WO2007097429A1 - Novel acylamidase gene and use thereof - Google Patents

Abstract

Disclosed is a method for amide or ester hydrolysis of various substrates under mild conditions. A screening is made using various bacteria isolated from soil and stock strains, and a microorganism having an acylamidase activity can be obtained. An enzyme having the activity can be successfully isolated and purified from the microorganism. A gene encoding the enzyme can be obtained by a genetic engineering technique and the nucleotide sequence of the gene can be identified. Further, by producing a transformant capable of producing the enzyme using the gene and culturing the transformant, a more highly active transformant can be produced. By using the more highly active transformant, a method for amide or ester hydrolysis under mild conditions can be established.

Description

 Novel isylamidase gene and its use

 Technical field

 [0001] The present invention relates to a novel acylamidase, a DNA encoding the acylamidase, a method for producing the acylamidase using the DNA, and an optically active compound using the acylamidase. It relates to a manufacturing method. The acylamidase is an industrially useful enzyme capable of hydrolyzing an amide bond or an ester bond under mild conditions.

 Background art

 [0002] In order to hydrolyze amide bonds chemically, reactions under intense conditions such as strong acidity and strong basicity are required. However, by using an amide hydrolase such as amidase, Hydrolysis reaction of amide bond is possible under mild conditions. It is also known that amidase can undergo 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 desired reaction. Known amidases with broad substrate specificity have been derived from Nocardia globerula (Patent Document 2, Non-Patent Document 1).

), Those derived from Arthrobacter aurescens (Non-Patent Document 2). The former acts on a wide range of compounds such as acetate-lides, amides such as benzamide, and esters such as phenyl acetate. The latter does not act on acyl groups other than the acetyl group, which acts on a wide range of N-acetylyl reelalkylamines. Thus, amide hydrolases with various substrate specificities have been found.

 Patent Document 1: Japanese Patent Application Laid-Open No. 61-88894

 Patent Document 2: JP-A-3-277281

 Non-Patent Document 1: Eur. J. Biochem, 199, 17-24 (1991)

 Non-Patent Document 2: Appl. Microbiol. Biotechnol, 47, 650-657 (1997) Disclosure of the Invention

Problems to be solved by the invention [0003] An object of the present invention is to obtain a novel acylamidase. In addition, DNA that encodes this novel acylamidase was isolated and used to provide a method for amide hydrolysis or ester hydrolysis under mild conditions for various substrates. is there.

 Means for solving the problem

[0004] As a result of screening for various soil-separated bacteria-preserving strains, the present inventors have obtained microorganisms having acylamidase activity. Moreover, the enzyme having the activity was successfully isolated and purified from the microorganism.

 Sarasako, the gene encoding the enzyme was obtained by genetic recombination techniques and the nucleotide sequence was clarified. Further, the transformant producing the enzyme was bred using the gene to produce the transformant with higher activity. Using this highly active transformant, we established a method for amide hydrolysis or ester hydrolysis under mild conditions.

 The present invention has the following features.

One feature of the present invention is a 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;

 (2) Substrate specificity: Substantially acts on each of formaldehyde, acetatetolide, and phenylacetate, and includes benzamide, n-butylamide, N-acetylethyl L-phenylalanine, N-acetylyl DL— Virtually no effect on tryptophan or N-acetyl-L-tyrosine;

 (3) Optimum pH: 6-9;

 (4) Optimum temperature of action: 25-45 ° C;

 (5) Thermal stability: 10-40 ° C;

 (6) Inhibitor: Activity is completely inhibited by HgCl, AgNO.

 twenty three

Another feature of the present invention is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, a deletion, substitution, or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 1. Alternatively, it has an added amino acid sequence ability and has a polypeptide having acylamidase activity, or an amino acid sequence having 70% identity or more with the amino acid sequence shown in SEQ ID NO: 1, and A polypeptide 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):

[0006] [Chemical 1]

[0007] (wherein, m and n each independently represents an integer of 0-7, heteroaryl independently R \ R ² are each, Ariru group having 6 to 14 carbon atoms, 4 to 14 to the carbon atoms Group, C6-C14 aryloxy group, C4-C14 heteroaryloxy group, C1-C5 alkoxy group, C2-C5 alkoxycarbonyl group, C3-C5 Indicates a branched alkyl group, 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 cyan group, a methyl group or a carboxyl group, and these groups are substituted. R ³ represents a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, X represents a nitrogen atom or an oxygen atom), or an ester compound represented by Production of the above-mentioned acylamidase, polypeptide or the enzyme into an amide compound General formula (2), characterized by acting a culture of microorganisms that have the ability:

[0008] [Chemical 2]

(Wherein, X, m, n, R \ R 2 is Formula (1)) to walk optically active alcohol represented by a method for producing an optically active Amin.

Other features and effects of the present invention will become apparent from the description of the following embodiments. Is done.

 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 include Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989), etc. It can be carried out by the method described in the book. Further,% used in the description of the present specification means% (wZv) unless otherwise specified. In addition, 1U of enzyme activity used in the description of the present specification was defined as the amount of enzyme that produces ammonia, which is a product of l / z mol per minute at 30 ° C, or alcohol.

 [0011] 1. Acylamidase

 The enzyme of the embodiment of the present invention is a acylamidase having the following physical properties (1) force (6):

 (1) Molecular weight: about 90,000 by gel filtration analysis, about 40,000 by SDS-polyacrylamide gel electrophoresis;

 (2) Substrate specificity: Substantially acts on each of formaldehyde, acetatetolide, and phenylacetate, and includes benzamide, n-butylamide, N-acetylethyl L-phenylalanine, N-acetylyl DL— Virtually no effect on tryptophan or N-acetyl-L-tyrosine;

 (3) Optimum pH: 6-9;

 (4) Optimum temperature of action: 25-45 ° C;

 (5) Thermal stability: 10-40 ° C;

 (6) Inhibitor: Activity is completely inhibited by HgCl, AgNO.

 twenty three

 (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 eluent, 0.05M potassium phosphate buffer (pH 7.0) containing 0.20M KC1 is used. The molecular weight of the subunit can be determined by relative mobility relative to the standard protein by 10% SDS-polyacrylamide gel electrophoresis.

[0012] (Substrate specificity) The amidase activity for 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, add 1 mL of methanol to stop the reaction, and quantify the produced amine, alcohol or ammonia.

 [Substrate solution composition]

 Substrate: 1 ~: L00mM,

 Phosphate buffer (ρΗ7.0): 50mM

In the present specification, “acylamidase” means a compound represented by P—CO—NH—Q (P is an arbitrary substituent, and Q is an arbitrary substituent other than a hydrogen atom). Anything that acts as an active agent and hydrolyzes to P COOH and Q—NH is meant. Example

 2

 For example, according to the classification of IUBMB, allylicylamidase (EC 3.5. 1.13), perillin acylase (EC 3.5. 5. 1. 76), (S) —N-acetyl-1-hydrophenylamine hydrolase (EC 3.5. 1. 1. 85), formamidase (EC 3.5. 1. 1. 9), peptide deformylase ( EC 3.5. 1.27), N Substituted Formamide Deformylase (EC 3.5. 1. 91).

 However, it is known that the classification of isylamidase 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, characterization was performed based on the difference in reactivity with a specific substrate.

 [0014] That is, the acylamidase of the present invention substantially acts on each of formaldehyde, acetonitrile, and acetic acid, and benzamide, n-butylamide, N-acetylene L-ferallanin. N-acetylyl DL-tryptophan and N-acetylyl L-tyrosylase which does not substantially act on each of 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. As the acylamidase of the present invention, those having a specific activity of 0.002 UZmg or less are preferred, and those having a specific activity of 0.0002 UZmg or less are more preferred. “Substantially acting” means that the specific activity of the purified enzyme is 0.2 UZmg or more when each substrate is used when the amidase activity is measured by the above method. The acylamidase of the present invention preferably has a specific activity of 2. OUZ mg or more.

[0015] (action optimal _P H)

 The optimum pH of the enzyme reaction is, for example, when the activity using acetate-lide as a substrate is measured in the range of pH 4.0 to L1. It is set as the range which shows activity. However, in the above measurement method, the following buffer solution is used in the base solution according to the pH to be measured.

 pH 4.0-6.0: 0.1 M citrate buffer

 pH 6.0-8.0: 0.1 M potassium phosphate buffer

 pH 7.5 to 9.0: 0.1 M Tris-HCl buffer

 pH 9.0 ~: L 1. 0: 0.1 M glycine sodium buffer

[0016] (Optimum temperature for action)

 The optimum temperature of the enzyme reaction is, for example, the activity using acetanilide as a substrate, the reaction temperature of 10 to

Measure in the range of 80 ° C. The activity at 30 ° C shall be 100%, and the relative activity should be over 80%.

[0017] (Thermal stability)

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

[0018] (Inhibitor)

 The effect of adding an inhibitor (eg, containing HgCl or AgNO) is as follows, for example:

 twenty three

Can be determined. 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 reaction solution is analyzed by high performance liquid chromatography (HPLC) to quantify the produced aline. Here, “completely inhibits amidase activity” means that when an inhibitor is added and the following measurement is performed, the activity is 1Z100 or less compared to when no inhibitor is added. That Say. The acylamidase of the present invention preferably has the above activity of 1Z1000 or less, more preferably 1Z10000 or less.

[0019] [Substrate solution composition]

 Acetanilide 5mM

 Phosphate buffer (ρΗ7.0) lOOmM

 Inhibitor ImM

 Total 0.9 mL

[0020] (Km for Asset-Lido)

 The Km of the enzyme for acetate-lid 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. Three

After 0 minutes, add 1 mL of methanol to stop the reaction, analyze the reaction mixture by high-performance liquid chromatography, quantify the amount of generated phosphorus, and determine Km by Lineweaver-Burk Plots.

 [Substrate solution composition]

 Acetanilide 0.1 to 2.5 mM

 Phosphate buffer (ρΗ7.0) lOOmM

 Total 0.9 mL

 The acylamidase of the present invention can hydrolyze racemic amido compounds or ester compounds in a stereoselective manner.

[0021] 2. Acquisition of enzyme and amino acid sequence

As long as the enzyme of the present invention is an enzyme exhibiting the above properties, 1S contained in any enzyme can be obtained, for example, microbial power of the genus Bacillus. The microorganism that is the source 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 preferred is Bacillus sp. KNK-MO1. This Bacillus sp. KNK—M01 is dated January 22, 2007, and has the accession number FERM BP—10765. 8566 Tsukuba, Ibaraki, Japan (Early 1-1-1 Central No. 6) Deposited (original deposit date: domestic deposit on January 26, 2006 transferred to international deposit under 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.

[0022] (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.

[0023] (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 bacterial cells are collected from the microorganism culture solution by centrifugation or filtration, and the obtained bacterial cells are crushed by a physical method using an ultrasonic crusher or glass beads, and then centrifuged. The cell-free extract is prepared by removing the cell residue in, and the cell-free extract is subjected to fractional precipitation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, ultrafiltration. It is possible to isolate the acylamidase.

[0024] (Amino acid sequence)

 The purified acylamidase can be clarified 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 having the amino acid sequence shown 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 As long as it has acylamidase activity (preferably stereoselective acylamidase activity), it is included in the present invention.

The acylamidase and polypeptide of the present invention may be natural or artificially modified. [0025] In the amino acid sequence represented by SEQ ID NO: 1, a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added is obtained using the amino acid sequence represented by SEQ ID NO: 1. , Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1989) and the like.

 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 refers to a plurality of enzymes with different origins having the same functions, for example, by using the multiple alignment function of the software GENETYX (manufactured by GENETYX) to optimize the amino acid sequence. When aligned and compared, amino acid matches between multiple sequences are indicated.

 Examples of the highly conserved region in SEQ ID NO: 1 include the 111th to 115th amino acid sequences, the 138th to 149th amino acid sequences, and the 163rd to 167th amino acid sequences.

[0026] 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, lie, Met, Cys, Pro, Phe (Group 2: neutral polar amino acids) Ser, Thr, Gin, Asn, Trp, Tyr

 (Group 3: acidic amino acids) Glu, Asp

 (Group 4: basic amino acids) His, Lys, Arg

In the present invention, an amino acid sequence having 70% or more, preferably 80% or more, more preferably 85% or more, and still more preferably 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1. Polypeptides having a sequence and having acylamidase activity are also polypeptides of the present invention.

As used herein, amino acid sequence identity is determined by BLAST (Altschul, Stephen F. et al., Nucleic Acids Res. 25, 3389-3402 (1997)), and can be determined by homology analysis.

 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 an acylamidase activity is, for example, 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 a acylamidase activity exhibiting the physicochemical properties (1) to (6) described above, or has at least a acylamidase activity that acts on formaldehyde.

[0028] 3. DNA

 The DNA of the present invention is the above-mentioned novel acylamidase and DNA encoding the 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 the microorganism that is the origin of the polypeptide by a known method.

[0029] In the following, as a method for obtaining the DNA of the present invention, the ability to describe an example using the above Bacillus sp. KNK-M01 is not limited to this.

 First, the polypeptide (enzyme) purified from the cell-free extract of the microorganism is digested with an appropriate endopeptidase, and the fragment cleaved by reverse-phase HPLC is purified. A part of the amino acid sequence is determined by the service (Applied Biosystems). 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, Murray et al. (Nucl., Acids Res., 8, 4 321 -4325, 1980). Using this chromosomal DNA as a saddle, PCR is performed using the PCR primers described above, a part of the DNA encoding the polypeptide is amplified, and the base sequence is determined. The base sequence is determined using, for example, ABI373A DNA Sequencer (Applied Biosystems). obtain. If the base sequence of a part of the DNA encoding the polypeptide is clarified, the entire sequence can be determined by, for example, the Southerno or hybridization method.

[0030] Examples of the DNA obtained in this manner include DNA having the base sequence set forth in SEQ ID NO: 2. However, the DNA of the present invention is not limited thereto, and all DNAs encoding the above-described polypeptides of the present invention are included in the present invention. For example, a DNA that hybridizes under stringent conditions with a DNA complementary to the base sequence shown in SEQ ID NO: 2 and that encodes a polypeptide having an acylamidase activity is included in the present invention. .

 Here, “DNA that hybridizes under stringent conditions with a DNA that also has a complementary base sequence ability to the base sequence shown in SEQ ID NO: 2” means the “Kouguchi-ichi” hybridization method, plaque hybrider. A DNA having a base sequence that is complementary to the base sequence shown in SEQ ID NO: 2 when a hybridization method or Southern hybridization test method is performed is a DNA that specifically forms a hybrid.

 The DNA of the present invention may be natural or artificially modified.

 [0031] In the present specification, "stringent conditions" refers to a filter in which a polynucleotide derived from a colony or plaque is immobilized, at a temperature of 65 ° C in the presence of 0.7 to 1. OM NaCl. After performing the hybridization, the filter was used under the condition of 65 ° C using a 2x SSC solution (composition of 1x SSC solution consisting of 150mM sodium chloride and 15mM sodium citrate). This is the condition for cleaning. Preferably, the hybridization is performed in the same manner as described above, and then washed with a 0.5-fold concentrated SSC solution at 65 ° C. More preferably, the hybridization is performed as described above. The condition is to wash with a 0.2 times concentrated SSC solution at 65 ° C. More preferably, after carrying out a noblerization in the same manner as described above, a 0.1 times concentration at 65 ° C is obtained. This is the condition for washing with the SSC solution.

 The DNA of the present invention encodes a acylamidase having the ability to code for the acylamidase exhibiting the physicochemical properties (1) to (6) described above, or at least the acylamidase activity acting on formaldehyde. I prefer that! /.

[0032] 4. Vector The vector DNA used for introducing the DNA of the present invention into a host microorganism and expressing it in the introduced host microorganism can express the gene encoding the DNA force S in an appropriate host microorganism. Any one can be used. Examples of such vector DNA include plasmid vectors, phage vectors, cosmid vectors, and the like. A shuttle vector capable of exchanging genes with other host strains can also be used.

 [0033] Such vectors may contain control elements such as operably linked promoters (lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter, etc.). And can be suitably used as a vector comprising an expression unit operably linked to the DNA of the present invention. For example, pUC18 (manufactured by Toyobo Co., Ltd.), pUC19 (manufactured by Toyobo Co., Ltd.), pUCNT etc. that can be prepared by those skilled in the art by the method described in the pamphlet of International Publication No. Can be preferably used.

 [0034] As used herein, the term "regulatory element" refers to a nucleotide sequence having a functional promoter and any related transcription elements (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).

 As used herein, the term “operably linked” refers to a gene in which various regulatory elements, such as a promoter that regulates the expression of a gene, an ensemble, and the like, are operably linked in a host cell. That means. It is a matter well known to those skilled in the art that the type and kind of the control factor can vary depending on the host.

[0035] 5. Host

 Examples of host cells into which the vector containing the DNA of the present invention is introduced include bacteria, yeasts, filamentous fungi, plant cells, animal cells and the like, and 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 the host cell, the vector can be introduced, for example, by the salted calcium method.

[0036] 6. Method for producing acylamidase or polypeptide

The present invention also provides a Bacillus sp. KNK-MOl (FERM BP-10765) or the above-mentioned transformant cultured in a nutrient medium, and from the obtained culture broth. The present invention relates to a method for producing a polypeptide by obtaining the above-mentioned acylamidase or polypeptide.

 In other words, by culturing Bacillus sp. KNK-M01 or the transformant of the present invention, the acylamidase can be produced efficiently. Bacillus sp. KNK-M01 and the culture of the transformant of the present invention can be cultured as long as it grows, usually liquid nutrients containing carbon source, nitrogen source, inorganic salts, organic nutrients, etc. It can be carried out using a medium. Bacillus sp. KNK-M01 and the acylamine accumulated in the culture medium of the transformant of the present invention can be used as a medium containing the acylamidase in the culture medium. It can also be used after purification or partial purification using generally known protein purification techniques.

 [0037] 7. Amide compound あ る is hydrolysis of ester compound

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

 [0038] 8. Method for optically active compounds

 Next, a method for producing a photoactive compound using the acylamidase of the present invention or a microorganism capable of producing the enzyme will be described.

 The method for producing an optically active substance of the present invention includes a racemic amidy compound, an ester compound, the above-mentioned acylamidase or polypeptide, Bacillus sp. KNK-MOL (FERM BP-10765 Or a culture of the transformant described above, and selectively hydrolyzing only one of the racemates.

Examples of the microorganism having the ability to produce the amidase of the present invention include, for example, the above-mentioned Bacillus sp. KNK-MOl (FERM BP-10765) and a transformant introduced with the above-described vector containing DNA. Is mentioned. According to this production method, the general formula (1)

 [0039] [Chemical 3]

 [0040] By reacting the ester compound or amide compound represented by [0040] with the enzyme or a culture of a microorganism capable of producing the enzyme, the general formula (2):

[0041] [Chemical 4]

 [0042] An optically active alcohol or an optically active amine represented by the formula:

In the formula (1) and (2), m and n each represent an integer of independently 0-7, independently each R \ R ² is Ariru group of carbon number 6-14, 4 carbon 14 heteroaryl groups, 6 to 14 carbonyloxy groups, 4 to 14 carbon heteroaryloxy groups, 1 to 5 alkoxy groups, 2 to 5 alkoxycarbonyl groups, carbon A branched alkyl group having 3 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 cyan group, a methyl group or a carboxyl group; These groups may be substituted. R ³ represents a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms. X represents a nitrogen atom or an oxygen atom.

[0043] Examples of the aryl group having 6 to 14 carbon atoms include a phenol group and a naphthyl group. Examples of the heteroaryl group having 4 to 14 carbon atoms include a pyridyl group, a cenyl 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 enyloxy 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 methoxy group and eth Examples thereof include a xy group and a tert butoxy group. Examples of the alkoxycarbon group having 2 to 5 carbon atoms include a methoxycarbol group, an ethoxycarboro group, a tertbutoxycarboro group, and the like. Examples of the branched alkyl group having 3 to 5 carbon atoms include isopropyl group, sec butyl group, and tert butyl group. Examples of the alkenyl group having 2 to 5 carbon atoms include a beryl group and an aryl 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 in the alkoxy carbo group is the number including 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 a substituted methyl group, n = 0 to 4 and R ² is a methyl group or a carbon number of 6 to 12 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 -phenyl) acetamide, 1-phenylethyl acetate and the like.

The compound force represented by the formula (1) m = 0-3 and R ¹ is substituted, but may be a methyl group, n = 0-4 and R ² cate group or an aryl having 6-12 carbon atoms. When R ³ is a linear alkyl group having 1 to ³ carbon atoms, is a branched chain alkyl group, the generated amine or alcohol force S37% ee, 51% ee, or even higher than 99% ee It can be obtained with optical purity.

In the production method of the present invention, the enzyme of the present invention or a culture of microorganisms capable of producing the enzyme is allowed to act on the amide or ester compound. Here, “cultured product” means a culture solution containing microbial cells, cultured microbial cells, or a processed product thereof. Here, “the processed 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 enzyme or immobilized bacteria by known means. The fixation can be performed by a method well known to those skilled in the art (for example, a crosslinking method, a physical adsorption method, a comprehensive method, etc.). [0045] (Substrate concentration)

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

[0046] (Reaction _P H)

 The pH at which the enzyme of the present invention is allowed to act is preferably a pH of 5.0 or more, more preferably pH 6.0 or more, and an upper limit of preferably from the viewpoint of the optimum pH of the enzyme action. Is pH 10.0 or less, more preferably pH 9.0 or less.

[0047] (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, from the viewpoint of the optimum temperature of action of the enzyme and thermal stability. C or lower, more preferably 40 ° C or lower.

[0048] (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; Halogenated hydrocarbon solvents such as methylene chloride and chloroform; ether solvents such as jetyl ether and diisopropyl ether; ester solvents such as ethyl acetate and butyl acetate; ketone solvents such as acetone and methyl ethyl ketone In addition, acetonitrile can be used as appropriate.

 The above reaction may be carried out in a one-phase system, or if necessary, the above organic solvent is added to a water solubility or higher and the reaction is carried out in a two-phase system of an aqueous phase and an organic solvent phase. You can also. By coexisting an organic solvent in the reaction system, selectivity, conversion rate, yield, etc. are often improved.

[0049] (Reaction time)

The reaction is usually carried out until about half of the racemic ester or amido 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 at 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 You may make it react excessively.

 [0050] (Extraction purification)

 By the above reaction, an optically active compound represented by the general formula (2) and an unreacted enantiomer substrate are formed. The produced optically active compound and the unreacted enantiomer substrate can be isolated by known methods such as reaction mixture liquid 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 jetyl ether and diisopropyl ether; esters such as ethyl acetate and butyl acetate; hydrocarbons such as hexane, octane and benzene ; With a common solvent such as 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 an ordinary method while maintaining the optical activity. The alcohol or amine of the opposite stereo to the active substance can be derived. Further, the optically active alcohol or amine obtained by the above reaction can be converted to an ester or amidy state while maintaining the 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.

 The invention's effect

 [0051] According to the present invention, a novel acylamidase, a DNA encoding the novel acylamidase, or any one of them can be used to react various substrates with amide compounds or under mild conditions. A method is provided for hydrolyzing an ester compound.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0052] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

(Example 1) Purification of acylamidase HBS Bacillus sp. KNK— M01 (FERM BP— 10765) in 5 L medium (thread and adult: Polypeptone 10 g / L, Meat Extract 10 g / L, NaCl 3 g / L, Yea st Extract 5 g / L (pH 7. In 2)), the cells were cultured at 28 ° C for 24 hours. Subsequently, the cells were collected from the culture solution by centrifugation, washed thoroughly with physiological saline, and then suspended in lOOmM phosphate buffer (pH 7.0) containing ImM DTT. The obtained suspension was crushed by ultrasonic crushing. Next, solids in the crushed material were removed by centrifugation, and a cell-free extract was prepared.

 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 the supernatant so as to be 60% saturation, and this was dissolved. Then, the precipitate generated by centrifugation was recovered.

 [0053] This precipitate was dissolved in an lOOmM phosphate buffer (pH 7.0) containing ImM 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 lOOmM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride and ImM DTT.

 [0054] The eluted active fractions were collected, and ammonium sulfate was dissolved in this to a final concentration of 20%, and 10 mM phosphate buffer containing 20% ammonium sulfate and ImM DTT. The solution was applied to an Octyl-Sepharose (Amersham Biosciences) column (size: 20 × 65 mm) pre-equilibrated with (pH 7.0) to adsorb the active fraction. Columns with 10 mM phosphate buffer (pH 7.0) containing 10% ammonium sulfate and ImM DTT, and then with 10 mM phosphate buffer (pH 7.0) containing 5% ammonium sulfate and ImM DTT. After washing, the active fraction was eluted with 25 mM phosphate buffer (pH 7.0) containing ImM DTT.

[0055] The eluted active fractions were collected and dialyzed against 5 mM phosphate buffer (pH 7.0) containing ImM DTT. This is applied to a Hydroxyapatite (Amershamno Science Co., Ltd.) column (size: 20x15mm) equilibrated with the same buffer solution. Adsorbed. After washing the column with 10 mM phosphate buffer (pH 7.0) containing ImM DTT, the active fraction was eluted with 25 mM phosphate buffer (pH 7.0) containing ImM DTT.

 [0056] The eluted active fractions were collected and sufficiently diluted with 10 OmM phosphate buffer (pH 7.0) containing 20% ammonium sulfate and ImM DTT. This was applied to a Butyl-Toyopearl (manufactured by Tosoichi Co., Ltd.) column (size: 20 × 20 mm) equilibrated with the same buffer to adsorb the active fraction. The column was washed with lOOmM phosphate buffer (PH 7.0) containing 15% ammonium sulfate and ImM DTT, and then lOOmM phosphate buffer (ρΗ7.0) containing 10% ammonium sulfate and ImMDTT. Thereafter, the active fraction was eluted with lOOmM phosphate buffer (pH 7.0) containing 5% ammonium sulfate and ImM DTT.

 [0057] The eluted active fractions were collected to obtain a single purified enzyme preparation electrophoretically. Hereinafter, this enzyme is referred to as HBS.

 In addition, the activity of the acylamidase 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, and the reaction solution was analyzed by high performance liquid chromatography to quantify the concentration of produced aline. The activity of the obtained acylamidase was 2.7 U per 1 mg of purified enzyme.

[0058] [Substrate Solution Composition]

 Acetanilide 5. OmM

 Phosphate buffer (ρΗ7.0) lOOmM

 Total 0.9 mL

 [0059] [Measurement conditions by high performance liquid chromatography]

 Column: Wakosil—II5C18AR 4.6 mm x 150 mm (manufactured by Wako Pure Chemical Industries, Ltd.) Mobile phase: 10 mM potassium phosphate buffer (pH 7.0): methanol = 4: 1

 Flow rate: 1. OmL, mm

 Detection wavelength: 265nm

[0060] (Example 2) Physicochemical matter of purified enzyme 1

 The HBS purified enzyme obtained in Example 1 was examined for its physical properties.

(1) Action The acylamidase activity of the HBS purified enzyme was measured in the same manner as in Example 1.

[0061] (2) Molecular weight

 When the purified enzyme was subjected to gel filtration analysis using TSK GEL-G-3000 (0.75 × 60 cm, manufactured by Tosohichi 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.

[0062] (3) Optimum pH

 In the range of pH 4.0 to: L I .0, the activity using acetonitrile 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, according to the measurement method described above, 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 to 9.0: 0.1 M Tris-HCl buffer

 pH 9.0 ~: L 1. 0: 0.1 M glycine sodium buffer

[0063] (4) Optimal temperature

 In the same manner as described above, the activity using acetate hydrochloride 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.

[0064] (5) Thermal stability

 The purified enzyme was added to 0.1 M potassium phosphate buffer (pH 7.0), and this was treated at 10 to 80 ° C. for 30 minutes, and then the amidase activity was measured. As a result, more than 90% of the activity remained after treatment at 10 ° C to 40 ° C compared to before treatment.

[0065] (6) Inhibitor

 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 reaction solution was analyzed by high-performance liquid chromatography to quantify the produced aline. As a result, HgCl or AgNO

 The reaction did not proceed even when the deviation of 2 3 was added.

[Substrate solution composition] Acetanilide 5mM

 Phosphate buffer (ρΗ7.0) lOOmM

 Inhibitor ImM

 Total 0.9 mL

 [0066] (Implementation 3) 2 mm ^ m

 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, ammine, or ammonia produced in the reaction solution was analyzed under the conditions shown in Tables 1 and 2. However, when p- -trophyl acetate, p- -trophyl propionate or p- -trophenylbutyrate is used as a substrate, the reaction solution is basified with NaHCO, and then 400

 Three

 The produced p--trophenol was measured by absorbance at nm.

 In the reaction using acetamide, n-butylamide, and benzamide as substrates, the generated ammonia was measured using Conway's diffusion analysis method. That is, 0.2 mL of the reaction supernatant and 2. OmL of saturated potassium carbonate solution were added to the outer chamber of Conway's diffusion tube. In addition, after placing 1.5 mL of 0.01N sulfuric acid in the inner chamber, it 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.

 Collected internal solution 0.5 mU 0.5 mL 330 mM sodium phenoxide and 0.5 mL 0.01% (wZv) -troprusside sodium 0.5 mL 0.84% (vZv) hypochlorous acid After adding aqueous sodium acid solution, the mixture was heated at 100 ° C for 5 minutes. Thereafter, 3 mL of distilled water was added, and the absorbance (A) of the reaction solution was compared with the absorbance of the standard sample to determine the activity value.

 640

 [0067] The amount of the enzyme used for measuring the substrate specificity varies depending on the substrate used, and the amount is shown in the column of enzyme usage in Table 1. The unit (mU) of the amount of enzyme used is shown as the enzyme activity when using acetate as a substrate. Relative activity was expressed as a relative value when the activity for each substrate per unit enzyme amount was defined as 100 for activity against acetate.

The results are shown in Table 1. This enzyme was highly active against acetate, formaldehyde, and phenyl acetate. It did not act on benzamide, n-butyramide, N-acetylyl L-phenylalanine, N-acetylyl DL-tryptophan, and N-acetylyl L-tyrosine. [0068] Therefore, the acylamidase of this example substantially acts on each of formaldehyde, acetonitrile, and acetic acid phenyl, and benzamide, n-butylamide, N-acetyl-L phenol. It is clear that it has a unique and useful substrate specificity, which is different from the basic specificity of conventional acylamidase, that it does not substantially act on each of lulanin, N-acetylyl DL tryptophan, and N-acetylyl-L tyrosine. became

[0069] [Substrate Solution Composition]

 Substrate 1 ~: LOOmM

 Phosphate buffer (ρΗ7.0) 50mM

[0070] [Table 1]

Relative activity Measurement conditions Enzyme consumption

 (mU) (mM) (nm)

Ethyl acetate 12370 GC 5 50 ―

Ethyl propionate 2744 GC 5 50

Ethyl butyrate 229 GC 25 50-

Phenyl acetate 3800 AS- 9 25 50 210

Benzyl acetate 730 AS-8 25 50 210

1-Phenylethyl acetate 398 AS— 10 75 50 210 p-Nitrophenyl acetate 122000 Abs 400nm 0.015 1

p-Nitrophenyl propionate 129000 Abs 400nm 0.015 1

p— Nitrophenyl butyrate 1860 Abs 400nm 15 1

 作 作

 Formanilide 260 AS- -3 5 50 240

Acetanilide 10 Sasese 0: AS- -3 5 5 240

Propanailide 12 AS- -3 5 50 240 o-Nitroacetanilide 5.3 AS- -5 1 20 5 240 m-Nitroacetanilide 347 AS- -5 20 1 240 p-Nitroacetanilide 21 AS- -5 60 20 240 o-Hydroxyacetanilide 2.1 AS-- 3 20 2 240 m-Hydroxyacetanilide 146 AS- -5 20 30 240 o-Chloroacetanilide 8.5 AS- -6 20 10 290 p-Chloroacetanilide 50 AS- -6 60 20 290

N-Benzylacetamide 11 AS- -1 1500 30 260

N- (1-Phenylethyl) acetamide 0.5 AS- -2 12000 50 210

N-(2-PhenylethyOacetamide 3.9 AS- 10 1500 30 210

N- (3-Phenylpropyl) acetamide 3.3 AS- -5 1500 50 260

N- (4-Phenylbutyl) acetamide 2.8 AS- -5 1500 50 260

N— Acetyl— o-toluidine 0.8 AS- -5 120 30 240

N— Acetyl—m—toluidine 366 AS- -5 20 10 240

N— Acetyl— p-toluidine 64 AS- -3 20 20 260

Melatonine 0.6 AS 1 4 240 225

Acetamide conway 80 30 I Butylamide conway 80 30 I Benzamide conway 80 30-

N—Acety and Phenylalanine AS-2 1500 20 260 N-Acety DL DL Tryptophan AS-2 1500 20 225 N—Acetyl— L—Tyrosine AS—1 1500 20 281] [AS-1 to 11]

 Column Wakosil-II 5C18RS (4.6 x 150mm) AS- 1 to 11

 (Wako Pure Chemical Industries, Ltd.) Mobile phase 73 mM phosphate buffer (pH 5.3) Acetonitrile = 19: 1 AS- -1

 73 mM phosphate buffer (pH 5.3) Phase 2 = 9: 1 AS- -2

73 mM phosphate buffer (pH 5.3) Phase 2 = 7: 1 AS- -3

73 mM phosphate buffer (pH 5.3) Phase 2 = 7: 2 AS- -4

73 mM phosphate buffer (pH 5.3) Phase 2 = 7: 3 AS- -5

73 mM phosphate buffer (pH 5.3) Phase 2 = 13: 7 AS- -6

73mM phosphate buffer (pH 2.5) Phase 1 = 99: 1 AS--7

73mM phosphate buffer (pH 2.8) Phase 2 = 9: 1 AS--8

73 mM phosphate buffer (pH 2.8) Phase 2 = 4: 1 AS- -9

73 mM phosphate buffer (pH 2.8) Phase 2 = 3: 1 AS- -10

73 mM phosphate buffer (pH 2.8) Phase 2 = 3-2 AS- -11

1.0 mL / mm AS— 1-11

[GC]

 Column TC-FFAP (15m x 0.25mm l.D.0.25 U m)

 (GL-Sciences) Column temperature 60 ° C_200 ° C gradient

 Injector temperature 200. C

 Detector temperature 200. C (Example 4) Physicochemical case of purified enzyme 3 (stereoselection case)

Of the reaction solution 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-phenol ethanol was 37% ee in R form, and 1-phenethylamine was 51% ee in R form.

 [Measurement conditions by high-performance liquid chromatography (1 phenylethanol)]

Column: Chiralcel OB 4.6mmx250mm

 (Daicel Chemical Industries)

Mobile phase: n— Hexane: 2— Propanol = 9: l Flow rate: 1. OmL / min

 Detection wavelength: 254nm

 [0073] [Measurement conditions by high performance liquid chromatography (1-phenethylamine)]

 Column: Chiralcel CR (+) 4. 6mmxl50mm

 (Daicel Chemical Industries)

 Mobile phase: HCIO (pHl. 5)

 Four

 Flow rate: 0.8 mL / min

 Detection wavelength: 210nm

 [0074] (Actual column 5) Physics and chemistry of purified enzyme 4 (Km needled to acetoanilide)

 For the HBS purified enzyme obtained in Example 1, the Km for acetate-lid was examined. Purified enzyme solution (0.1 mL) was added to a substrate solution (0.9 mL) having the following composition and reacted at 30 ° C. After 30 minutes, 1 mL of methanol was added to stop the reaction, and the reaction solution was analyzed by high performance liquid chromatography. there were. High performance liquid chromatography was performed under the same conditions as in Example 1.

 [Substrate solution composition]

 Acetanilide 0.1 to 2.5 mM

 Phosphate buffer (ρΗ7.0) lOOmM

 Total 0.9 mL

 [0075] (Example 6) Closing of HBS message

 (Create PCR primers)

 The N-terminal amino acid sequence of the purified HBS obtained in Example 1 was determined with an ABI492 type protein sequencer (PerkinElmer Biosystems). Further, the purified HBS obtained in Example 1 was digested with lysyl endobeptidase, and the amino acid sequence of the obtained peptide fragment was determined in the same manner as the N-terminal amino acid sequence. Considering the nucleotide sequence that is also expected to have 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 according to the method described in the culture solution of Bacillus sp. KNK-M01 (FERM BP-10765), Murray et al. (Nucl. Acids Res., 8, 4321, 1980). PCR was performed on the obtained chromosomal DNA in a saddle type using the primer synthesized above. As a result, an approximately 134 bp DNA fragment considered to be a part of the HBS gene was obtained.

 This DNA fragment was cloned into plasmid pT7Blue T—Vector (Novagen), and ABI PRI¾M Dye Terminator Cycle sequencing Ready Reaction Kit (Perkin Elmer) and ABI 310 DNA Sequencer (Perkin Elmer) were used. The base sequence was determined.

 [0076] (Southern Hybridization)

 The chromosomal DNA was treated with the restriction enzyme Hindlll to prepare a DNA fragment. Southern hybridization was performed using the determined DNA sequence as a probe, and DNA fragments that hybridized with the probe were extracted from the gel. The extracted DNA fragment was inserted into the pBluescript vector to create a mini-library, and then transformed into E. coli CFM109). From the transformed E. coli library, positive clones that hybridize with the probe are selected by colony hybridization, and the DNA sequence of the insert is determined using the ABI 310 DNA Sequencer (Perkin Elmer). did. As a result, a 1050 bp ORF (SEQ ID NO: 2) was revealed.

 [0077] (ORF database analysis)

 When the homology search by BLAST was performed on the DNA sequence of the obtained ORF, the top three genes with high homology and their homologies were as follows.

 Homo serine ac etyltransf era se (C oryneb ac terium glutamicum ATCC 1 3032): 64%

 Homo serine acetyltransferase (Rhodococcus sp. DK 17): 63%

 Deacetylcephalosporin and acetyltransferase (Streptomyces clavuligerus): 34%

 (Example 7) Construction of a plasmid containing an HBS gene

Based on the ORF information obtained in Example 6, the site of the restriction enzyme Ndel at the 5 'end of the ORF Primer 3 (SEQ ID NO: 5) designed so as to add, and primer 4 (SEQ ID NO: 6) designed to add the restriction enzyme Hindm site to the 3 ′ end of ORF were synthesized. Using this primer, PCR was carried out using the resulting plasmid as a positive clone, and the amplified DNA fragment was treated with the restriction enzymes Ndel and Hindm, and pET21a (Takara Bio) was used. +) It was incorporated into a vector (Novagen) to obtain a recombinant vector PET21HBS.

(Example 8) Production of Escherichia coli containing HBS gene Expression of HBS

Using the recombinant vector pET21HBS obtained in Example 7, E. coli BL21 (DE3) competent cells (Novagen) were transformed according to the instruction manual, and recombinant E. coli BL21 (DE3 ) (pET21HBS) was obtained. The resulting recombinant E. coli BL21 (DE3) (pET21HBS) is inoculated into LB medium, cultured at 37 ° C for 4-6 hours, and then IP TG is aseptically added to a final concentration of ImM. The culture was further performed at 20 ° C for 20 hours. The obtained culture broth was collected, suspended in lOOmM 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 acetate-lid as a substrate in the same manner as in Example 3, the activity was found to be 0.73 U per mL of culture solution.

Claims

Claims [1] A acylamidase having the following physical properties:  (1) Molecular weight: about 90,000 by gel filtration analysis, about 40,000 by SDS-polyacrylamide gel electrophoresis;  (2) Substrate specificity: Substantially acts on each of formaldehyde, acetatetolide, and phenylacetate, and includes benzamide, n-butylamide, N-acetylethyl L-phenylalanine, N-acetylyl DL— Virtually no effect on tryptophan or N-acetyl-L-tyrosine;  (3) Optimum pH: 6-9;  (4) Optimum temperature of action: 25-45 ° C;  (5) Thermal stability: 10-40 ° C;  (6) Inhibitor: Activity is completely inhibited by HgCl, AgNO.  twenty three  [2] The acylamidase according to claim 1, which is an enzyme having a microbial strength belonging to the genus Bacillus.  [3] The Bacillus sp. Force The acylamidase according to claim 1, which is an obtained enzyme.  [4] The acylamidase according to claim 1, which is an enzyme obtained from Bacillus sp. KNK-M01 (FERM BP-10765). [5] 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 set forth in SEQ ID NO: 1 and having an acylamidase activity.  [6] The polypeptide according to claim 5, which has acylamidase activity exhibiting physicochemical properties according to claim 1. [7] The method according to claim 5, having at least a acylamidase activity acting on formaldehyde. Polypeptide.  [8] A DNA encoding the polypeptide according to any one of claims 5 to 7. [9] DNA shown in (A) or (B) below:  (A) DNA comprising the base sequence set forth in SEQ ID NO: 2;  (B) A DNA that is hybridized under stringent conditions with a DNA complementary to the nucleotide sequence set forth in SEQ ID NO: 2 and that encodes an enzyme having acylamidase activity. [10] according to claim 1 DNA ₀ of claim 9, wherein the encoding Ashiruamidaze indicating the physical I inhibit biological properties described  11. The DNA according to claim 9, which encodes a acylamidase having an acylamidase activity that acts on at least formaldehyde. [12] Claim 9-: A vector comprising the DNA according to any one of L1. [13] A transformant obtained by transforming a host cell with the vector according to claim 12. 14. The transformant according to claim 13, wherein the host cell is E. coli. [15] Bacillus sp. KNK-MOl (FERM BP-10765) or the transformant according to claim 13 or 14 is cultured in a nutrient medium, and the obtained culture solution is used to claim Any one of the forces 1 to 4 or the Vacylamid of claim 5 to 7, or the polypeptide of claim 1 is obtained. Production method. [16] The amide compound or the ester compound, the acylamidase according to any one of claims 1 to 4, or the polypeptide according to any one of claims 5 to 7, the Bacillus sp. ) KNK-MOl (FERM BP-10765), or a culture of the transformant according to claim 13 or 14, wherein the amidy compound or esteri compound is hydrolyzed. To produce carboxylic acid, alcohol or amine. [17] The racemic amidy compound or the ester compound, the force according to any one of claims 1 to 4, the acylamidase according to claim 1, or the polypeptide according to any one of claims 5 to 7, Bacillus sp. Bacillus sp. KNK-MOl (FERM BP-10765), or a culture of the transformant according to claim 13 or 14, wherein one of the racemates A method for producing an optically active substance, wherein only the three-dimensional structure is selectively hydrolyzed. General formula (1):  [Chemical 1]

(In the formula, m and n each independently represent an integer of 0 to 7,

R ² is independently an aryl group having 6 to 14 carbon atoms, a heteroaryl group having 4 to 14 carbon atoms, an aryloxy group having 6 to 14 carbon atoms, a heteroaryloxy group having 4 to 14 carbon atoms, or carbon. An alkoxy group having 1 to 5 carbon atoms, an alkoxycarbonyl group having 2 to 5 carbon atoms, a branched alkyl group having 3 to 5 carbon atoms, an alkenyl group having 2 to 5 carbon atoms, an alkynyl group having 2 to 5 carbon atoms, and a carbon number 5 to 7 alkyl groups, cyano groups, methyl groups or carboxyl groups are shown, 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. The ester compound or amide compound represented by formula (1) is any one of claims 1 to 4 and the polypeptide according to any one of claims 5 to 7 or the Bacillus sp (Bacillus sp.). sp.) KNK-M01 (FERM BP—10765), or the general formula (2) by acting the transformant culture of claim 13 or 14: [Chemical 2]

(Wherein, X, m, n, R \ R 2 is Formula (1)) to the production method of the walking optically active alcohol represented by the optically active Amin. In the compound represented by the general formula (1), m = 0 to 3 and R ¹ is substituted !, may! /, A methyl group, 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 hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms. The manufacturing method as described.