nitrile hydratase>
According to the method described in Example 79 of International Publication No. 2004/56990, No. 119 clone bacterial cells were obtained and cultured by the method described in Example 1 of the same document to obtain wet bacterial cells containing mutant nitrile hydratase.

<Preparation of microbial cells containing mutant nitrile hydratase 8>
According to the method described in Example 80 of International Publication No. 2004/56990, No. 120 cloned bacterial cells were obtained and cultured by the method described in Example 1 of the same document to obtain wet bacterial cells containing mutant nitrile hydratase.

<Preparation of microbial cells containing mutant nitrile hydratase 9>
Amino-substituted product 5 (clone bacterial cell) was obtained according to the method described in Example 2 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to obtain a cell containing mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation 10 of microbial cells containing mutant nitrile hydratase>
Amino-substituted product 12 (clone bacterial cell) was obtained according to the method described in Example 6 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 11>
Amino-substituted product 33 (clone bacterial cell) was obtained according to the method described in Example 14 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 12>
Amino-substituted cell 41 (clone bacterial cell) was obtained according to the method described in Example 19 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 13>
Amino-substituted cell 45 (clone bacterial cell) was obtained according to the method described in Example 21 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 14>
Amino-substituted cell 58 (clone bacterial cell) was obtained according to the method described in Example 29 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 15>
Amino-substituted cell 59 (clone bacterial cell) was obtained according to the method described in Example 30 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 16>
Amino substituted product 60 (clone bacterial cell) was obtained according to the method described in Example 31 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 17>
Amino-substituted product 85 (clone bacterial cell) was obtained according to the method described in Example 46 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 18>
Amino-substituted cell 93 (clone bacterial cell) was obtained according to the method described in Example 52 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 19>
Amino-substituted cell 104 (clone bacterial cell) was obtained according to the method described in Example 60 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 20>
Amino-substituted product 111 (clone bacterial cell) was obtained according to the method described in Example 65 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 21>
Amino-substituted product 117 (clone bacterial cell) was obtained according to the method described in Example 71 of International Publication No. 2010/55660, and cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 22>
Transformant 21 (clone bacterial cell) was obtained according to the method described in Example 19 of International Publication No. 2018/124247, and was cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 23>
Transformant 23 (clone bacterial cell) was obtained according to the method described in Example 20 of International Publication No. 2018/124247, and was cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 24>
Transformant 23 (clone bacterial cell) was obtained according to the method described in Example 21 of International Publication No. 2018/124247, and was cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 25>
Transformant 26 (clone bacterial cell) was obtained according to the method described in Example 24 of International Publication No. 2018/124247, and was cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 26>
Transformant 28 (clone bacterial cell) was obtained according to the method described in Example 26 of International Publication No. 2018/124247, and was cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

<Preparation of microbial cells containing mutant nitrile hydratase 27>
Transformant 30 (clone bacterial cell) was obtained according to the method described in Example 28 of International Publication No. 2018/124247, and was cultured according to the method described in Example 1 of the same document to contain mutant nitrile hydratase. We obtained wet bacterial cells.

In the same manner as in Example 15, except that instead of the wet cells containing the mutant nitrile hydratase in Example 15, the wet cells prepared in Preparation of Microbial Cells 2 to 27 were used, respectively. 4-chloro-3-hydroxybutanoic acid was produced.
Preparation of microbial cells No matter which of the suspensions containing wet bacterial cells prepared in steps 2 to 27 are used, 4-chloro-3-hydroxybutyronitrile can be converted to 4-chloro-3-hydroxybutyramide. The conversion rate was 99% or more, and no production of 4-chloro-3-hydroxybutanoic acid was observed.
In addition, in the subsequent hydrolysis reaction using Pth-amidase producing bacteria, the 4-chloro-3-hydroxybutyramide concentration was below the detection limit (10 mass ppm or below). The conversion rate of 4-chloro-3-hydroxybutyramide to 4-chloro-3-hydroxybutanoic acid was 99% or more.

In Tables 22 to 24, for example, "α6" at the mutation site means that the 6th position from the N-terminus of the amino acid sequence corresponding to the α subunit shown in SEQ ID NO: 1 is substituted. For example, "Thr" in the amino acid residue after substitution means that the 6th amino acid residue from the N-terminus of the amino acid sequence corresponding to the α subunit shown in SEQ ID NO: 1 is replaced with "Thr". It means that it was done.
Similarly, for example, "β10" means that the 10th position from the N-terminus of the amino acid sequence corresponding to the β subunit shown by SEQ ID NO: 2 is substituted. For example, "Asp" in the amino acid residue after substitution means that the amino acid residue corresponding to the 10th position from the N-terminus of the amino acid sequence corresponding to the β subunit shown in SEQ ID NO: 2 is replaced with "Asp". It means that

From the above results, according to the method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to the present disclosure of Examples 1 to 42, 3-hydroxycarboxylic acid obtained by hydrolyzing a 3-hydroxyamide compound It can be seen that the compound or its salt can be obtained efficiently.

Claims (4)

At least one species selected from the group consisting of bacterial cells and bacterial cell-treated products containing amidase derived from at least one bacterium selected from the genus Pseudonocardia and the genus Acidophilium, and a 3-hydroxyamide compound; A method for producing a 3-hydroxycarboxylic acid compound or a salt thereof, the method comprising reacting the compound with a 3-hydroxycarboxylic acid compound or a salt thereof in an aqueous medium. The 3-hydroxyamide compound is obtained by reacting the 3-hydroxynitrile compound with at least one member selected from the group consisting of nitrile hydratase-containing microbial cells and bacterial cell-treated products in an aqueous medium. The method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to claim 1, further comprising: The production of a 3-hydroxycarboxylic acid compound or a salt thereof according to claim 2, which is a method for producing a 3-hydroxycarboxylic acid compound or a salt thereof from a 3-hydroxynitrile compound in the presence of the nitrile hydratase and the amidase. Method. The method for producing a 3-hydroxycarboxylic acid compound or a salt thereof according to claim 2 or 3, wherein the nitrile hydratase is derived from a bacterium of the genus Pseudonocardia.