Amidase, gene for the same, vector, transformant, and method for production of optically active carboxylic acid amide and optically active carboxylic acid by using any one of those items

Abstract

The present invention has its object to provide a novel polypeptide having amidase activity to selectively hydrolyze S-enantiomer in racemic nipecotamide, a DNA encoding the polypeptide, a vector containing the DNA, a transformant transformed with the vector, and a method for producing an optically active carboxylic acid amide and an optically active carboxylic acid in which a racemic carboxylic acid amide is hydrolyzed with the polypeptide or the transformant.

Claims

1. An isolated DNA which encodes a polypeptide which is any one of the following polypeptides (b), and (c): (b) a polypeptide comprising an amino acid sequence derived from the amino acid sequence set forth in SEQ ID NO:1 by substitution, insertion, deletion, and/or addition of 90 or less amino acids, and having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide and wherein the polypeptide is not SEQ ID NO: 1; and (c) a polypeptide having sequence identity of 80% or higher to the amino acid sequence set forth in SEQ ID NO:1, and having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide and wherein the polypeptide is not SEQ ID NO: 1.

2. An isolated DNA which is any one of the following DNAs (a), (b), and (c): (b) a DNA hybridizable under a stringent condition with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO:2, and encoding a polypeptide having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide, and wherein the DNA does not comprise SEQ ID NO: 2, wherein the stringent condition is a condition where hybridization is carried out in the presence of 0.7 to 1.0 M NaCl, at 65° C., followed by washing the filter with 0.2-fold concentration of SSC solution at 65° C.; and (c) a DNA having sequence identity of 80% or higher to the nucleotide sequence set forth in SEQ ID NO:2, and encoding a polypeptide having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide, and wherein the DNA does not comprise SEQ ID NO: 2.

3. The DNA according to claim 2, which has sequence identity of 85% or higher to the nucleotide sequence set forth in SEQ ID NO:2, and encodes the polypeptide having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide.

4. A vector comprising an isolated DNA which encodes a polypeptide which is any one of the following polypeptides (a), (b), and (c): (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1; (b) a polypeptide comprising an amino acid sequence derived from the amino acid sequence set forth in SEQ ID NO:1 by substitution, insertion, deletion, and/or addition of 90 or less amino acids, and having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide; and (c) a polypeptide having sequence identity of 80% or higher to the amino acid sequence set forth in SEQ ID NO:1, and having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide.

5. A transformant which is producible by transformation of a host microorganism with the vector according to claim 4, wherein said host microorganism is not Cupriavidus sp.

6. The transformant according to claim 5, wherein the host microorganism is Escherichia coli.

7. The isolated DNA according to claim 1 which encodes the polypeptide which has sequence identity of 85% or higher to the amino acid sequence set forth in SEQ ID NO:1, and has activity to selectively hydrolyze S-enantiomer in racemic nipecotamide.

8. The DNA according to claim 1, which is isolated from a microorganism belonging to the genus Cupriavidus.

9. The DNA according to claim 8, wherein the microorganism belonging to the genus Cupriavidus is Cupriavidus sp. KNK-J915 strain (FERM BP-10739).

10. A vector comprising an isolated DNA which is any one of the following DNAs (a), (b), and (c): (a) a DNA comprising the nucleotide sequence set forth in SEQ ID NO:2; (b) a DNA hybridizable under a stringent condition with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO:2, and encoding a polypeptide having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide, wherein the stringent condition is a condition where hybridization is carried out in the presence of 0.7 to 1.0 M NaCl, at 65° C., followed by washing the filter with 0.2-fold concentration of SSC solution at 65° C.; and (c) a DNA having sequence identity of 80% or higher to the nucleotide sequence set forth in SEQ ID No:2, and encoding a polypeptide having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide.

Description

EXAMPLES

(1) The following examples illustrate the present invention in detail. However, the present invention is not limited to these examples.

(Example 1) Purification of Amidase Derived from Cupriavidus sp. KNK-J915 (FERM BP-10739)

(2) In the following examples, the amidase activity was determined by the following procedure. To 100 mM phosphate buffer (pH 7.0) were added 1% N-benzylnipecotamide and an enzyme solution, and allowed to react for 1 hour at 30° C. Then, the solution was analyzed under high speed liquid chromatography analysis conditions A. Enzyme activity which produces 1 μmol of N-benzylnipecotic acid per 1 minute under these conditions was defined as 1 unit.

(3) High Speed Liquid Chromatography Analysis Condition A

(4) Column: YMC-A303 (4.6 mmφ×250 mm, product of YMC Co., Ltd.)

(5) Eluant: 20 mM phosphate aqueous solution (pH 2.5)/acetonitrile=9/1

(6) Flow rate: 1.0 ml/min

(7) Column temperature: 30° C.

(8) Measurement wavelength: 210 nm

(9) An amount of 100 ml of a liquid medium (pH 7.0) (composition: 1.0% meat extract; 1.5% polypeptone; 0.5% bacto yeast extract; and 0.3% NaCl) was poured into a 500-ml Sakaguchi flask, and steam-sterilized for 20 minutes at 120° C. The liquid medium was aseptically inoculated with Cupriavidus sp. KNK-J915 strain, and the strain was cultured with shaking at 35° C. for 72 hours to yield a culture solution in an amount of 4.2 L. The obtained culture solution was centrifuged to collect cells, and then the cells were washed with 500 ml of 100 mM phosphate buffer (pH 7.0), and suspended in 200 ml of 100 mM phosphate buffer (pH 7.0). The cells in the suspension were ultrasonically disrupted by an ultrasonic disintegrator (SONIFIER 250, product of BRANSON), and centrifuged to remove cell residues. Thus, a cell-free extract was obtained.

(10) Ammonium sulfate was added to the cell-free extract to reach a saturation concentration of 20%. After being stirred for 30 minutes at 4° C., the mixture was centrifuged to remove formed precipitates. Additionally, ammonium sulfate was added to the supernatant to reach a saturation concentration of 40%. After being stirred for 30 minutes at 4° C., the mixture was centrifuged to obtain formed precipitates, and the precipitates were suspended in 100 mM phosphate buffer (pH 7.0), and dialyzed with 100 mM phosphate buffer (pH 7.0).

(11) The dialyzed enzyme liquid was supplied to a 400-ml column (DEAE-TOYOPEARL 650M, product of Tosoh Corporation) that had been equilibrated in advance with 10 mM phosphate buffer (pH 8.0), and the enzyme was allowed to adsorb on the column. The column was washed with the same buffer, and an active fraction was eluted with a linear gradient of sodium chloride from 0 M to 0.3 M.

(12) Ammonium sulfate was added to the active fraction to give a final concentration of 0.8 M. The obtained solution was supplied to a 75-ml column (Phenyl-TOYOPEARL 650M, product of Tosoh Corporation) that had been equilibrated with 10 mM phosphate buffer (pH 7.0) containing 0.8 M ammonium sulfate, and the enzyme was allowed to adsorb thereon. The column was washed with the same buffer, and an active fraction was eluted with a linear gradient of ammonium sulfate from 0.8 M to 0 M.

(13) Ammonium sulfate was added to the active fraction to give a final concentration of 0.8 M. The obtained solution was supplied to a 25-ml column (Butyl-TOYOPEARL 650S, product of Tosoh Corporation) that had been equilibrated with 10 mM phosphate buffer (pH 7.0) containing 0.8 M ammonium sulfate, and the enzyme was allowed to adsorb thereon. The column was washed with the same buffer, and an active fraction was eluted with a linear gradient of ammonium sulfate from 0.8 M to 0 M.

(14) After dialysis with 10 mM phosphate buffer (pH 8.0), the active fraction was supplied to a 6-ml column (Resource Q, product of Pharmacia Biotech) that had been equilibrated with 10 mM phosphate buffer (pH 8.0), and the enzyme was allowed to adsorb thereon. The column was washed with the same buffer solution, and an active fraction was eluted with a linear gradient of sodium chloride from 0 M to 0.5 M.

(15) The enzyme contained in this active fraction was determined as a purified enzyme because the result of dodecyl-sodium-sulfate-polyacrylamide electrophoresis analysis of the active fraction showed a single band.

(16) The specific activity of the purified enzyme was 1.7 U/mg-protein. Hereinafter, this enzyme is referred to as HCS.

(Example 2) Enzymological Property of Purified HCS

(17) The Optimum pH and optimum temperature of an amidase reaction of the purified enzyme obtained in Example 1, and thermal stability of the enzyme were determined by quantifying N-benzylnipecotic acid produced in the hydrolysis reaction using N-benzylnipecotamide as a substrate, by HPLC. For determination of the optimum pH, the reaction was allowed to proceed at each pH for 1 hour at 30° C., and stopped by addition of acetonitrile. The resultant solution was analyzed under the high speed liquid chromatography analysis conditions A described in Example 1. For determination of the optimum temperature, the reaction was allowed to proceed at each temperature for 1 hour, and stopped by addition of acetonitrile. The resultant solution was analyzed under the high speed liquid chromatography analysis conditions A described in Example 1. For determining the thermal stability, after incubation at each temperature for 30 minutes, the reaction was allowed to proceed for 1 hour, and stopped by addition of acetonitrile. The resultant solution was analyzed under the high speed liquid chromatography analysis conditions A described in Example 1.

(18) Table 1 shows the results.

(19) TABLE-US-00001 TABLE 1 Optimum reaction pH pH 8.0 to 9.0 Optimum reaction temperature 50° C. Thermal stability Remaining activity 100% at 50° C. or below

(20) Next, the substrate specificity of HCS was analyzed. The substrate compound was added in a form of a 1% solution. The enzyme reaction was allowed to proceed at 30° C. in 100 mM phosphate buffer (pH 7.0). The resultant solution was analyzed under the high speed liquid chromatography analysis conditions A described in Example 1 or the high speed liquid chromatography analysis conditions B shown below.

(21) High Speed Liquid Chromatography Analysis Condition B

(22) Column: SUMICHIRAL OA-5000 (4.6 mmφ×150 mm, product of Sumika Chemical Analysis Service, Ltd.)

(23) Eluant: 2 mM CuSO.sub.4 aqueous solution

(24) Flow rate: 1.0 ml/min

(25) Column temperature: 30° C.

(26) Measurement wavelength: 254 nm

(27) Table 2 shows the activities relative to the activity on (R,S)-nipecotamide set to 100.

(28) TABLE-US-00002 TABLE 2 Substrate Relative activity (%) (R,S)-nipecotamide 100 (R,S)-N-benzylnipecotamide 49 (R,S)-pipecolic acid amide 34 isonipecotamide 21 indoline carboxylic acid amide 64 D,L-phenylalanine amide 3.4 D,L-β-phenylalanine amide 0 D,L-proline amide 0 D,L-alanine amide 0 D,L-leucine amide 0 benzamide 5.4 propionic acid amide 59 isobutylamide 230 (R,S)-mandelic acid amide 49 (R,S)-phenyl propionic acid amide 309

(29) HCS showed particularly strong amidase activity on heterocyclic compounds such as nipecotamide, N-benzylnipecotamide, pipecolic acid amide, isonipecotamide and indoline carboxylic acid amide, and aliphatic amides such as propionic acid amide, isobutylamide, mandelic acid amide and phenyl propionic acid amide, and weak amidase activity on phenylalanine amide and benzamide.

(Example 3) Cloning of HCS

(30) The N-terminal-amino-acid sequence of the purified HCS obtained in Example 1 was analyzed using a protein sequencer (ABI 492, produced by Applied Biosystems). The purified HCS was denatured in the presence of 8 M urea, and digested with lysyl endopeptidase derived from an achromobacter species (product of Wako Pure Chemical Industries, Ltd.). The amino acid sequence of the obtained peptide fragments was determined. In consideration of the DNA sequence deduced from the amino acid sequence, primer 1 (SEQ ID NO:3 in the sequence listing) and primer 2 (SEQ ID NO:4 in the sequence listing) were synthesized. An amount of 50 μl of a buffer for ExTaq was prepared. This buffer contained the two primers (primer 1 and primer 2, each 40 pmol), chromosomal DNA derived from Cupriavidus sp. KNK-J915 strain (100 ng), dNTP (each 10 nmol) and ExTaq (2.5 U, product of TAKARA SHUZO Co., Ltd.). Heat denaturation (95° C., 1 minute), annealing (50° C., 1 minute), and an extension reaction (72° C., 0.5 minutes) were repeated for 30 cycles. The reaction fluid was cooled to 4° C., and then subjected to agarose gel electrophoresis to confirm amplification of the DNA. The chromosomal DNA of Cupriavidus sp. KNK-J915 strain used in the reaction was prepared in accordance with the small-scale preparation of bacterial genomic DNA taught in “Bunshiseibutugaku Jikkenn Protocol 1 (Current Protocols in Molecular Biology)” (Maruzen) p. 36.

(31) The amplified DNA was subcloned into pT7Blue Vector (product of Novagen), and the base sequence thereof was determined. The result revealed that the amplified DNA has 196 bases except for the primer sequence. This sequence is hereinafter referred to as a “core sequence”.

(32) Based on a part close to the 5′ end of the core sequence, primer 3 (SEQ ID NO:5 in the sequence listing) having a base sequence complementary to the base sequence of the part close to the 5′ end of the core sequence was prepared, and primer 4 (SEQ ID NO:6 in the sequence listing) was prepared based on the base sequence of a part close to the 3′ end of the core sequence. The chromosomal DNA of Cupriavidus sp. KNK-J915 strain was digested with restriction enzyme PstI, and the digested fragment was self-closed with T4 DNA ligase to obtain a circular DNA used as a template for inverse PCR. An amount of 50 μl of a buffer for ExTaq was prepared. This buffer contained the self-closed circular DNA (200 ng), the two primers (primer 3 and primer 4, each 50 pmol), dNTP (each 10 nmol) and ExTaq (2.5 U, product of TAKARA SHUZO Co., Ltd.). Heat denaturation (97° C., 1 minute), annealing (60° C., 1 minute), and an extension reaction (72° C., 5 minutes) were repeated for 30 cycles. The reaction fluid was cooled to 4° C., and then subjected to agarose gel electrophoresis to confirm amplification of the DNA.

(33) The amplified DNA was subcloned into pT7Blue Vector (product of Novagen), and the base sequence thereof was determined. Based on the base sequence determined above and the core sequence, the entire base sequence of the gene encoding HCS derived from Cupriavidus sp. KNK-J915 strain was determined. The entire base sequence of the gene encoding HCS is shown as SEQ ID NO:2, and the deduced amino acid sequence encoded by the gene is shown as SEQ ID NO:1.

(Example 4) Construction of Recombinant Vector Containing HCS Gene

(34) In order to obtain an Escherichia coli cell capable of expressing HCS, a recombinant vector used for transformation was constructed. First, a double strand DNA containing an NdeI site added to the initiation codon site of the HCS gene, and a new termination codon and an EcoRI site inserted immediately downstream of the original termination codon was prepared as follows.

(35) Based on the base sequence determined in Example 3, primer 5 having the NdeI site added to the initiation codon site of the HCS gene (SEQ ID NO:7 in the sequence listing), and primer 6 having the EcoRI site inserted immediately downstream of the termination codon of the HCS gene (SEQ ID NO:8 in the sequence listing) were synthesized. An amount of 50 μl of a buffer for ExTaq was prepared. This buffer contained the two primers (primer 5 and primer 6, each 50 pmol), chromosomal DNA derived from Cupriavidus sp. KNK-J915 strain (10 ng), dNTP (each 10 nmol) and ExTaq (2.5 U, product of TAKARA SHUZO Co., Ltd.). Heat denaturation (97° C., 1 minute), annealing (60° C., 1 minute), and an extension reaction (72° C., 1.5 minutes) were repeated for 30 cycles. The reaction fluid was cooled to 4° C., and then subjected to agarose gel electrophoresis to confirm amplification of the DNA. The DNA fragment obtained by the PCR was digested with NdeI and EcoRI, and inserted between the NdeI recognition site and the EcoRI recognition site downstream of the lac promoter of the plasmid pUCN18 to construct recombinant vector pNCS. Here, the plasmid pUCN18 is a plasmid having a base sequence in which the NdeI site is destroyed by a T to A substitution at nucleotide position 185 of pUC18 (product of Takara Bio, Inc., GenBank Accession No. L09136), and a new NdeI site is introduced by a GC to TG substitution at nucleotide positions 471 and 472, by PCR.

(Example 5) Preparation of Transformant

(36) The recombinant vector pNCS constructed in Example 4 was transformed into competent cells of E. coli HB 101 (product of Takara Bio, Inc.) to obtain E. coli HB101 (pNCS).

(37) The bacteriological properties of E. coli HB101 are shown in various publications including “BIOCHEMICALS FOR LIFE SCIENCE” (Toyobo Co., Ltd., 1993, p. 116-119), and are known to those skilled in art. E. coli HB101 (pNCS) has acquired activity to produce the specific enzyme by gene recombination in addition to the same bacteriological properties as those of E. coli HB101.

(Example 6) Expression of HCS in Transformant

(38) The transformant obtained in Example 5, and the transformant E. coli HB101 (pUCN18) containing vector plasmid pUCN18 (Comparative Example) were separately inoculated into 5 ml of a 2xYT culture medium (triptone 1.6%, yeast extract 1.0% and NaCl 0.5%; pH 7.0) containing 200 μg/ml of ampicillin, and cultured with shaking for 24 hours at 37° C. Cells were collected by centrifugation, and suspended in 5 ml of 100 mM phosphate buffer (pH 7.0). The cells were disrupted using an ultrasonic homogenizer (UH-50, product of SMT Co., Ltd), and cell residues were removed by centrifugation to obtain a cell-free extract. Table 3 shows the specific activity determined based on the measured amidase activity of the cell-free extracts.

(39) TABLE-US-00003 TABLE 3 Specific activity of cell-free extract (U/mg-protein) E. coli HB101 (pUCN18) 0 E. coli HB101 (pNCS) 1.2

(40) As shown in Table 3, expression of amidase activity was found in the transformant obtained in Example 5. The amidase activity was measured by the procedure described in Example 1.

(Example 7) Selective Hydrolysis of S-Enantiomer in Racemic Nipecotamide

(41) An amount of 10.1 g of racemic nipecotamide was dissolved in water to prepare a substrate solution having a pH adjusted to 8.0. To 200 ml of the prepared substrate solution was added 2 ml of a culture solution obtained by culturing E. coli HB101 (pNCS) as in Example 6, and stirred for 25 hours at 45° C. After completion of the reaction, the reaction liquid was heated for 30 minutes at 70° C., and centrifuged to remove solid matters including cells. Subsequently, the substrate and the product in the reaction liquid were converted into derivatives with benzyl chloroformate. The obtained derivatives were analyzed by high speed liquid chromatography to determine the conversion ratio (%) and optical purity (% e.e.). The results showed that the conversion rate was 50.2%, the optical purity of (R)-nipecotamide was 98.3% e.e., and the optical purity of (S)-nipecotic acid was 97.1% e.e.
Conversion ratio (%)=P/(S.sub.1+P)×100

(42) (P: amount of product (mol), S.sub.1: amount of residual substrate (mol))
Optical purity (% e.e.)=(A−B)/(A+B)×100

(43) (A represents the amount of the target enantiomer, and B represents the amount of the corresponding enantiomer.)

(44) High Speed Liquid Chromatography Analysis Condition

(45) [Analysis of Conversion Ratio]

(46) Column: YMC-A303 (4.6 mmφ×250 mm, product of YMC Co., Ltd.)

(47) Eluant: 20 mM phosphate aqueous solution (pH 2.5)/acetonitrile=7/3

(48) Flow rate: 1.0 ml/min

(49) Column temperature: 35° C.

(50) Measurement wavelength: 210 nm

(51) [Optical Purity Analysis]

(52) Column: CHIRALPAK AD-RH (4.6 mmφ×150 mm, product of DAICEL CHEMICAL INDUSTRIES, LTD.)

(53) Eluant: 20 mM phosphate aqueous solution (pH 2.5)/acetonitrile=7/3

(54) Flow rate: 0.5 ml/min

(55) Column temperature: room temperature

(56) Measurement wavelength: 210 nm

(Example 8) Selective Hydrolysis of R-Enantiomer in Racemic Pipecolic Acid Amide

(57) To 100 mM phosphate buffer (pH 7.0) were added 1% racemic pipecolic acid amide and the purified enzyme solution obtained in Example 1, and allowed to react at 30° C. Then high speed liquid chromatography analysis was conducted to determine the conversion ratio (%) and optical purity (% e.e.). The results showed that the conversion ratio was 18.3%, and the optical purity of (R)-pipecolic acid was 80.1% e.e.
Conversion ratio (%)=P/(S.sub.1+P)×100

(58) (P: amount of product (mol), S.sub.1: amount of residual substrate (mol))
Optical purity (% e.e.)=(A−B)/(A+B)×100

(59) (A represents the amount of the target enantiomer, and B represents the amount of the corresponding enantiomer.)

(60) High Speed Liquid Chromatography Analysis Condition

(61) [Analysis of Conversion Ratio and Optical Purity]

(62) Column: SUMICHIRAL OA-5000 (4.6 mmφ×150 mm, product of Sumika Chemical Analysis Service, Ltd.)

(63) Eluant: 2 mM CuSO.sub.4 aqueous solution

(64) Flow rate: 1.0 ml/min

(65) Column temperature: 30° C.

(66) Measurement wavelength: 254 nm

(Example 9) Selective Hydrolysis of R-Enantiomer in Racemic Indoline Carboxylic Acid Amide

(67) To 100 mM phosphate buffer solution (pH 7.0) were added 1% racemic indoline carboxylic acid amide and the purified enzyme solution obtained in Example 1. The obtained mixture was allowed to react at 30° C., and then the substrate and product in the reaction liquid were converted into derivatives with acetic anhydride. The conversion ratio (%) and optical purity (% e.e.) were determined by high speed liquid chromatography analysis of the obtained derivatives. The results showed that the conversion ratio was 39.2%, and the optical purity of (R)-indoline carboxylic acid was 97.8% e.e.

(68) High Speed Liquid Chromatography Analysis Condition

(69) [Analysis of Conversion Ratio]

(70) Column: YMC-A303 (4.6 mmφ×250 mm, product of YMC Co., Ltd.)

(71) Eluant: 20 mM phosphate aqueous solution (pH 2.5)/acetonitrile=9/1

(72) Flow rate: 1.0 ml/min

(73) Column temperature: 30° C.

(74) Measurement wavelength: 210 nm

(75) [Analysis of Optical Purity]

(76) Column: SUMICHIRAL OA-5000 (4.6 mmφ×150 mm, product of Sumika Chemical Analysis Service, Ltd.)

(77) Eluant: 2 mM CuSO.sub.4 aqueous solution/methanol=7/3

(78) Flow rate: 2.0 ml/min

(79) Column temperature: 35° C.

(80) Measurement wavelength: 254 nm

(Example 10) Selective Hydrolysis of S-Enantiomer of Racemic Phenyl Propionic Acid Amide

(81) To 100 mM phosphate buffer (pH 7.0) were added 1% racemic phenyl propionic acid amide and the purified enzyme solution obtained in Example 1, and allowed to react at 30° C. The conversion ratio (%) and optical purity (% e.e.) were determined by high speed liquid chromatography analysis. The results showed that the conversion ratio was 36.0%, and the optical purity of (S)-phenyl propionic acid was 89.1% e.e.

(82) High Speed Liquid Chromatography Analysis Condition

(83) [Analysis of Conversion Ratio]

(84) Column: YMC-A303 (4.6 mmφ×250 mm, product of YMC Co., Ltd.)

(85) Eluant: 20 mM phosphate aqueous solution (pH 2.5)/acetonitrile=7/3

(86) Flow rate: 1.0 ml/min

(87) Column temperature: 35° C.

(88) Measurement wavelength: 210 nm

(89) [Analysis of Optical Purity]

(90) Column: CHIRALPAK AD-H (4.6 mmφ×250 mm, product of DAICEL CHEMICAL INDUSTRIES, LTD.)

(91) Eluant: Hexane/isopropanol/TFA=95/5/0.02

(92) Flow rate: 1.0 ml/min

(93) Column temperature: 30° C.

(94) Measurement wavelength: 254 nm

(Example 11) Preparation of Escherichia coli Transformed with DNA Encoding Putative Amidase Derived from Ralstonia eutropha JMP134 Strain

(95) Primer 7 (SEQ ID NO:10 in the sequence listing) having the NdeI site added to the initiation codon site, and primer 8 (SEQ ID NO:11 in the sequence listing) having an SacI site immediately downstream of the termination codon were synthesized based on the base sequence (SEQ ID NO:9 in the sequence listing) encoding the putative amidase derived from Ralstonia eutropha JMP134 strain, which has high sequence identity to the base sequence of the present invention derived from Cupriavidus sp. KNK-J915 (FERM BP-10739) determined in Example 3. An amount of 50 μl of a buffer for ExTaq was prepared. This buffer contained the two primers (primer 7 and primer 8, each 50 pmol), chromosomal DNA derived from Ralstonia eutropha JMP134 strain (10 ng), dNTP (each 10 nmol) and ExTaq (2.5 U, product of TAKARA SHUZO Co., Ltd.). Heat denaturation (97° C., 1 minute), annealing (60° C., 1 minute), and an extension reaction (72° C., 1.5 minutes) were repeated for 30 cycles. The reaction fluid was cooled to 4° C. and subjected to agarose gel electrophoresis to confirm amplification of the DNA. The DNA fragment obtained by the PCR was digested with NdeI and SacI, and inserted between the NdeI recognition site and the SacI recognition site downstream of the lac promoter of the plasmid pUCN18 to construct a recombinant vector pNRE. The recombinant vector pNRE thus constructed was transformed into competent cells of E. coli HB 101 (product of Takara. Bio, Inc.) to obtain E. coli HB101 (pNRE).

(Example 12) Selective Hydrolysis of S-Enantiomer in Racemic N-Benzylnipecotamide Using Escherichia coli Transformed with DNA Encoding Putative Amidase Derived from Ralstonia eutropha JMP134 Strain

(96) To 100 mM phosphate buffer (pH 7.0) were added 1% racemic N-benzylnipecotamide and the culture solution of E. coli HB101 (pNRE) obtained in Example 11, and allowed to react at 30° C. Thereafter, the conversion ratio (%) and optical purity (% e.e.) were determined by high speed liquid chromatography analysis. The results showed that the conversion ratio was 50.1%, and the optical purity of residual (R)—N-benzylnipecotamide was 99.4% e.e.
Conversion ratio (%)=P/(S.sub.1+P)×100

(97) (P: amount of product (mol), S.sub.1: amount of residual substrate (mol))
Optical purity (% e.e.)=(A−B)/(A+B)×100

(98) (A represents the amount of the target enantiomer, and B represents the amount of the corresponding enantiomer.)

(99) High Speed Liquid Chromatography Analysis Condition

(100) [Analysis of Conversion Rate]

(101) Column: YMC-A303 (4.6 mmφ×250 mm, product of YMC Co., Ltd.)

(102) Eluant: 20 mM phosphate aqueous solution (pH 2.5)/acetonitrile=9/1

(103) Flow rate: 1.0 ml/min

(104) Column temperature: 35° C.

(105) Measurement wavelength: 210 nm

(106) [Analysis of Optical Purity]

(107) Column: CHIRALPAK AD-RH (4.6 mmφ×150 mm, product of DAICEL CHEMICAL INDUSTRIES, LTD.) Eluant: 20 mM potassium phosphate buffer (pH 8.0)/acetonitrile=7/3

(108) Flow rate: 0.5 ml/min

(109) Column temperature: room temperature

(110) Measurement wavelength: 210 nm