JPWO2007097429A1 - Novel acylamidase gene and its use - Google Patents

Abstract

An object of the present invention is to provide a method for hydrolyzing an amide compound or an ester compound under various conditions on various substrates. As a result of screening for various soil isolates and stored strains, microorganisms with acylamidase activity were obtained. Moreover, the enzyme having the activity was successfully isolated and purified from the microorganism. Furthermore, the gene encoding the enzyme was obtained by a genetic recombination technique, and the base sequence was clarified. Furthermore, the highly active transformant was produced by breeding a transformant that produces the enzyme using the gene. A method for performing an amide hydrolysis reaction or an ester hydrolysis reaction under mild conditions using the highly active transformant was established.

Description

The present invention relates to a novel acylamidase, a DNA encoding the acylamidase, a method for producing an acylamidase using the DNA, and a method for producing an optically active compound using the acylamidase. Acylamidase is an industrially useful enzyme capable of hydrolyzing amide bonds or ester bonds under mild conditions.

Chemical hydrolysis of an amide bond requires a reaction under intense conditions such as strong acidity and strong basicity. However, by using an amide hydrolase such as amidase, Hydrolysis reaction of amide bond is possible. It is also known that amidase can perform an enantioselective reaction and can produce an optically active compound (Patent Document 1).
However, enzyme reactions generally have strict substrate specificity and are often not applicable to the target reaction. As the amidase having a wide substrate specificity known so far, those derived from Nocardia globulara (Patent Document 2, Non-Patent Document 1), derived from Arthrobacter aurescens Thing (nonpatent literature 2) etc. are mentioned. The former acts on a wide range of compounds such as acetanilides, amides such as benzamide, and esters such as phenyl acetate. The latter acts on a wide range of N-acetylarylalkylamines, but not on acyl groups other than acetyl groups. Thus, amide hydrolases having various substrate specificities have been found.
JP-A-61-88894 JP-A-3-277281 Eur. J. et al. Biochem, 199, 17-24 (1991) Appl. Microbiol. Biotechnol, 47, 650-657 (1997)

An object of the present invention is to obtain a novel acylamidase. In addition, DNA encoding this novel acylamidase is isolated and used to provide a method for amide hydrolysis reaction or ester hydrolysis reaction under various conditions for various substrates. is there.

As a result of screening for various soil isolates and storage strains, the present inventors have obtained microorganisms having acylamidase activity. Moreover, the enzyme having the activity was successfully isolated and purified from the microorganism.
Furthermore, a gene encoding the enzyme was obtained by a genetic recombination technique, and the base sequence was clarified. Furthermore, the highly active transformant was produced by breeding a transformant that produces the enzyme using the gene. A method for conducting an amide hydrolysis reaction or an ester hydrolysis reaction under mild conditions using the highly active transformant was established.
The present invention has the following features.

One feature of the present invention is an acylamidase having the following physicochemical properties (1) to (6):
(1) Molecular weight: about 90,000 by gel filtration analysis, about 40,000 by SDS-polyacrylamide gel electrophoresis analysis;
(2) Substrate specificity: It substantially acts on each of formanilide, acetanilide, and phenyl acetate, and benzamide, n-butyramide, N-acetyl-L-phenylalanine, N-acetyl-DL-tryptophan, N-acetyl Substantially does not act on each of the L-tyrosines;
(3) Optimum pH of action: 6-9;
(4) Optimum temperature of action: 25 to 45 ° C;
(5) Thermal stability: 10-40 ° C;
(6) Inhibitor: Activity is completely inhibited by HgCl ₂ and AgNO ₃ .
Another feature of the present invention is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence set forth in SEQ ID NO: 1. And a polypeptide having acylamidase activity, or having a 70% or higher identity with the amino acid sequence shown in SEQ ID NO: 1 and having acylamidase activity.
Another feature of the present invention is DNA encoding the polypeptide, a vector containing the DNA, and a transformant transformed with the vector.
Another feature of the present invention is the general formula (1):

(In the formula, m and n each independently represent an integer of 0 to 7, and R ¹ and R ² each independently represents an aryl group having 6 to 14 carbon atoms, a heteroaryl group having 4 to 14 carbon atoms, C6-C14 aryloxy group, C4-C14 heteroaryloxy group, C1-C5 alkoxy group, C2-C5 alkoxycarbonyl group, C3-C5 branched alkyl group Represents an alkenyl group having 2 to 5 carbon atoms, an alkynyl group having 2 to 5 carbon atoms, a cycloalkyl group having 5 to 7 carbon atoms, a cyano group, a methyl group or a carboxyl group, and these groups may be substituted. R ³ represents a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, and X represents a nitrogen atom or an oxygen atom). A general formula (2) characterized in that a culture of microorganisms capable of producing a repeptide or the enzyme is allowed to act:

(Wherein, X, m, n, R ¹ and R ² are the same as those in the formula (1)).
Other features and effects of the present invention will become apparent from the description of the embodiments below.

Hereinafter, the present invention will be described in detail based on embodiments. Unless otherwise specified, genetic manipulations such as DNA isolation, vector preparation, and transformation described in this specification are written documents such as Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989). Can be carried out by the method described in. Further,% used in the description of the present specification means% (w / v) unless otherwise specified. In addition, 1 U of enzyme activity used in the description of the present specification was defined as the amount of enzyme that produces amine, alcohol or ammonia as 1 μmol of product per minute at 30 ° C.

1. Acylamidase The enzyme of the embodiment of the present invention is an acylamidase having the following physicochemical properties (1) to (6):
(1) Molecular weight: about 90,000 by gel filtration analysis, about 40,000 by SDS-polyacrylamide gel electrophoresis analysis;
(2) Substrate specificity: It substantially acts on each of formanilide, acetanilide, and phenyl acetate, and benzamide, n-butyramide, N-acetyl-L-phenylalanine, N-acetyl-DL-tryptophan, N-acetyl Substantially does not act on each of the L-tyrosines;
(3) Optimum pH of action: 6-9;
(4) Optimum temperature of action: 25 to 45 ° C;
(5) Thermal stability: 10-40 ° C;
(6) Inhibitor: Activity is completely inhibited by HgCl ₂ and AgNO ₃ .
(Molecular weight)
The molecular weight of the enzyme can be determined by, for example, gel filtration analysis using TSK GEL-G-3000 (0.75 × 60 cm, manufactured by Tosoh Corporation) and the relative elution time with respect to the standard protein. As an eluent, 0.05 M potassium phosphate buffer (pH 7.0) containing 0.20 M KCl is used. The molecular weight of the subunit can be determined from the relative mobility with respect to the standard protein by 10% SDS-polyacrylamide gel electrophoresis.

(Substrate specificity)
The amidase activity with respect to each substrate of the enzyme can be measured, for example, by the method described in Example 3 described later. That is, 0.1 mL of the purified enzyme solution is added to 0.9 mL of a substrate solution having the following composition and reacted at 30 ° C. After 30 minutes, 1 mL of methanol is added to stop the reaction, and the produced amine, alcohol or ammonia is quantified.
[Substrate solution composition]
Substrate: 1-100 mM,
Phosphate buffer solution (pH 7.0): 50 mM

In the present specification, “acylamidase” substantially acts on a compound represented by P—CO—NH—Q (P represents an arbitrary substituent, and Q represents an optional substituent other than a hydrogen atom). , P-COOH and Q-NH ₂ have any activity to hydrolyze. For example, according to the classification of IUBMB, allyl acylamidase (EC 3.5.1.13), penicillin acylase (EC 3.5.1.11), allyl alkyl acylamidase (EC 3.5.1.7), (S) -N-acetyl-1-phenethylamine hydrolase (EC 3.5.1.85), formamidase (EC 3.5.1.9). ), Peptide deformylase (EC 3.5.1.1.27), N-substituted formamide deformylase (EC 3.5.1.91) and the like.
However, it is known that the classification of acylamidase may be unclear in the IUBMB classification (Enzyme Handbook, Asakura Shoten, 1982, p. 585). Therefore, in order to distinguish the enzyme of the present invention from other enzymes, the enzyme was characterized from the difference in reactivity with a specific substrate.

That is, the acylamidase of the present invention substantially acts on each of formanilide, acetanilide, and phenyl acetate, and benzamide, n-butylamide, N-acetyl-L-phenylalanine, N-acetyl-DL-tryptophan, N An acylamidase that does not substantially act on each of acetyl-L-tyrosine.
Here, “substantially does not act” means that the specific activity of the purified enzyme when each substrate is used is 0.02 U / mg or less when the amidase activity is measured by the method of Example 3 described later. It means that. The acylamidase of the present invention preferably has a specific activity of 0.002 U / mg or less, more preferably 0.0002 U / mg or less.
In addition, “substantially act” means that when the amidase activity is measured by the above method, the specific activity of the purified enzyme when each substrate is used is 0.2 U / mg or more. The acylamidase of the present invention preferably has a specific activity of 2.0 U / mg or more.

(Optimum pH for action)
The optimum pH of the enzyme reaction is, for example, a relative activity of approximately 80% or more when the activity using acetanilide as a substrate is measured in the range of pH 4.0 to 11.0, with the maximum activity being 100%. Range. However, in the above measurement method, the following buffer solution is used in the substrate solution depending on the pH at which the measurement is performed.
pH 4.0-6.0: 0.1 M citrate buffer pH 6.0-8.0: 0.1 M potassium phosphate buffer pH 7.5-9.0: 0.1 M Tris-HCl buffer pH 9.0 ˜11.0: 0.1 M glycine sodium buffer

(Optimum temperature for action)
The optimum temperature for the enzyme reaction is, for example, a range in which the activity using acetanilide as a substrate is measured in the reaction temperature range of 10 to 80 ° C., and the activity at 30 ° C. is 100%, and the relative activity is approximately 80% or more. And

(Thermal stability)
The thermal stability of the enzyme can be determined, for example, by adding the purified enzyme to 0.1 M potassium phosphate buffer (pH 7.0), treating it at 10-80 ° C. for 30 minutes, and then measuring the amidase activity. Can be determined.

(Inhibitor)
The effect of adding an inhibitor (for example, containing HgCl ₂ or AgNO ₃ ) can be determined, for example, as follows. That is, 0.1 mL of the purified enzyme solution is added to 0.9 mL of a substrate solution having the following composition and reacted at 30 ° C. After 30 minutes, 1 mL of methanol is added to stop the reaction, the reaction solution is analyzed by high performance liquid chromatography (HPLC), and the produced aniline is quantified. Here, “completely inhibits amidase activity” means that when an inhibitor is added and the following measurement is performed, the activity is 1/100 or less compared to the case where an inhibitor is not added. That means. In the acylamidase of the present invention, the activity is preferably 1/1000 or less, more preferably 1/10000 or less.

[Substrate solution composition]
Acetanilide 5 mM
Phosphate buffer solution (pH 7.0) 100 mM
Inhibitor 1 mM
0.9 mL total

(Km for acetanilide)
The Km of the enzyme for acetanilide can be determined, for example, as follows. That is, 0.1 mL of the purified enzyme solution is added to 0.9 mL of a substrate solution having the following composition and reacted at 30 ° C. After 30 minutes, 1 mL of methanol is added to stop the reaction, the reaction solution is analyzed by high performance liquid chromatography, the produced aniline is quantified, and Km is determined by Lineweaver-Burk Plots.
[Substrate solution composition]
Acetanilide 0.1-2.5 mM
Phosphate buffer solution (pH 7.0) 100 mM
0.9 mL total
The acylamidase of the present invention can stereoselectively hydrolyze a racemic amide compound or ester compound.

2. Acquisition of enzyme and amino acid sequence The enzyme of the present invention may be any enzyme as long as it exhibits the above properties, and can be acquired from, for example, a microorganism belonging to the genus Bacillus. The microorganism that is the origin of the enzyme of the present invention is preferably Bacillus sp., Which can be easily obtained by a person skilled in the art from a public preservation agency (for example, National Institute of Technology and Evaluation (NBRC)). More preferably, Bacillus sp (Bacillus sp.) KNK-M01 is mentioned. The Bacillus sp. KNK-M01 is dated January 22, 2007, and has the accession number FERM BP-10765, the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (〒305-8866 Japan) It has been deposited at Tsukuba City 1-1-1 Chuo No. 6 in Tsukuba City, Ibaraki Prefecture (original deposit date: domestic deposit on January 26, 2006 has been transferred to an international deposit based on the Budapest Treaty). The bacteriological properties of the Bacillus sp. Are well known to those skilled in the art. The bacteriological properties of the Bacillus sp. KNK-M01 are the same as the bacteriological properties of Bacillus sp. In addition to having a predetermined acylamidase activity.

(Medium components)
As the culture medium for the microorganism producing the enzyme of the present invention, a liquid nutrient medium containing a normal carbon source, nitrogen source, inorganic salts, organic nutrients and the like can be used as long as the microorganism grows.

(Enzyme purification)
Purification of the enzyme from the microorganism producing the enzyme of the present invention can be performed by protein purification methods well known to those skilled in the art. For example, the cells are collected from the microorganism culture solution by centrifugation or filtration, and the obtained cells are crushed by a physical method using an ultrasonic crusher or glass beads, and then centrifuged. A cell-free extract is prepared by removing cell residues, and this cell-free extract is subjected to fractional precipitation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, ultrafiltration, etc. Thus, acylamidase can be isolated.

(Amino acid sequence)
The purified acylamidase can reveal the DNA sequence encoding it by the method described later, and the amino acid sequence of the acylamidase can be determined from the DNA sequence. Examples of the acylamidase thus obtained include a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. However, the enzyme of the present invention is not limited to this, and a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 1 Any acylamidase activity (preferably stereoselective acylamidase activity) is included in the present invention.
The acylamidase and polypeptide of the present invention may be natural or artificially modified.

In the amino acid sequence represented by SEQ ID NO: 1, a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added is obtained by using Current Protocols shown in SEQ ID NO: 1. In Molecular Biology (John Wiley and Sons, Inc., 1989), it can prepare according to the well-known method as described in experiment documents.
The place where amino acids are substituted, inserted, deleted or added is not particularly limited, but it is preferable to avoid highly conserved regions. Here, the highly conserved region is the optimal alignment of amino acid sequences using multiple alignment functions of software GENETYX (manufactured by Genetics Co., Ltd.) for a plurality of enzymes with different origins having the same function. When compared, the positions where amino acids are identical among a plurality of sequences are shown.
Examples of the highly conserved region in SEQ ID NO: 1 include the 111st to 115th amino acids, the 138th to 149th amino acids, and the 163rd to 167th amino acid sequences.

The number of amino acids to be substituted, inserted, deleted or added (the above “several amino acids”) is preferably 10 or less, more preferably 5 or less, and even more preferably 3 or less. The modified amino acid sequence may include only one type (for example, substitution), or may include two or more types of modification (for example, substitution and insertion). In the case of substitution, the amino acid after substitution is preferably a homologous amino acid of the original amino acid.
In the present specification, amino acids in the same group of the following groups are regarded as homologous amino acids.
(Group 1: neutral nonpolar amino acids) Gly, Ala, Val, Leu, Ile, Met, Cys, Pro, Phe
(Group 2: neutral polar amino acids) Ser, Thr, Gln, Asn, Trp, Tyr
(Group 3: acidic amino acids) Glu, Asp
(Group 4: basic amino acids) His, Lys, Arg

In the present invention, the amino acid sequence of SEQ ID NO: 1 has an amino acid sequence having 70% or more, preferably 80% or more, more preferably 85% or more, and further preferably 90% or more, and an acyl A polypeptide having amidase activity is also a polypeptide of the present invention.
In the present specification, amino acid sequence identity can be determined by amino acid sequence homology analysis using BLAST (Altschul, Stephen F. et al., Nucleic Acids Res. 25, 3389-3402 (1997)). it can.
Thus, a polypeptide having an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and having acylamidase activity is, for example, a polypeptide to the polypeptide shown in SEQ ID NO: 1. It can be obtained by random mutagenesis or removal of sequences unnecessary for activity.
The polypeptide of the present invention preferably has acylamidase activity exhibiting the physicochemical properties (1) to (6) described above, or has at least acylamidase activity that acts on formanilide.

3. DNA
The DNA of the present invention is a DNA encoding the above-mentioned novel acylamidase and polypeptide, and any DNA can be used as long as it can express the polypeptide in a host cell introduced according to the method described below. Any untranslated region may be included. If the purified polypeptide can be obtained, those skilled in the art can obtain the DNA from a microorganism that is the origin of the polypeptide by a known method.

Hereinafter, examples of using the Bacillus sp. KNK-M01 as a method for obtaining the DNA of the present invention will be described, but the present invention is not limited thereto.
First, the polypeptide (enzyme) purified from the cell-free extract of the microorganism is digested with an appropriate endopeptidase, and a fragment cleaved by reverse-phase HPLC is purified. For example, an ABI492 type protein sequencer (Applied Biosystems) is used. A part of the amino acid sequence is determined. Then, based on the obtained partial amino acid sequence information, a PCR (Polymerase Chain Reaction) primer for amplifying a part of the DNA encoding the polypeptide is synthesized.
Next, the chromosomal DNA of the microorganism is prepared by a conventional DNA isolation method, for example, a method such as Murray (Nucl., Acids Res., 8, 4321-4325, 1980). Using this chromosomal DNA as a template, PCR is performed using the above-described PCR primers, a part of the DNA encoding the polypeptide is amplified, and the base sequence is determined. The determination of the base sequence can be performed using, for example, ABI373A type DNA Sequencer (Applied Biosystems). If the partial base sequence of DNA encoding the polypeptide is clarified, the entire sequence can be determined by, for example, Southern hybridization.

Examples of the DNA thus obtained include DNA having the base sequence set forth in SEQ ID NO: 2. However, the DNA of the present invention is not limited to this, and all DNAs encoding the above-described polypeptides of the present invention are included in the present invention. For example, DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 2 and encodes a polypeptide having acylamidase activity is included in the present invention.
Here, “DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 2” means colony hybridization, plaque hybridization, or Southern high When the hybridization method or the like is carried out, DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 2 refers to DNA that specifically forms a hybrid.
The DNA of the present invention may be natural or artificially modified.

In this specification, “stringent conditions” means hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which a colony or plaque-derived polynucleotide is immobilized. Thereafter, the filter is washed under a condition of 65 ° C. using a 2 × concentration SSC solution (composition of a 1 × concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate). Preferably, hybridization is performed in the same manner as described above, followed by washing with an SSC solution having a concentration of 0.5 times at 65 ° C. More preferably, hybridization is performed at 65 ° C. Conditions for washing with a 0.2 times concentrated SSC solution, more preferably conditions for washing with a 0.1 times concentrated SSC solution at 65 ° C. after hybridization as described above.
The DNA of the present invention preferably encodes an acylamidase exhibiting the above physicochemical properties (1) to (6), or at least an acylamidase having an acylamidase activity that acts on formanilide.

4). Vector A vector DNA used for introducing the DNA of the present invention into a host microorganism and expressing it in the introduced host microorganism includes a gene encoded by the DNA in an appropriate host microorganism. Any one can be used as long as it can express. Examples of such vector DNA include a plasmid vector, a phage vector, and a cosmid vector. A shuttle vector capable of gene exchange with other host strains can also be used.

Such a vector contains control elements such as an operably linked promoter (lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter, etc.), and the DNA of the present invention. And a vector containing an expression unit operably linked to each other. For example, pUC18 (manufactured by Toyobo Co., Ltd.), pUC19 (manufactured by Toyobo Co., Ltd.), pUCNT or the like that can be prepared by a person skilled in the art by the method described in International Publication No. 94/03613 pamphlet in a state in which the DNA of the present invention can be expressed. Can be preferably used.

As used herein, the term “regulatory element” refers to a base sequence having a functional promoter and any associated transcription element (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).
As used herein, the term “operably linked” means that a gene is operably linked to various regulatory elements such as promoters and enhancers that regulate the expression of the gene. It is well known to those skilled in the art that the type and kind of the control factor can vary depending on the host.

5). Host Host cells into which a vector containing the DNA of the present invention is introduced include bacteria, yeasts, filamentous fungi, plant cells, animal cells, etc., but Escherichia coli is particularly preferred. A vector containing the DNA of the present invention can be introduced into a host cell by a known method. When Escherichia coli is used as a host cell, the vector can be introduced by, for example, the calcium chloride method.

6). Method for producing acylamidase or polypeptide The present invention also comprises culturing Bacillus sp. KNK-M01 (FERM BP-10765) or the above transformant in a nutrient medium, The present invention relates to a method for producing an acylamidase or polypeptide by obtaining the above-mentioned acylamidase or polypeptide from the obtained culture broth.
That is, acylamidase can be efficiently produced by culturing Bacillus sp. KNK-M01 or the transformant of the present invention. As long as the Bacillus sp. KNK-M01 and the transformant of the present invention are grown, a normal liquid nutrient medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients and the like is used. Can be implemented. The acylamidase accumulated in the culture solution of Bacillus sp. KNK-M01 and the transformant of the present invention can be used as an acylamidase-containing product in the culture solution. It can also be used after purification or partial purification using known protein purification techniques.

7). Hydrolysis of amide compound or ester compound The amide compound or ester compound is added to the above-mentioned novel acylamidase or polypeptide, Bacillus sp. KNK-M01 (FERM BP-10765), or the above-mentioned trait. Carboxylic acid, alcohol, or amine can be produced by allowing the culture of the transformant to act and hydrolyzing the amide compound or ester compound. The acylamidase of the present invention or a microorganism capable of producing the enzyme is suitable for producing an optically active compound.

8). Production method <br/> Next optically active compounds, a method for producing an optically active compound by using a microorganism having an ability to produce acylamidase or the enzyme of the present invention.
The method for producing an optically active substance of the present invention comprises a racemic amide compound or an ester compound, the aforementioned acylamidase or polypeptide, Bacillus sp. KNK-M01 (FERM BP-10765), or the aforementioned trait. It is characterized in that a culture of a transformant is allowed to act to selectively hydrolyze only one of the racemates.
Examples of the microorganism having the ability to produce the amidase of the present invention include, for example, the Bacillus sp. KNK-M01 (FERM BP-10765) and a transformant introduced with the above-described vector containing DNA. It is done.
According to this production method, the general formula (1):

The above-mentioned enzyme or a culture of a microorganism capable of producing the enzyme is allowed to act on the ester compound or amide compound represented by general formula (2):

An optically active alcohol or an optically active amine represented by the formula can be produced.
In the above formulas (1) and (2), m and n each independently represent an integer of 0 to 7, and R ¹ and R ² each independently represent an aryl group having 6 to 14 carbon atoms and 4 carbon atoms. -14 heteroaryl group, C6-C14 aryloxy group, C4-C14 heteroaryloxy group, C1-C5 alkoxy group, C2-C5 alkoxycarbonyl group, C3-C3 Represents a branched alkyl group having 5 to 5 carbon atoms, an alkenyl group having 2 to 5 carbon atoms, an alkynyl group having 2 to 5 carbon atoms, a cycloalkyl group having 5 to 7 carbon atoms, a cyano group, a methyl group or a carboxyl group, and these groups May be substituted. R ³ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms. X represents a nitrogen atom or an oxygen atom.

Examples of the aryl group having 6 to 14 carbon atoms include a phenyl group and a naphthyl group. Examples of the heteroaryl group having 4 to 14 carbon atoms include a pyridyl group, a thienyl group, an oxadiazolyl group, an imidazolyl group, a thiazolyl group, a furyl group, and a pyrrolyl group. Examples of the aryloxy group having 6 to 14 carbon atoms include a phenoxy group and a naphthoxy group. Examples of the heteroaryloxy group having 4 to 14 carbon atoms include a pyridyloxy group, a thienyloxy group, an oxadiazolyloxy group, an imidazolyloxy group, a thiazolyloxy group, a furyloxy group, and a pyrrolyloxy group. Examples of the alkoxy group having 1 to 5 carbon atoms include a methoxy group, an ethoxy group, and a tert-butoxy group. Examples of the alkoxycarbonyl group having 2 to 5 carbon atoms include a methoxycarbonyl group, an ethoxycarbonyl group, and a tert-butoxycarbonyl group. Examples of the branched alkyl group having 3 to 5 carbon atoms include isopropyl group, sec-butyl group, tert-butyl group and the like. Examples of the alkenyl group having 2 to 5 carbon atoms include a vinyl group and an allyl group. An acetylene group etc. are mentioned as a C2-C5 alkynyl group. Examples of the cycloalkyl group having 5 to 7 carbon atoms include a cyclopentyl group, a cyclohexyl group, and a cycloheptyl group. The number of carbon atoms of the alkoxycarbonyl group includes the carbonyl carbon.
These groups may be further substituted, and examples of the substituent include a halogen atom, a hydroxyl group, a cyano group, a methoxy group, an ethoxy group and an alkoxy group having 1 to 4 carbon atoms such as methylenedioxy.

Among the compounds represented by the formula (1), m = 0 to 3 and R ¹ may be substituted methyl group, n = 0 to 4 and R ² is methyl group or 6 to 12 carbon atoms. It is an aryl group, and R ³ is preferably a hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms. Specific examples include N- (1-phenylethyl) acetamide, 1-phenylethyl acetate and the like.
The compound represented by the formula (1) is a methyl group in which m = 0 to 3 and R ¹ may be substituted, n = 0 to 4 and R ² is a methyl group or an aryl group having 6 to 12 carbon atoms. And R ³ is a linear or branched alkyl group having 1 to ³ carbon atoms, the resulting amine or alcohol is 37% e.e. e. 51% e. e. And 99% e.e. e. It can be obtained with the above high optical purity.
In the production method of the present invention, the amide or ester compound is allowed to act on the enzyme of the present invention or a culture of a microorganism capable of producing the enzyme. Here, the “culture product” means a culture solution containing microbial cells, cultured microbial cells, or a processed product thereof. Here, “the treated product” means, for example, a cell-free extract, freeze-dried cells, acetone-dried cells, or a ground product of these cells. Furthermore, these enzymes and cultures can also be used in the form of immobilized enzymes or immobilized cells by known means. Immobilization can be performed by methods well known to those skilled in the art (for example, a crosslinking method, a physical adsorption method, a comprehensive method, etc.).

(Substrate concentration)
The concentration of the substrate used in the reaction is 0.1 to 50% by weight, preferably 1 to 20% by weight, of the amide or ester compound in the reaction solution composition.

(Reaction pH)
From the viewpoint of the optimum pH of the enzyme action, the pH when the enzyme of the present invention is allowed to act is preferably pH 5.0 or more, more preferably pH 6.0 or more, and the upper limit is preferably pH 10. 0.0 or less, more preferably pH 9.0 or less.

(Reaction temperature)
The temperature at which the enzyme of the present invention is allowed to act is preferably 20 ° C. or higher, more preferably 25 ° C. or higher, and preferably 45 ° C. or lower, from the viewpoint of the optimal temperature and thermal stability of the enzyme. More preferably, it is 40 ° C. or lower.

(solvent)
As the reaction solvent, an aqueous medium such as ion-exchanged water or a buffer solution is usually used, but the reaction can also be performed in a system containing an organic solvent. Examples of the organic solvent include alcohol solvents such as methanol, ethanol, propanol, isopropanol, and butanol; aliphatic hydrocarbon solvents such as pentane and hexane; aromatic hydrocarbon solvents such as benzene and toluene; methylene chloride and chloroform. Halogenated hydrocarbon solvents such as: ether solvents such as diethyl ether and diisopropyl ether; ester solvents such as ethyl acetate and butyl acetate; ketone solvents such as acetone and methyl ethyl ketone;
The above reaction may be carried out in a one-phase system, or if necessary, the above organic solvent may be added to a solubility higher than water to carry out the reaction in a two-phase system of an aqueous phase and an organic solvent phase. it can. The coexistence of an organic solvent in the reaction system often improves the selectivity, conversion rate, yield, and the like.

(Reaction time)
The reaction is usually carried out until about half of the racemic ester or amide compound is hydrolyzed. Usually, the reaction time is 1 hour to 1 week, preferably 1 to 72 hours, and it is preferable to select reaction conditions for completing the reaction in such time. Depending on the optical purity and yield of the desired product, the reaction may be interrupted at an early stage of the reaction or may be allowed to react excessively.

(Extraction purification)
By the above reaction, an optically active compound represented by the general formula (2) and an unreacted enantiomer substrate are generated. The produced optically active compound and the unreacted enantiomer substrate can be isolated from the reaction mixture by known methods such as extraction, distillation, recrystallization and column separation.
For example, when the product is an optically active amine, after adjusting the pH to acidic, ethers such as diethyl ether and diisopropyl ether; esters such as ethyl acetate and butyl acetate; hydrocarbons such as hexane, octane and benzene; With a general solvent such as a halogenated hydrocarbon such as methylene chloride, the unreacted enantiomer substrate can be selectively extracted while leaving the generated optically active amino compound in the aqueous phase. Thereafter, the pH is adjusted to basic, and an optically active amino compound produced using a common organic solvent can be extracted.
The unreacted enantiomer substrate in the above reaction can hydrolyze the ester or amide moiety by a conventional method while maintaining the optical activity, and the optically active substance obtained in the above reaction, if necessary. The opposite stereo alcohol or amine can be derived.
The optically active alcohol or amine obtained by the above reaction can be esterified or amidated while maintaining optical activity. By repeating the enzyme reaction of the embodiment with the substrate thus obtained a plurality of times, it is possible to obtain a target compound with higher optical purity as required.

According to the present invention, a novel acylamidase, a DNA encoding the novel acylamidase, or any of them is used to hydrolyze an amide compound or an ester compound under various conditions on various substrates. A method is provided.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by these.
Example 1 Purification of acylamidase HBS Bacillus sp. Bacillus sp. KNK-M01 (FERM BP-10765) in 5 L of medium (composition: polypeptone 10 g / L, meat extract 10 g / L, NaCl 3 g) / L, Yeast Extract 5 g / L (pH 7.2)) at 28 ° C. for 24 hours. Next, the cells were collected from the culture solution by centrifugation, washed thoroughly with physiological saline, and then suspended in a 100 mM phosphate buffer (pH 7.0) containing 1 mM DTT. The obtained suspension was crushed by ultrasonic crushing. Next, solids in the crushed material were removed by centrifugation to prepare a cell-free extract.
Ammonium sulfate was added to the obtained cell-free extract so as to be 30% saturated, this was dissolved, and the resulting precipitate was removed by centrifugation. Ammonium sulfate was added to this supernatant so as to be 60% saturated, and this was dissolved, and then the precipitate produced by centrifugation was collected.

This precipitate was dissolved in a 100 mM phosphate buffer (pH 7.0) containing 1 mM DTT, and dialyzed against the same buffer. This was applied to a DEAE-Sephacel (Amersham Biosciences) column (size: 30 × 215 mm) equilibrated with the same buffer to adsorb the active fraction. After washing the column with the same buffer, the active fraction was eluted with 100 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride and 1 mM DTT.

The eluted active fractions were collected, dissolved in ammonium sulfate to a final concentration of 20%, and Octyl previously equilibrated with 10 mM phosphate buffer (pH 7.0) containing 20% ammonium sulfate and 1 mM DTT. -Sepharose (manufactured by Amersham Biosciences) column (size: 20 x 65 mm) was used to adsorb the active fraction. After washing the column with 10 mM phosphate buffer (pH 7.0) containing 10% ammonium sulfate and 1 mM DTT, and then with 10 mM phosphate buffer (pH 7.0) containing 5% ammonium sulfate and 1 mM DTT, 25 mM containing 1 mM DTT The active fraction was eluted with a phosphate buffer (pH 7.0).

The eluted active fractions were collected and dialyzed against 5 mM phosphate buffer (pH 7.0) containing 1 mM DTT. This was applied to a Hydroxyapatite (manufactured by Amersham Biosciences) column (size: 20 × 15 mm) equilibrated with the same buffer to adsorb the active fraction. After washing the column with 10 mM phosphate buffer (pH 7.0) containing 1 mM DTT, the active fraction was eluted with 25 mM phosphate buffer (pH 7.0) containing 1 mM DTT.

The eluted active fractions were collected and fully diluted with 100 mM phosphate buffer (pH 7.0) containing 20% ammonium sulfate and 1 mM DTT. This was applied to a Butyl-Toyopearl (manufactured by Tosoh Corporation) column (size: 20 × 20 mm) equilibrated with the same buffer to adsorb the active fraction. The column was washed with 100 mM phosphate buffer (pH 7.0) containing 15% ammonium sulfate and 1 mM DTT, and then with 100 mM phosphate buffer (pH 7.0) containing 10% ammonium sulfate and 1 mM DTT, and then 5% ammonium sulfate and 1 mM DTT. The active fraction was eluted with 100 mM phosphate buffer (pH 7.0) containing.

The eluted active fractions were collected to obtain a single purified enzyme preparation electrophoretically. Hereinafter, this enzyme is referred to as HBS.
Acylamidase activity was measured as follows. That is, 0.1 mL of the enzyme solution was added to 0.9 mL of a substrate solution having the following composition and reacted at 30 ° C. After 30 minutes, 1 mL of methanol was added to stop the reaction, the reaction solution was analyzed by high performance liquid chromatography, and the produced aniline concentration was quantified. The activity of the obtained acylamidase was 2.7 U per 1 mg of the purified enzyme.

[Substrate solution composition]
Acetanilide 5.0 mM
Phosphate buffer solution (pH 7.0) 100 mM
0.9 mL total

[Measurement conditions by high performance liquid chromatography]
Column: Wakosil-II5C18AR 4.6 mm × 150 mm (Wako Pure Chemical Industries, Ltd.)
Mobile phase: 10 mM potassium phosphate buffer (pH 7.0): methanol = 4: 1
Flow rate: 1.0 mL / min
Detection wavelength: 265 nm

Example 2 Physicochemical properties 1 of purified enzyme
The physicochemical properties of the purified HBS enzyme obtained in Example 1 were examined.
(1) Action The acylamidase activity of the purified HBS enzyme was measured in the same manner as in Example 1.

(2) When the molecular weight purified enzyme was analyzed by gel filtration analysis using TSK GEL-G-3000 (0.75 × 60 cm, manufactured by Tosoh Corporation), the molecular weight was about 90,000. The molecular weight of the subunit was measured by 10% SDS-polyacrylamide gel electrophoresis, and the molecular weight was about 40,000.

(3) Optimum pH
In the range of pH 4.0 to 11.0, the activity using acetanilide as a substrate was measured in the same manner as described above, and the optimum pH of HBS was examined. As a result, the optimum pH was 6-9. However, in the above measurement method, the following buffer solution was used in the substrate solution according to the pH at which the measurement was performed.
pH 4.0-6.0: 0.1 M citrate buffer pH 6.0-8.0: 0.1 M potassium phosphate buffer pH 7.5-9.0: 0.1 M Tris-HCl buffer pH 9.0 ˜11.0: 0.1 M glycine sodium buffer

(4) Optimum temperature In the same manner as described above, the activity using acetanilide as a substrate was measured in the reaction temperature range of 10 to 80 ° C. As a result, the optimum temperature was 25 to 45 ° C.

(5) A thermostable purified enzyme was added to 0.1 M potassium phosphate buffer (pH 7.0), and this was treated at 10-80 ° C. for 30 minutes, and then the amidase activity was measured. As a result, 90% or more of the activity remained in the treatment at 10 ° C. to 40 ° C. as compared with that before the treatment.

(6) 0.1 mL of the purified inhibitor enzyme solution was added to 0.9 mL of a substrate solution having the following composition, and reacted at 30 ° C. After 30 minutes, 1 mL of methanol was added to stop the reaction, the reaction solution was analyzed by high performance liquid chromatography, and the produced aniline was quantified. As a result, the reaction did not proceed even when either HgCl ₂ or AgNO ₃ was added.
[Substrate solution composition]
Acetanilide 5 mM
Phosphate buffer solution (pH 7.0) 100 mM
Inhibitor 1 mM
0.9 mL total

(Example 3) Physicochemical property 2 of purified enzyme (substrate specificity)
0.1 mL of the purified enzyme solution was added to 0.9 mL of a substrate solution having the following composition and reacted at 30 ° C. After 30 minutes, 1 mL of methanol was added to stop the reaction, and the alcohol, amine, or ammonia produced in the reaction solution was analyzed under the conditions shown in Tables 1 and 2.
However, when p-nitrophenyl acetate, p-nitrophenyl propionate, and p-nitrophenyl butyrate were used as substrates, the reaction solution was made basic with NaHCO ₃ , and the generated p- Nitrophenol was measured.
In the reaction using acetamide, n-butylamide, and benzamide as substrates, the produced ammonia was measured using Conway's diffusion analysis method. That is, 0.2 mL of the reaction solution supernatant and 2.0 mL of saturated potassium carbonate solution were added to the outer chamber of the Conway diffusion unit, respectively. Further, after adding 1.5 mL of 0.01N sulfuric acid to the inner chamber, the lid was covered, and the two liquids in the outer chamber were contacted and left for 3 hours. Thereafter, 0.5 mL of the reaction solution in the inner chamber was collected.
0.5 mL of the collected internal solution 0.5 mL of 330 mM sodium phenoxide and 0.5 mL of 0.01% (w / v) sodium nitroprusside, 0.5 mL of 0.84% (v / v) hypochlorous acid After adding an aqueous sodium solution, the mixture was heated at 100 ° C. for 5 minutes. Thereafter, 3 mL of distilled water was added, and the absorbance (A ₆₄₀ ) of the reaction solution was compared with the absorbance of the standard sample to determine the activity value.

The amount of enzyme used for the measurement of substrate specificity varies depending on the substrate used, and the amount is listed in the column of enzyme usage in Table 1. The unit (mU) of the amount of enzyme used is indicated by the enzyme activity when acetanilide is used as a substrate. The relative activity was expressed as a relative value when the activity for each substrate per unit enzyme amount was defined as 100 for the activity against acetanilide.
The results are shown in Table 1. This enzyme showed high activity on acetanilide, formanilide, and phenyl acetate. Moreover, it did not act on benzamide, n-butylamide, N-acetyl-L-phenylalanine, N-acetyl-DL-tryptophan, and N-acetyl-L-tyrosine.

Therefore, the acylamidase of this example substantially acts on each of formanilide, acetanilide, and phenyl acetate, and benzamide, n-butylamide, N-acetyl-L-phenylalanine, N-acetyl-DL-tryptophan, It has been found that it has a unique and useful substrate specificity that differs from the substrate specificity of conventional acylamidases in that it does not substantially act on each of N-acetyl-L-tyrosine.

[Substrate solution composition]
Substrate 1-100 mM
Phosphate buffer solution (pH 7.0) 50 mM

(Example 4) Physicochemical property 3 (stereoselectivity) of purified enzyme
Among the reaction solutions obtained in Example 3, the optical purity of the product was measured under the following measurement conditions for 1-phenylethyl acetate and N- (1-phenylethyl) acetamide. As a result, 1-phenylethanol was R-form with 37% e.e. e. 1-phenethylamine is 51% e.e. in R form. e. Met.
[Measurement conditions by high performance liquid chromatography (1-phenylethanol)]
Column: Chiralcel OB 4.6mm x 250mm
(Daicel Chemical Industries)
Mobile phase: n-Hexane: 2-Propanol = 9: 1
Flow rate: 1.0 mL / min
Detection wavelength: 254 nm

[Measurement conditions by high performance liquid chromatography (1-phenethylamine)]
Column: Chiralcel CR (+) 4.6 mm x 150 mm
(Daicel Chemical Industries)
Mobile phase: HClO ₄ (pH 1.5)
Flow rate: 0.8 mL / min
Detection wavelength: 210 nm

(Example 5) Physicochemical property 4 of purified enzyme (Km for acetanilide)
For the HBS purified enzyme obtained in Example 1, the Km for acetanilide was examined. 0.1 mL of the purified enzyme solution was added to 0.9 mL of a substrate solution having the following composition and reacted at 30 ° C. After 30 minutes, 1 mL of methanol was added to stop the reaction, the reaction solution was analyzed by high performance liquid chromatography, the produced aniline was quantified, and the Km was determined by Lineweaver-Burk Plots to be 0.21 mM. High performance liquid chromatography was performed under the same conditions as in Example 1.
[Substrate solution composition]
Acetanilide 0.1-2.5 mM
Phosphate buffer solution (pH 7.0) 100 mM
0.9 mL total

(Example 6) Cloning of HBS gene (creation of PCR primers)
The N-terminal amino acid sequence of the purified HBS obtained in Example 1 was determined using an ABI492 type protein sequencer (PerkinElmer Biosystems). Further, the purified HBS obtained in Example 1 was digested with lysyl endopeptidase, and the amino acid sequence of the obtained peptide fragment was determined in the same manner as the N-terminal amino acid sequence. In consideration of the base sequence predicted from this amino acid sequence, primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4) for amplifying a part of the HBS gene by PCR were synthesized.
(Amplification of HBS gene by PCR)
Chromosomal DNA was extracted from the culture solution of Bacillus sp. KNK-M01 (FERM BP-10765) according to the method described in Murray et al. (Nucl. Acids Res., 8, 4321, 1980). PCR was performed using the obtained chromosomal DNA as a template and the primers synthesized above. As a result, a DNA fragment of about 134 bp considered to be a part of the HBS gene was obtained.
This DNA fragment was cloned into plasmid pT7Blue T-Vector (Novagen), and ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) and ABI 310 DNA Sequen (Sequen). The sequence was determined.

(Southern hybridization)
The chromosomal DNA was treated with the restriction enzyme HindIII to prepare a DNA fragment. Southern hybridization was performed using the determined DNA sequence as a probe, and a DNA fragment hybridizing with the probe was extracted from the gel. The extracted DNA fragment was inserted into a pBluescript vector to create a mini library, and then E. coli E. coli. E. coli (JM109) was transformed. A positive clone that hybridizes with the probe was selected from the transformed E. coli library by colony hybridization, and the DNA sequence of the insert was determined using ABI 310 DNA Sequencer (manufactured by Perkin Elmer). As a result, a 1050 bp ORF (SEQ ID NO: 2) was revealed.

(ORF database analysis)
When the homology search by BLAST was performed on the DNA sequence of the obtained ORF, the top three genes having high homology and their homology were as follows.
Homoserine acyltransferase (Corynebacterium glutamicum ATCC 13032): 64%
Homoserine acyltransferase (Rhodococcus sp. DK 17): 63%
Deacetycephalosporin C acyltransferase (Streptomyces cvululigerus): 34%

(Example 7) Preparation of recombinant plasmid containing HBS gene Based on the ORF information obtained in Example 6, designed to add a site of restriction enzyme NdeI to the 5 'end of ORF. Primer 3 (SEQ ID NO: 5) and primer 4 (SEQ ID NO: 6) designed to add a restriction enzyme HindIII site to the 3 ′ end of the ORF were synthesized. Using this primer, PCR was carried out using the plasmid obtained from the positive clone as a template, the amplified DNA fragment was treated with restriction enzymes NdeI and HindIII, and pET21a (+) using DNA ligation Kit (Takara Bio Inc.). ) Incorporated into a vector (manufactured by Novagen) to obtain a recombinant vector pET21HBS.

(Example 8) Production of recombinant E. coli containing HBS gene and expression of HBS E. coli E. coli using recombinant vector pET21HBS obtained in Example 7. E. coli BL21 (DE3) competent cells (Novagen) were transformed according to the instruction manual. coli BL21 (DE3) (pET21HBS) was obtained. The resulting recombinant E. coli E. coli BL21 (DE3) (pET21HBS) was inoculated into LB medium and cultured at 37 ° C. for 4-6 hours, then IPTG was aseptically added to a final concentration of 1 mM, and further cultured at 20 ° C. for 20 hours. . The obtained culture broth was collected, suspended in 100 mM phosphate buffer (pH 7.0), and a cell-free extract was obtained by ultrasonic disruption. When the amidase activity of this cell-free extract was measured using acetanilide as a substrate in the same manner as in Example 3, the activity was found to be 0.73 U per 1 mL of the culture solution.

Claims (19)

Acylamidases with the following physicochemical properties:
(1) Molecular weight: about 90,000 by gel filtration analysis, about 40,000 by SDS-polyacrylamide gel electrophoresis analysis;
(2) Substrate specificity: It substantially acts on each of formanilide, acetanilide, and phenyl acetate, and benzamide, n-butyramide, N-acetyl-L-phenylalanine, N-acetyl-DL-tryptophan, N-acetyl Substantially does not act on each of the L-tyrosines;
(3) Optimum pH of action: 6-9;
(4) Optimum temperature of action: 25 to 45 ° C;
(5) Thermal stability: 10-40 ° C;
(6) Inhibitor: Activity is completely inhibited by HgCl ₂ and AgNO ₃ . The acylamidase according to claim 1, which is an enzyme obtained from a microorganism belonging to the genus Bacillus. The acylamidase according to claim 1, which is an enzyme obtained from Bacillus sp. The acylamidase according to claim 1, which is an enzyme obtained from Bacillus sp. KNK-M01 (FERM BP-10765). Any of the following polypeptides:
(1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1;
(2) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence set forth in SEQ ID NO: 1 and having acylamidase activity;
(3) A polypeptide having an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and having acylamidase activity. The polypeptide according to claim 5, which has an acylamidase activity exhibiting the physicochemical properties according to claim 1. 6. The polypeptide according to claim 5, which has an acylamidase activity that acts at least on formanilide. DNA encoding the polypeptide according to any one of claims 5 to 7. DNA shown in the following (A) or (B):
(A) DNA comprising the base sequence set forth in SEQ ID NO: 2;
(B) A DNA that hybridizes with a DNA complementary to the base sequence described in SEQ ID NO: 2 under stringent conditions and encodes an enzyme having acylamidase activity. The DNA according to claim 9, which encodes an acylamidase exhibiting the physicochemical properties according to claim 1. The DNA according to claim 9, which encodes an acylamidase having an acylamidase activity that acts at least on formanilide. A vector comprising the DNA according to any one of claims 9 to 11. A transformant obtained by transforming a host cell with the vector according to claim 12. The transformant according to claim 13, wherein the host cell is Escherichia coli. Bacillus sp. KNK-M01 (FERM BP-10765) or the transformant according to claim 13 or 14 is cultured in a nutrient medium, and from the obtained culture broth, claims 1-4 A method for producing an acylamidase or polypeptide, comprising obtaining the acylamidase according to any one of claims 1 to 5 or the polypeptide according to any one of claims 5 to 7. The acylamidase according to any one of claims 1 to 4 or the polypeptide according to any one of claims 5 to 7, Bacillus sp. KNK-M01 (FERM) BP-10765) or a culture of the transformant according to claim 13 or 14 to act to hydrolyze the amide compound or ester compound, thereby producing a carboxylic acid, alcohol, or amine Method. The acylamidase according to any one of claims 1 to 4 or the polypeptide according to any one of claims 5 to 7, Bacillus sp. KNK- An optical activity characterized in that M01 (FERM BP-10765) or a culture of the transformant according to claim 13 or 14 is allowed to act to selectively hydrolyze only one of the racemates. Body manufacturing method. General formula (1):

(In the formula, m and n each independently represent an integer of 0 to 7, and R ¹ and R ² each independently represents an aryl group having 6 to 14 carbon atoms, a heteroaryl group having 4 to 14 carbon atoms, C6-C14 aryloxy group, C4-C14 heteroaryloxy group, C1-C5 alkoxy group, C2-C5 alkoxycarbonyl group, C3-C5 branched alkyl group Represents an alkenyl group having 2 to 5 carbon atoms, an alkynyl group having 2 to 5 carbon atoms, a cycloalkyl group having 5 to 7 carbon atoms, a cyano group, a methyl group or a carboxyl group, and these groups may be substituted. R ³ represents a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, and X represents a nitrogen atom or an oxygen atom). Any one of The acylamidase according to claim 8, or the polypeptide according to any one of claims 5 to 7, Bacillus sp. KNK-M01 (FERM BP-10765), or the claim 13 or 14, General formula (2) by allowing a culture of transformants to act:

(Wherein, X, m, n, R ¹ and R ² are the same as those in the above formula (1)). In the compound represented by the general formula (1), m = 0 to 3 and R ¹ may be a substituted methyl group, n = 0 to 4 and R ² is a methyl group or an aryl group having 6 to 12 carbon atoms. The production method according to claim 18, wherein R ³ is a hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms.