JP2006340630A - New amidase and its gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein having high ammonia resistance and amidase activities; and to provide an amidase gene encoding the protein. <P>SOLUTION: The new amidase is isolated from Pseudonocardia thermophila JCM3095, and the gene thereof is identified. The amidase has the high ammonia resistance and exhibits very high specific activities on 2-hydroxy-4-methylthiobutylamide. The amidase has the following characteristics: (a) exhibiting ≥50% of the amidase activities in the absence of the ammonia in a reaction condition of pH 9.0 even in the presence of 0.2 M ammonia; (b) exhibiting higher specific activities on the 2-hydroxy-4-methylthiobutylamide than methionineamide; (c) having an apparent molecular mass of 53 kDa on SDS-PAGE; (d) having 60-70°C optimum temperature of the activities; (e) stable at a temperature of ≤60°C at a nearly neutral pH; and (f) exhibiting high activities in a wide pH range of pH 5.5-10.0. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel amidase having high ammonia resistance, a protein having amidase activity, and an amidase gene encoding the protein.

  Amidases are known as enzymes that hydrolyze amides into the corresponding carboxylic acids. By using amidase, a carboxylic acid useful as a raw material for medicines and agricultural chemicals can be produced from amide. When an amidase is used, for example, an α-amino acid can be produced from an α-amino acid amide, and an α-hydroxy acid can be produced from an α-hydroxy acid amide.

Microbial-derived amidase is purified from Arthrobacter sp. J-1, Brevibacterium sp. R 312, Aspergillus nidulans, Pseudomonas chlororaphis B23, Pseudonocardia thermophila, etc. 7).
On the other hand, Pseudomonas aeruginosa, Aspergillus oryzae, Rhodococcus sp., Pseudomonas chlororaphis B23, Rhodococcus sp.N-774, Brevibacterium sp. R312, Rhodococcus rhodochrous J1, Pseudomonas putida, Bacillus stearothermophilus BR From these microorganisms, amidase genes have been cloned, and as a result, amino acid sequences of various amidases have been clarified (for example, see Non-Patent Documents 8 to 19).

  Patent documents relating to the amidase gene include Brevibacterium R312 and Rhodococcus (Patent Document 1), Comamonas testosteroni NI 1 (Patent Document 2), Comamonas acidovorans KPO-2771-4 (Patent Document 3), Rhodococcus rhodochrous IFO 15564 (Patent Document 4) , Enterobacter cloacae (patent document 5), Pseudomonas putida (patent document 6), Rhodococcus sp. (Patent document 7), Brevibacillus borstelensis (patent document 8), Pseudomonas azotoformans IAM 1603 (patent document 9), Ochrobactrum anthropi NCIMB 40321 (patent) Literature 10), Thermus sp. (Patent Literature 11), Pseudomonas sp. MCI 3434 and Pseudomonas aeruginosa PAO1 (Patent Literature 12), Thermococcus GU5L5 (Patent Literature 13), Variovarax paradoxus DSM 14468 (Patent Literature 14), and Comamonas testosteroni 5 -There is a thing about MGAM-4D (patent document 15). In addition, an amidase derived from Pseudonocardia thermophila that is a collection source of the amidase gene of the present invention and its gene are also known (Patent Document 16), but this amidase is clearly different in structure from the amidase of the present invention. The enzymological properties are also clearly different.

  Thus, although amidase and amidase gene were known in various microorganisms, these microorganisms or enzymes were industrially used to convert α-amino acid amide or α-hydroxy acid amide to α-amino acid or α. -When trying to produce a carboxylic acid such as a hydroxy acid, there is a problem that the amidase activity is strongly inhibited by the by-product ammonia, and the production efficiency of the carboxylic acid is lowered or the production of the carboxylic acid is stopped ( For example, refer nonpatent literature 20). In addition, although the manufacturing method of DL-methionine using the cell body of Pseudonocardia thermophila was known (refer patent document 17), the knowledge about the amidase which has ammonia tolerance was not acquired at all.

US Pat. No. 5,260,208 WO94 / 17190 pamphlet Japanese Patent Laid-Open No. 8-256771 JP-A-9-9973 US Pat. No. 6,617,139 US Pat. No. 6,251,650 International Publication No. 01/30994 Pamphlet International Publication No. 03/008569 Pamphlet JP 2003-250558 A International Publication No. 03/010312 Pamphlet International Publication No. 03/020929 Pamphlet JP 2004-105152 A International Publication No. 03/027315 Pamphlet US Patent Application Publication No. 2003/0186423 US Patent Application Publication No. 2004/0225116 International Publication No. 2004/083423 Pamphlet JP 2004-254690 A Y. Asano et al., 1982, Agric. Biol. Chem. 46: 1175-1181 A. Thiery et al., 1986, J. Basic Microbiol. 26: 299-311 J-F. Mayaux et al., 1990, J. Bacteriol. 172: 6764-6773 C. M. Corrick et al., 1987, Gene 53: 63-71 J. A. Fraser et al., 2002, Fungal Genet. Biol. 35: 135-146 L. M. Ciskanik et al., 1995, Appl. Environ. Microbiol. 61: 998-1003 K. Egorova et al., 2004, Appl. Microbiol. Biotechnol. 65: 38-45 W. J. Brammar et al., 1987, FEBS Lett. 215: 291-294 K. Gomi et al., 1991, Gene 108: 91-98 J-F. Mayaux et al., 1991, J. Bacteriol. 173: 6694-6704 M. Nishiyama et al., 1991, J. Bacteriol. 173: 2465-2472 Y. Hashimoto et al., 1991, Biochim. Biophys. Acta 1088: 225-233 F. Soubrier et al., 1992, Gene 116: 99-104 M. Kobayashi et al., 1993. Eur. J. Biochem. 217: 327-336 S. Wu et al., 1998, DNA Cell Biol. 17: 915-920 T. K. Cheong and P. J. Oriel, 2000, Enzyme Microb. Technol. 26: 152-158 S. Trott et al., 2001, Microbiology 147: 1815-1824 A. S. d’ Abusco et al., 2001, Extremophiles 5: 183-192 S. Trott et al., 2002, Appl. Environ. Microbiol. 68: 3279-3286 M. J. Woods et al., 1979, Biochim. Biophys. Acta 567: 225-237

  As described above, by using a microorganism exhibiting a known amidase activity or an amidase enzyme obtained therefrom, an α-amino acid amide or an α-hydroxy acid amide is converted into an carboxylic acid such as an α-amino acid or an α-hydroxy acid. When an acid is to be produced, there is a problem that amidase activity is strongly inhibited by ammonia produced as a by-product, so that the production efficiency of carboxylic acid is reduced or the production of carboxylic acid is stopped. An object of the present invention is to provide a protein having amidase activity, which has high ammonia resistance, and an amidase gene encoding the protein.

  As a result of intensive studies to solve the above problems, the present inventor isolated a novel amidase from Pseudocardia thermophila JCM3095, found that the amidase has high ammonia resistance, and identified its gene. As a result, the present invention has been completed.

  That is, the present invention shows (1) (a) 50% or more amidase activity in the presence of 0.2 M ammonia even in the presence of 0.2 M ammonia under the reaction conditions of pH 9.0, and (b) 2% more than methionine amide. Higher specific activity for -hydroxy-4-methylthiobutyramide, (c) apparent molecular mass on SDS-PAGE is 53 kDa, and (d) optimum temperature for activity is 60 to 70 (E) an amidase that is stable at a temperature of about 60 ° C. or less at a neutral pH, and (f) an amidase that exhibits high activity in a wide pH range of pH 5.5 to pH 10.0; The present invention relates to the amidase described in (1) above, which is derived from Pseudonocardia thermophila.

  In the present invention, (3) (g) a protein comprising the amino acid sequence shown in SEQ ID NO: 10 or (h) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 10 is deleted, substituted or added. A protein having an amino acid sequence and having amidase activity, (4) (i) a protein having an amino acid sequence comprising amino acid sequences 24 to 95 of the amino acid sequence represented by SEQ ID NO: 10 and having amidase activity, or (J) It consists of an amino acid sequence comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of positions 24 to 95 of the amino acid sequence shown in SEQ ID NO: 10, and has amidase activity It relates to protein. In addition, the present invention shows (5) (a) 50% or more amidase activity in the absence of ammonia even in the presence of 0.2M ammonia under the reaction conditions of pH 9.0, and (b) 2% more than methionine amide. -The protein according to any one of the above (3) to (4), which has a higher specific activity with respect to hydroxy-4-methylthiobutyramide, and (6) (c) an apparent molecule on SDS-PAGE The mass is 53 kDa, (d) the optimum temperature of activity is 60 to 70 ° C., (e) at a neutral pH, and stable at a temperature of 60 ° C. or less, (f) pH 5.5 to pH 10 (3) to (3), which are derived from the protein according to any one of (3) to (5) above, which shows high activity in a wide pH range of 0.0, or (7) Pseudonocardia thermophila. Any of 6) On the protein described.

  Furthermore, the present invention relates to (8) an amidase gene DNA encoding the protein according to any one of (3) to (7) above, or (9) (k) an amidase gene DNA comprising the base sequence represented by SEQ ID NO: 9 Or (l) an amidase gene DNA consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 9 and encoding a protein having amidase activity; ) It relates to an amidase gene DNA which hybridizes under stringent conditions with a DNA comprising a sequence complementary to the DNA comprising the base sequence shown in SEQ ID NO: 9 and encodes a protein having amidase activity. The present invention also provides that (11) a protein having amidase activity exhibits (a) 50% or more amidase activity in the absence of ammonia even in the presence of 0.2 M ammonia under the reaction conditions of pH 9.0 (b) A) amidase gene DNA according to (9) or (10) above, which is a protein having a property of higher specific activity with respect to 2-hydroxy-4-methylthiobutyramide than methionine amide; (12) a protein having amidase activity has (c) an apparent molecular mass on SDS-PAGE of 53 kDa, (d) an optimal temperature of activity of 60 to 70 ° C., (e) It is stable at a temperature of 60 ° C. or less at a pH close to that of the water, and (f) has a property of exhibiting high activity in a wide pH range of pH 5.5 to pH 10.0. The amidase gene DNA according to any one of (9) to (11) above, which is a protein, and (13) a protein having amidase activity is derived from Pseudonocardia thermophila The amidase gene DNA according to any one of (9) to (12) above.

  The present invention also relates to (14) a recombinant vector into which the amidase gene DNA described in any of (8) to (13) above is incorporated.

  Furthermore, the present invention relates to (15) a transformed microorganism, wherein the recombinant vector described in (14) is introduced.

  The present invention also relates to (16) the amidase described in the above (1) or (2) or its producing bacterium or a processed product thereof, the protein described in any of the above (3) to (7), or the above (15 ) A method for producing a carboxylic acid, wherein the transformed microorganism or treated product thereof is allowed to act on an amide, or (17) the amide is an α-amino acid amide or an α-hydroxy acid amide, and the carboxylic acid is α -It is an amino acid or alpha-hydroxy acid, It is related with the manufacturing method of the carboxylic acid of the said (16) description characterized by the above-mentioned.

  When using the amidase with high ammonia resistance of the present invention, when industrially producing carboxylic acids from amides, the reaction rate is less likely to be lowered by the by-product ammonia, so an efficient production process can be constructed. It becomes.

  The amidase of the present invention shows (a) 50% or more amidase activity in the absence of ammonia even in the presence of 0.2 M ammonia under the reaction conditions of pH 9.0, and (b) 2-methionine amide. Higher specific activity for hydroxy-4-methylthiobutyramide, (c) apparent molecular mass on SDS-PAGE is 53 kDa, and (d) optimum temperature for activity is 60-70 ° C. (E) At a neutral pH, stable at a temperature of 60 ° C. or lower, and (f) Amidase exhibiting high activity in a wide pH range of pH 5.5 to pH 10.0 is particularly limited. The amidase derived from Pseudonocardia thermophila can be preferably exemplified as such amidase. Hereinafter, the amidase of the present invention may be collectively referred to as “the present amidase”.

  In the present invention, “amidase derived from Pseudonocardia thermophila” refers to a method such as transformation as long as it is the same or similar to the amidase actually present in Pseudocardia thermophila. It is used in the meaning including amidase expressed by microorganisms other than Pseudonocardia thermophila.

  In the present invention, “showing 50% or more amidase activity in the absence of ammonia even in the presence of 0.2 M ammonia under the reaction conditions of pH 9.0” is preferable for conditions other than pH and ammonia concentration. Based on the conditions, the specific activity of amidase with respect to methionine amide shown at pH 9.0 and ammonia concentration 0.2M is the specific activity of amidase with respect to methionine amide shown at pH 9.0 and ammonia concentration 0M. It means that it is 50% or more. For example, it can be examined according to the method described in (6) of Example 6 described later. In addition, “showing a higher specific activity for 2-hydroxy-4-methylthiobutyramide than methionine amide” is compared with the specific activity of amidase for methionine amide under suitable conditions. This means that the specific activity of amidase shown against 2-hydroxy-4-methylthiobutyramide is higher, and more preferably, the amidase shown above against 2-hydroxy-4-methylthiobutyramide It means that the specific activity is 2 times or more, more preferably 5 times or more, still more preferably 10 times or more than the specific activity of amidase shown for the aforementioned methionine amide.

  The method for producing the amidase is not particularly limited as long as it is a method of culturing the amidase-producing bacterium belonging to the genus Pseudonocardia in a medium and collecting the amidase, and the amidase-producing bacterium belonging to the genus Pseudonocardia Pseudonocardia thermophila can be preferably exemplified, and more specifically, Pseudocardia thermophila JCM3095 can be mentioned. The JCM3095 strain is Japan Collection of Microorganisms (JCM: Microbial strain of RIKEN). Can be obtained from the preservation facility. The culture medium may be any medium as long as the microorganism can grow. For example, by adding an amide compound such as ε-caprolactam or methacrylamide, amidase is induced and generated. .

  The protein of the present invention includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 10, an amino acid sequence shown in SEQ ID NO: 10 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and A protein having an amidase activity, consisting of an amino acid sequence comprising amino acid sequences 24 to 95 of the amino acid sequence shown in SEQ ID NO: 10, and having amidase activity, or 24 to 95 positions of the amino acid sequence shown in SEQ ID NO: 10 The amino acid sequence is not particularly limited as long as it is a protein having an amino acid sequence including an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having amidase activity. Even in the presence of 0.2M ammonia under the reaction conditions 50% or more amidase activity in the absence of ammonia, (b) higher specific activity to 2-hydroxy-4-methylthiobutyramide than methionine amide, (c) SDS-PAGE The apparent molecular mass above is 53 kDa, (d) the optimum temperature of activity is 60 to 70 ° C., (e) is stable at a temperature below 60 ° C. at a neutral pH, ( f) Those exhibiting high activity in a wide pH range of pH 5.5 to pH 10.0 are preferred, those having all the properties (a) to (f) are more preferred, and preferably Pseudonocardia thermo The thing derived from a filler can be mentioned. Hereinafter, these proteins of the present invention may be collectively referred to as “the present protein”.

  The amidase gene DNA of the present invention includes an amidase gene DNA encoding the present protein, an amidase gene DNA consisting of the base sequence shown in SEQ ID NO: 9, and one or several bases in the base sequence shown in SEQ ID NO: 9. A DNA comprising an amidase gene DNA encoding a protein having an amidase activity, or a sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 9 There is no particular limitation as long as it is an amidase gene DNA that hybridizes under stringent conditions and encodes a protein having amidase activity, but the protein having the amidase activity is (a) pH 0 under reaction conditions of 9.0. Even in the presence of 2M ammonia, 50% or more amidase activity in the absence of monia, (b) higher specific activity to 2-hydroxy-4-methylthiobutyramide than methionine amide, (c) SDS-PAGE The apparent molecular mass above is 53 kDa, (d) the optimum temperature of activity is 60 to 70 ° C., (e) is stable at a temperature below 60 ° C. at a neutral pH, ( f) Those exhibiting high activity in a wide pH range of pH 5.5 to pH 10.0 are preferred, those having all the properties (a) to (f) are more preferred, and preferably Pseudonocardia thermo The thing derived from a filler can be mentioned. Hereinafter, these amidase gene DNAs of the present invention may be collectively referred to as “the present gene DNA”.

  The above “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 5. , Most preferably 1 to 3 amino acid sequences with any number of amino acids deleted, substituted or added, and the above-mentioned “one or several bases deleted, substituted or added bases” “Sequence” means, for example, 1-20, preferably 1-15, more preferably 1-10, even more preferably 1-5, most preferably 1-3, any number of bases deleted. Means a substituted or added base sequence.

  For example, DNA (mutant DNA) consisting of a base sequence in which one or several bases are deleted, substituted or added can be obtained by any method known to those skilled in the art such as chemical synthesis, genetic engineering techniques, mutagenesis, etc. Can also be produced. Specifically, the DNA consisting of the base sequence shown in SEQ ID NO: 9 is mutated into these DNAs using a method of contacting with a mutagen agent, a method of irradiating with ultraviolet rays, a genetic engineering technique, etc. Mutant DNA can be obtained by introducing. Site-directed mutagenesis, which is one of genetic engineering techniques, is useful because it can introduce a specific mutation at a specific position. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 (hereinafter abbreviated as "Molecular Cloning 2nd Edition") and the like. By expressing this mutant DNA using an appropriate expression system, a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added can be obtained.

  In the present invention, “having amidase activity” means having an enzyme activity to hydrolyze an amide to produce carboxylic acid and ammonia. Whether the obtained protein has amidase activity is determined by, for example, contacting the protein with an amide in an appropriate buffer or salt solution and examining the production of carboxylic acid, as described in Example 2 below. Can be easily confirmed. Whether or not the obtained protein exhibits a higher specific activity with respect to 2-hydroxy-4-methylthiobutyramide than with methionine amide as described in Example 6 (4) described later. The specific activity of the protein with respect to methionine amide and the specific activity with respect to 2-hydroxy-4-methylthiobutyramide can be measured, and the measured values can be easily compared.

  The above-mentioned “stringent conditions” include, for example, the conditions including washing at 68 ° C. in 0.5 × SSC containing 0.1% SDS in the Southern blot hybridization method. The hybridization method includes conditions including washing at 68 ° C. in 1 × SSC containing 0.1% SDS. Hybridization can be performed according to the method described in Molecular Cloning 2nd edition.

  For example, DNA that can hybridize under stringent conditions can include DNA having a certain degree of homology with the base sequence of DNA used as a probe. For example, the amino acid represented by SEQ ID NO: 10 A DNA encoding an amino acid sequence having a homology of 86% or more, preferably 90% or more, more preferably 93% or more, still more preferably 95% or more, and most preferably 98% or more, is preferably exemplified. it can.

  The method for obtaining and preparing the gene DNA is not particularly limited, and an appropriate probe based on the nucleotide sequence information shown in SEQ ID NO: 9 or the amino acid sequence information shown in SEQ ID NO: 10 disclosed in the present specification. For example, Pseudonocardia thermophila, genomic DNA library of organisms other than Pseudonocardia thermophila, etc. are used to isolate the target gene or prepared by chemical synthesis according to conventional methods can do. For example, a genomic DNA library is prepared from Pseudonocardia thermophila according to a conventional method, and then a desired clone is selected from the library using an appropriate probe specific to the gene DNA. DNA can be obtained. Moreover, acquisition of genomic DNA and its cloning can all be carried out according to conventional methods. Examples of the method for screening the present gene DNA from a genomic DNA library include methods commonly used by those skilled in the art, such as the method described in Molecular Cloning 2nd Edition, in addition to the method described in the Examples below. it can. In addition, as a mutated gene or a homologous gene, a DNA fragment having the base sequence shown in SEQ ID NO: 9 or a part thereof is used, and DNA having a base sequence having high homology with the DNA is appropriately used from other organisms. Can be isolated by screening under mild conditions. In addition, it can also be prepared by the aforementioned method for producing mutant DNA.

  The recombinant vector of the present invention is not particularly limited as long as it is a recombinant vector into which the present gene DNA is incorporated. The recombinant vector of the present invention is constructed by appropriately recombining the present gene DNA into an expression vector. be able to. For example, a configuration in which the present gene DNA is connected downstream of a suitable promoter can be preferably exemplified. The expression vector is preferably one that can replicate autonomously in the host cell, or one that can be integrated into the chromosome of the host cell, and a control sequence such as a promoter or terminator involved in the expression of the gene of the present invention and What contains the gene of a transcriptional regulatory factor can be used conveniently.

As expression vectors for bacterial, e.g., T7 phage promoter, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, SPO1 promoter, SPO2 promoter, can be specifically exemplified penP promoter.

  In addition, the transformed microorganism of the present invention is not particularly limited as long as it is a transformed microorganism into which the above recombinant vector of the present invention has been introduced. Examples of the host include microorganisms such as bacteria and yeasts. Examples of such bacteria include Escherichia bacteria, Pseudonocardia bacteria, Streptomyces bacteria, Bacillus bacteria, Streptococcus bacteria, Staphylococcus bacteria, and the like. As a method for introducing the above recombinant vector into a host microorganism, a method described in many standard laboratory manuals such as Molecular Cloning Second Edition, for example, electroporation, transduction, transformation, etc. Can do. Moreover, the present protein can be produced and accumulated in the culture by culturing the transformed microorganism of the present invention, and the protein can be produced in large quantities by collecting the protein from the culture.

  The origin of the present protein is not particularly limited, and may be a naturally derived protein, a chemically synthesized protein, or a recombinant protein produced by a gene recombination technique. When a naturally derived protein is obtained, the protein of the present invention can be obtained by appropriately combining protein isolation / purification methods from cells or tissues expressing such protein. For example, the isolation and purification of the protein of the present invention from Pseudonocardia thermophila is carried out by using a strain of Pseudonocardia thermophila, for example, a medium supplemented with an amide compound such as ε-caprolactam or methacrylamide. The cells can be cultured, collected from the culture, and the collected cells can be disrupted to obtain a cell extract. There is no particular restriction on the method for disrupting the cells, but in the case of ultrasonic treatment, it is effective to add glass beads to the cell suspension for treatment. The method for purifying the protein from the cell extract thus obtained is not particularly limited, but ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography and hydroxyapatite chromatography. Any known method including graphy can be used. In particular, as a column used for affinity chromatography, for example, a column in which an antibody against the protein is bound, or a column in which a substance having an affinity for the peptide tag is bound when a normal peptide tag is added to the protein. By using, purified protein can be obtained.

  The amidase activity of the fraction obtained at each stage of purification can be quantified as follows, for example. When the sample solution is diluted with an appropriate buffer or salt solution and an amide such as α-amino acid amide or α-hydroxy acid amide is added, a hydrolysis reaction occurs, and a carboxylic acid such as α-amino acid or α-hydroxy acid is produced. Generate. Reaction conditions are not particularly limited, but 40 to 60 ° C. and pH 6 to 9 are suitable. Carboxylic acids such as α-amino acids or α-hydroxy acids produced in the reaction solution can be quantified by HPLC. In addition, the protein of the fraction obtained at each stage of purification is obtained by the Lowry method (OH Lowry et al., 1951, J. Biol. Chem. 193: 265-275) using bovine serum albumin as a standard. It can be quantified. The purity of the amidase at each purification step can be examined by SDS-polyacrylamide gel electrophoresis (U. K. Laemmli, 1970, Nature 227: 680-685).

  When the protein of the present invention is prepared by chemical synthesis, for example, the protein of the present invention is synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). can do. In addition, the protein of the present invention can be synthesized using various commercially available peptide synthesizers.

When a protein is prepared by a gene recombination technique, the protein of the present invention can be prepared by introducing DNA consisting of a base sequence encoding the protein into a suitable expression system. This method by gene recombination technology can prepare a target protein in a large amount by a relatively easy operation.

  Furthermore, a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 10, or has 86% or more homology with the amino acid sequence shown in SEQ ID NO: 10. A protein comprising an amino acid sequence can be appropriately prepared or obtained by those skilled in the art based on the information of the base sequence shown in SEQ ID NO: 9 showing an example of the base sequence encoding the amino acid sequence shown in SEQ ID NO: 10. . For example, by screening a DNA obtained from an organism other than Pseudocardia thermophila using a DNA having the base sequence shown in SEQ ID NO: 9 or a part thereof as a probe under appropriate conditions, High DNA can be isolated. The gene DNA thus obtained is incorporated into an expression vector and expressed in an appropriate host, whereby a protein encoded thereby can be produced.

  The amino acid sequence at positions 24 to 95 of the amino acid sequence shown in SEQ ID NO: 10 is between the amidase actually obtained in the present application and the amidase actually obtained in WO 2004/083423, This is a very different part (see FIG. 2). It is predicted that a protein having the same properties as the amidase of SEQ ID NO: 10 of the present application exists in a protein comprising a sequence containing an amino acid sequence in which one or several amino acids of this part of the amino acid are deleted, substituted or added. Is done.

  The method for producing the carboxylic acid of the present invention is not particularly limited as long as the method comprises the action of cells of the transformed microorganism of the present invention or a processed product thereof on an amide. The “transformed microorganism cell” referred to here may be a living cell or a dead cell. The “processed cell product” is a product obtained by treating cells of the transformed microorganism of the present invention, and is not particularly limited as long as it can be used for the production of carboxylic acid from an amide. About cells of transformed microorganisms, those subjected to mechanical crushing treatment, ultrasonic treatment, freeze-thaw treatment, drying treatment, solvent treatment, surfactant treatment, enzyme treatment, pressure reduction treatment, osmotic pressure treatment, etc., and The thing which fixed the cell or its processed material with alginic acid etc. is mentioned. Further, the protein of the present invention and its immobilization product purified from the cell extract of the transformed microorganism of the present invention by, for example, ammonium sulfate salting-out, hydrophobic chromatography, ion exchange chromatography, gel filtration chromatography, etc. Are included in “Processed cells”. In addition, although the cell or its processed material in the manufacturing method of carboxylic acid of this invention does not need to be fix | immobilized, it is more preferable that it is fix | immobilized. This is because, when immobilized cells or processed products are used, separation of the cells or processed products from the products is facilitated. The carboxylic acid such as α-amino acid or α-hydroxy acid thus produced can be concentrated and purified by a usual method.

  Moreover, about the culture | cultivation method of the transformed microorganism of this invention used for the manufacturing method of the carboxylic acid of this invention, or the manufacturing method of its processed material, as long as the cell or its processed material can be used for the manufacturing method of carboxylic acid. There is no restriction | limiting in particular and a conventionally well-known method can be used.

  In the carboxylic acid production method of the present invention, the amidase of the present invention can be allowed to act on the amide in, for example, a suitable buffer or salt solution. The buffer or salt is not particularly limited as long as it does not inhibit the hydrolysis reaction from amide to carboxylic acid.

  The “amide” in the method for producing carboxylic acid of the present invention is not particularly limited as long as the protein of the present invention can be used as a substrate, but α-amino acid amide or α-hydroxy acid amide is preferable. When cells of the transformed microorganism of the present invention or processed products thereof are allowed to act on, for example, α-amino acid amide, α-hydroxy acid amide, 2-hydroxy-4-methylthiobutyramide, α-amino acid, α-hydroxy Acid and 2-hydroxy-4-methylthiobutyric acid are produced respectively.

  Although there is no restriction | limiting in particular about the density | concentration of the amide in the reaction liquid in the manufacturing method of carboxylic acid of this invention, Usually, it is about 0.1 to 30 weight%. The amide may be added to the reaction system continuously or intermittently. Although there is no restriction | limiting in particular in the addition amount to the reaction system of the cell of the transformed microorganism of this invention, or its processed material, It can add in 0.1-60 g / L in conversion of dry cell weight. The reaction temperature, pH, and the like are not particularly limited as long as hydrolysis reaction from amide to carboxylic acid occurs, but can be, for example, 40 to 60 ° C. and pH 6 to 9. The produced carboxylic acid such as α-amino acid or α-hydroxy acid can be confirmed and quantified by HPLC.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the technical scope of this invention is not limited by these Examples.

[Culture of Pseudocardia thermophila JCM3095]
A 3000 ml conical flask with a baffle was charged with 500 ml of TY medium (1% tryptone, 0.5% yeast extract, and 40 μg / ml FeSO ₄ · 7H ₂ O), and Pseudonocardia thermophila JCM3095 (Japan Collection of (Available from Microorganisms) and inoculated at 50 ° C. for 2 days with shaking. Next, another 3000 ml baffled Erlenmeyer flask was charged with 500 ml of TYC medium (TY medium containing 10 μg / ml CoCl ₂ .6H ₂ O and 0.5% ε-caprolactam), and 10 ml of the above preculture. And shake-cultured at 50 ° C. for 2 days. The cells were collected by centrifugation (5 krpm, 10 min, 4 ° C.) to obtain 9.15 g of wet cells. As a control, Pseudonocardia thermophila JCM3095 was similarly cultured using a TYC medium not containing ε-caprolactam, and collected to obtain 14.5 g of wet cells.

[Measurement of amidase activity of bacterial cells]
The wet cells obtained in Example 1 were washed with 200 ml of 100 mM potassium phosphate buffer (pH 7.5), added with the same buffer, and suspended using a Teflon homogenizer to make a total volume of 50 ml. 50 to 100 μl of this bacterial cell suspension was placed in an Eppendorf tube, and 100 mM potassium phosphate buffer (pH 7.5) was added to make a total volume of 500 μl, which was then kept warm in a 30 ° C. water bath for 5 minutes. To this, 50 μl of 1M L-methionine amide hydrochloride was added and stirred, and reacted in a 30 ° C. water bath for 30 minutes. The reaction was stopped by adding 100 μl of 2N hydrochloric acid thereto, and the supernatant obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was analyzed by HPLC (ion-exchanged water / acetonitrile / TFA = 950/50/1). did. The amount of microbial cells producing 1 μmol of methionine per minute was 1 U. The amidase activity was 19.1 U / g wet cells when using TYC medium, whereas the amidase activity was 3.14 U / g wet cells when using TYC medium without ε-caprolactam. Met. From this result, it was found that the addition of ε-caprolactam increased the amidase activity of JCM3095 by about 6 times.

[Influence of ammonia on cell reaction at pH 7.5]
Pseudonocardia thermophila JCM3095 was cultured in the same manner as in Example 1, and the cells were collected by centrifugation. To the collected cells, physiological saline (0.8% NaCl) was added and suspended to prepare a cell suspension. On the other hand, Rhodococcus rhodochrous IFO 15564 (available from NBRC, National Institute of Technology and Evaluation) can be used with CSL medium (2% corn steep liquor, 1% sucrose, and 0.5% ε-caprolactam). The cells were cultured with shaking at 28 ° C., and the cells were collected by centrifugation (16 krpm, 10 min, 4 ° C.). The collected bacterial cells were suspended by adding physiological saline to prepare a bacterial cell suspension (control). Take a certain amount of these cell suspensions into an Eppendorf tube, add 50 μl of 1M potassium phosphate buffer (pH 7.5) and various amounts of 2M aqueous ammonia (pH 7.5 adjusted with hydrochloric acid), Water was added to bring the total volume to 450 μl. This was kept warm in a 30 ° C. water bath for 5 minutes, 50 μl of 1M DL-methionine amide hydrochloride was added and stirred, and reacted in a 30 ° C. water bath for 30 minutes. The reaction was stopped by adding 100 μl of 2N hydrochloric acid thereto, and the supernatant obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was analyzed by HPLC (ion-exchanged water / acetonitrile / TFA = 950/50/1). did. The amount of microbial cells producing 1 μmol of methionine per minute was 1 U. As a result, as shown in FIG. 1, the relationship between the ammonia concentration and the cell activity (the cell activity when no ammonia was added was taken as 100%) was determined. The amidase activity of Rhodococcus rhodochrous IFO 15564 was also strongly inhibited by low concentrations of ammonia, whereas the amidase activity of Pseudonocardia thermophila JCM3095 was hardly affected by ammonia.

[Influence of ammonia on cell reaction at pH 9.0]
In the same manner as in Example 3, a cell suspension of Pseudocardia thermophila JCM3095 and Rhodococcus rhodochrous IFO 15564 was prepared. A certain amount of these cell suspensions are taken in an Eppendorf tube, and 50 μl of 1M Tris-HCl buffer (pH 9.0) and various amounts of 2M aqueous ammonia (adjusted to pH 9.0 with hydrochloric acid) are added. Water was added to bring the total volume to 450 μl. This was kept warm in a 30 ° C. water bath for 5 minutes, 50 μl of 1M DL-methionine amide hydrochloride was added and stirred, and reacted in a 30 ° C. water bath for 30 minutes. The reaction was stopped by adding 100 μl of 2N hydrochloric acid thereto, and the supernatant obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was analyzed by HPLC (ion-exchanged water / acetonitrile / TFA = 950/50/1). did. The amount of microbial cells producing 1 μmol of methionine per minute was 1 U. As a result, as shown in FIG. 3, the relationship between the ammonia concentration and the cell activity (the cell activity when no ammonia was added was taken as 100%) was determined. The amidase activity of Rhodococcus rhodochrous IFO 15564 was strongly inhibited even by low concentrations of ammonia, but the amidase activity of Pseudocardia thermophila JCM3095 was found to be less susceptible to inhibition by ammonia.

[Purification of amidase]
In the same manner as in Example 1, Pseudocardia thermophila JCM3095 was cultured using 500 ml of TYC medium. The cells are collected by centrifugation (5 krpm, 10 min, 4 ° C.), and the cells are washed with 200 ml of TED buffer (50 mM Tris-HCl buffer (pH 7.5), 1 mM EDTA, and 1 mM DTT), and 50 ml of the same buffer is washed. In addition, it was suspended using a Teflon homogenizer. To this, 20 g of glass beads (particle size 50 μm) was added, treated with an ultrasonic homogenizer (Seiko Denshi Kogyo; Model 7500) (maximum output, effective time 3 min, ice-cooled), and centrifuged (16 krpm, 30 min, 4 The supernatant was obtained. To this supernatant, add ammonium sulfate to 30% saturation and leave it in ice for 1 hour or more. Then, remove the formed precipitate by centrifugation (same as above), and add ammonium sulfate to 60% saturation to the supernatant. After being placed in ice for 1 hour or longer, the resulting precipitate was obtained by centrifugation (same as above).

  The precipitate obtained above was dissolved in 20 ml of TED buffer containing 1 M ammonium sulfate and loaded onto a column (2.2 × 16.5 cm) of phenyl sepharose CL-4B (Pharmacia) equilibrated with the same buffer. Thereafter, the column was washed with 150 ml of TED buffer containing 1 M ammonium sulfate, and 1 to 0 M ammonium sulfate gradient elution (total amount of 300 ml) was performed. Finally, elution with 150 ml of TED buffer was performed. The eluate from the column was fractionated by 15 g. The amidase activity of each fraction was measured as follows. An appropriate amount of the sample solution was placed in an Eppendorf tube, and 100 mM potassium phosphate buffer (pH 7.5) was added to make a total volume of 500 μl. This was incubated for 5 minutes in a 50 ° C. water bath, 50 μl of 1M DL-methionine amide hydrochloride was added and stirred, and reacted in a 50 ° C. water bath for 30 minutes. The reaction was stopped by adding 100 μl of 2N hydrochloric acid thereto, and the supernatant obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was analyzed by HPLC (ion-exchanged water / acetonitrile / TFA = 950/50/1). did. The amount of enzyme that produces 1 μmol of methionine per minute was 1 U. The amidase activity was eluted when the ammonium sulfate concentration was approximately 0M.

  The amidase activity fraction obtained by the above phenyl sepharose chromatography was precipitated with ammonium sulfate (60% saturation), dissolved by adding 10 ml of TED buffer, and dialyzed overnight against 1000 ml of TED buffer. This was loaded onto a DEAE Sepharose Fast Flow (Amersham Bioscience) column (1.6 × 13 cm) equilibrated with TED buffer, washed with 75 ml of TED buffer, and gradient elution from 0 to 0.5 M NaCl. (Total amount 150 ml) was performed, and finally, elution was performed with 75 ml of TED buffer containing 0.5 M NaCl. The eluate from the column was fractionated by 15 g, and the amidase activity of each fraction was measured by the method described above. Amidase activity was found in the fraction eluted with approximately 0.2M NaCl.

  The fraction of amidase activity obtained by DEAE Sepharose chromatography above was salted out with ammonium sulfate (60% saturation), dissolved in 5 ml of TED buffer, and equilibrated with TED buffer (Toyopearl HW-55S (TOSOH) column) 2.6 × 88 cm). The eluate from the column was fractionated by 5 g, and the amidase activity of each fraction was measured by the method described above. The amidase activity was eluted in fractions corresponding to 100 to 200 kDa. Catalase (240 kDa), lactate dehydrogenase (140 kDa), albumin (67 kDa), α-chymotrypsinogen A (25 kDa), and lysozyme (14 kDa) were used as molecular mass markers.

  The amidase activity fraction obtained by the above gel filtration chromatography was salted out with ammonium sulfate (60% saturation), dissolved by adding 2 ml of TED buffer, dialyzed against 500 ml of TED buffer (twice), and -30 Cryopreserved at ℃. Table 1 shows the specific activity and the like of the amidase activity fraction at each purification step. Protein was quantified by the Lowry method using bovine serum albumin as a standard. The amidase was purified 6.7 times with a yield of 12%. The specific activity of the purified amidase finally obtained was 14.7 U / mg. FIG. 4 shows the results of analysis of the amidase activity fraction at each purification stage by SDS-polyacrylamide gel electrophoresis. The molecular mass of the amidase was determined to be 53 kDa. In the gel filtration chromatography described above, considering that the amidase activity was eluted in a fraction corresponding to 100 to 200 kDa, the natural amidase is considered to be a dimer to tetramer.

[Enzymatic properties of purified amidase]
(1) Reaction temperature 500 μl of 100 mM potassium phosphate buffer (pH 7.5) containing 36 μg of purified amidase was placed in an Eppendorf tube and incubated for 5 minutes in a constant temperature water bath. To this, 50 μl of 1M DL-methionine amide hydrochloride was added, stirred and allowed to react for 10-30 minutes in a water bath at the same temperature. The reaction was stopped by adding 100 μl of 2N hydrochloric acid thereto, and the supernatant obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was obtained by HPLC (ion-exchanged water / acetonitrile / TFA = 950/50/1). analyzed. The amount of enzyme that produces 1 μmol of methionine per minute was 1 U. As a result, as shown in FIG. 5, the relationship between the reaction temperature and the enzyme activity was determined. The optimum temperature for enzyme activity was 65 ° C.

(2) Thermal stability 500 μl of 100 mM potassium phosphate buffer (pH 7.5) containing 36 μg of purified amidase was placed in an Eppendorf tube and heat-treated in a constant temperature water bath for 30 minutes. Thereafter, the mixture was kept in a 50 ° C. water bath for 5 minutes, 50 μl of 1M DL-methionine amide hydrochloride was added and stirred, and the mixture was reacted in a 50 ° C. water bath for 10 minutes. The reaction was stopped by adding 100 μl of 2N hydrochloric acid thereto, and the supernatant obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was analyzed by HPLC (ion-exchanged water / acetonitrile / TFA = 950/50/1). did. The amount of enzyme that produces 1 μmol of methionine per minute was 1 U. As a result, as shown in FIG. 6, the relationship between the heat treatment temperature and the enzyme activity was determined. The purified enzyme was stable up to 60 ° C.

(3) Reaction pH
250 μl of 200 mM buffer at various pH was taken in an Eppendorf tube, 36 μg of purified amidase was added, and water was added to make a total volume of 500 μl. This was kept warm in a 50 ° C. water bath for 5 minutes, 50 μl of 1M DL-methionine amide hydrochloride was added and stirred, and reacted in a 50 ° C. water bath for 10 minutes. The reaction was stopped by adding 100 μl of 2N hydrochloric acid thereto, and the supernatant obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was analyzed by HPLC (ion-exchanged water / acetonitrile / TFA = 950/50/1). did. The amount of enzyme that produces 1 μmol of methionine per minute was 1 U. As a result, as shown in FIG. 7, the relationship between reaction pH and enzyme activity was determined. The purified enzyme showed high activity over a wide pH range from pH 5.5 to 10.

(4) Substrate specificity 500 μl of 100 mM potassium phosphate buffer (pH 7.5) containing various amide compounds at a concentration of 100 mM is placed in an Eppendorf tube and kept in a 50 ° C. water bath for 5 minutes to obtain 36 μg (20 μl) of purified amidase. The mixture was stirred and reacted in a 50 ° C. water bath for 10 minutes. The reaction was stopped by adding 100 μl of 2N hydrochloric acid thereto, and the supernatant obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was analyzed by HPLC (ion-exchanged water / acetonitrile / TFA = 950/50/1). did. The amount of enzyme that produces 1 μmol of acid per minute was 1 U. The results are shown in Table 2. The specific activity for HMB-amide was about 12 times the specific activity for methionine amide.

(5) Reaction kinetic analysis Reaction kinetic analysis was performed using methionine amide, HMB-amide (2-hydroxy-4-methylthiobutyramide), or crotonamide as a substrate. Reaction of 500 μl of a reaction mixture consisting of various concentrations of amide compound, a fixed amount (0.5 to 10 μg) of purified amidase and 100 mM potassium phosphate buffer (pH 7.5) in a water bath at 50 ° C. for 1 to 10 minutes I let you. The reaction was stopped by adding 100 μl of 2N hydrochloric acid thereto, and the supernatant obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was analyzed by HPLC (ion-exchanged water / acetonitrile / TFA = 950/50/1). did. From the result, a Lineweaver-Burk plot was created, and K _m and V _max were obtained as shown in Table 3. HMB- _{V max} when the amide substrate was about 12 times the _{V max} when the methionine amide substrates.

(6) Effect of ammonia In an Eppendorf tube, 50 μl of 1M Tris-HCl buffer (pH 9.0), 50 μl of 1M DL-methionine amide hydrochloride, and various amounts of 2M ammonia (pH adjusted to 9.0 with hydrochloric acid) The total volume was adjusted to 490 μl by adding ion exchange water. This was incubated in a 30, 40, or 50 ° C. water bath for 5 minutes, 10 μl (18 μg) of purified amidase was added, and allowed to react in the same temperature water bath for 30 minutes. The reaction was stopped by adding 100 μl of 2N hydrochloric acid thereto, and the supernatant obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was analyzed by HPLC (ion-exchanged water / acetonitrile / TFA = 950/50/1). . The amount of enzyme that produces 1 μmol of methionine per minute was 1 U. As a result, as shown in FIG. 8, the relationship between ammonia concentration and enzyme activity (specific activity when no ammonia was added was defined as 100%) was determined. Inhibition by ammonia increased slightly with increasing reaction temperature. However, this amidase retained 57% of enzyme activity even in the presence of 0.2M ammonia at 50 ° C.

[Determining N-terminal amino acid sequence and internal amino acid sequence of amidase]
Multiplying purified amidase obtained in Example 5 to SDS- polyacrylamide gel electrophoresis, after electrophoresis, by electroblotting from the polyacrylamide gel PVDF (polyvinylidene fluoride) ^membrane; the (Immobilon -P SQ Millipore), protein bands Moved. Thereafter, the PVDF membrane was rinsed lightly with water and shaken in CBB staining solution (0.1% Coomassie Brilliant Blue R-250, 45% methanol, 10% acetic acid) for 5 minutes. This was shaken in decoloring solution 1 (45% methanol, 7% acetic acid) for 15 minutes, rinsed three times with water, shaken in decoloring solution 2 (90% methanol, 7% acetic acid) for 40 seconds, and air dried. The 53 kDa protein band was cut out and the N-terminal amino acid sequence was determined by the Edman method. As a result, the amino acid sequence of SEQ ID NO: 1 was obtained.

  In addition, the purified amidase obtained in Example 5 was subjected to SDS-polyacrylamide gel electrophoresis, and after electrophoresis, a 53 kDa protein band was cut out from the polyacrylamide gel. This gel slice was used for digestion with lysyl endopeptidase, and the resulting peptides were separated by HPLC. For some of these peptides, the N-terminal amino acid sequence was determined by the Edman method. As a result, the amino acid sequences of SEQ ID NOs: 2, 3, and 4 were obtained.

[Preparation of genomic DNA]
250 ml of TY medium (1% tryptone, 0.5% yeast extract, and 40 μg / ml FeSO ₄ .7H ₂ O) was placed in a 2000 ml baffled Erlenmeyer flask. Pseudonocardia thermophila JCM3095 was inoculated into the medium and cultured with shaking at 50 ° C. for 2 days. The obtained culture broth was collected by centrifugation (5 krpm, 10 min, 4 ° C.), and the collected cells were washed with 100 ml of TEN buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 10 mM NaCl). did. Then, the same buffer was added to the cells and suspended using a Teflon homogenizer to obtain a suspension of 20 ml in total.

To this suspension was added 200 μl of TEN buffer in which 40 mg of lysozyme was dissolved, and the mixture was gently shaken at 37 ° C. for 1 hour. Furthermore, 2 ml of 10% SDS and 100 μl of 20 mg / ml proteinase K were added, and after gently shaking at 37 ° C. for 1 hour, 440 μl of 5 M NaCl was added, and further 12 ml of 10 mM Tris containing 1% SDS and 0.1 M NaCl. -HCl buffer (pH 8.0) was added and it was transferred to a centrifuge tube. To this was added the same volume of buffer equilibrated phenol (about 0.1% of 8-hydroxyquinoline was added to the phenol crystal, and the same volume of 1M Tris-HCl buffer [pH 8.0] was added to the solution and stirred to dissolve the phenol crystal. The upper aqueous layer was removed by decantation, and the lower phenol layer was equilibrated with 0.1 M Tris-HCl buffer (pH 8.0) containing 0.2% β-mercaptoethanol. This phenol layer was referred to as “buffer-equalized phenol”), stirred, and separated into two layers by centrifugation (3000 rpm, 10 min, room temperature). Transfer the upper aqueous layer to a new centrifuge tube, add 1/2 volume of buffer equilibrated phenol and 1/2 volume of chloroform containing isoamyl alcohol (chloroform containing 4% isoamyl alcohol) and stir. Separated into layers. Transfer the upper aqueous layer to a new centrifuge tube, add the same volume of chloroform containing isoamyl alcohol, stir, and separate into two layers by centrifugation (same as above) (hereinafter, this series of operations will be called phenol / chloroform extraction). ). The upper aqueous layer was transferred to a new centrifuge tube, 2 volumes of ethanol was added, and the mixture was stirred with a glass rod to wind up the DNA to be insolubilized. This DNA was dissolved in 5 ml of TEN buffer and dialyzed twice against 1000 ml of TEN buffer. After dialysis, 25 μl of 10 mg / ml ribonuclease A was added and incubated at 37 ° C. for 2 hours. 25 μl of 20 mg / ml proteinase K was added thereto, and the mixture was incubated at 37 ° C. for 1 hour, and then 5 ml of TEN buffer and 200 μl of 5M NaCl were added to perform phenol / chloroform extraction. The insoluble DNA was wound up by adding 2 volumes of ethanol to the aqueous layer and stirring with a glass rod. This DNA was dissolved in 5 ml of TEN buffer, dialyzed twice against 1000 ml of TEN buffer, and the resulting genomic DNA solution was stored at 4 ° C. _{A 260} of the DNA solution since it was 0.200, DNA concentration was determined to 0.500 mg / ml.

[Amplification of amidase gene by PCR]
Based on the sequence of Leu-Gly-Ala-Trp-Ala-Val-Gln-Thr among the internal amino acid sequences of the amidase of SEQ ID NO: 3 obtained in Example 7, the PCR sense primer shown in SEQ ID NO: 5 Was synthesized. Among the internal amino acid sequences of the amidase of SEQ ID NO: 4, the Lys-Val-Ala-Asn-Ala-Phe-Glu-Gln sequence (because it is a peptide generated by lysyl endopeptidase digestion, Lys is present on the N-terminal side. Based on that, an antisense primer of PCR shown in SEQ ID NO: 6 was synthesized. In the design of the two PCR primers above, the third base of the codon was G or C (S).

  In an ice-cooled 0.2 ml PCR tube, 0.5 μl (2.5 U) Taq DNA polymerase (Takara Bio), 10 μl 10-fold reaction buffer, 8 μl dNTP mixture (2.5 mM each), 4 μl of genomic DNA (0.1 μg / μl; a 5-fold dilution of the genomic DNA solution obtained in Example 7), 1 μl of the sense primer shown in SEQ ID NO: 5 (100 μM), 1 μl of SEQ ID NO: 6 Antisense primer (100 μM), 5 μl dimethyl sulfoxide, and 70.5 μl water were added. This was set in a PCR apparatus and the DNA was denatured by treatment at 94 ° C. for 5 minutes, followed by 30 temperature cycles (denaturation step at 94 ° C. for 20 seconds, annealing step at 59 ° C. for 30 seconds, and 72 ° C. The DNA was amplified by a 120 second extension reaction step) and finally the reaction was completed by treatment at 72 ° C. for 2 minutes. After completion of the reaction, the reaction solution was subjected to 1% agarose gel electrophoresis, a gel containing about 1320 bp DNA fragment was cut out, and DNA was extracted from the gel using a QIAquick gel extraction kit (Qiagen).

[Determining the base sequence of DNA amplified by PCR]
The DNA amplified by PCR obtained in Example 9 was cloned as follows using pGEM-T Easy Vector System (Promega). In an Eppendorf tube, 5 μl of 2 × rapid ligation buffer, 1 μl (50 ng) of pGEM-T Easy Vector, 1 μl of DNA amplified by PCR of Example 9, 1 μl (3 U) of T4 DNA ligase, and water 2 μl was added and reacted at room temperature for 1 hour. 5 μl of this reaction solution was taken out, added to 200 μl of E. coli JM103 competent cell, placed in ice for 30 minutes, and then heat-treated at 42 ° C. for 45 seconds. Thereafter, the mixture was ice-cooled, 800 μl of LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) was added, and the mixture was gently shaken at 37 ° C. for 1 hour. 100 μl of this was applied to a selective agar medium (LB agar medium containing 50 μg / ml ampicillin, 0.5 mM IPTG, and 40 μg / ml X-gal), and the selective agar medium was incubated at 37 ° C. overnight. .

  White colonies appearing on the selective agar medium were inoculated into 50 ml of LB medium containing 50 μg / ml ampicillin and cultured overnight at 37 ° C. with shaking. The cells were collected by centrifugation (16 krpm, 10 min, 4 ° C.), washed with 10 ml of TEN buffer, and then 2.5 ml of solution I (25 mM Tris-HCl buffer [pH 8.0], 50 mM glucose, and 10 mM). EDTA) was added and suspended. To this, 250 μl of TE buffer (10 mM Tris-HCl buffer [pH 8.0], 1 mM EDTA) in which 2.5 mg of lysozyme was dissolved was added, and the mixture was allowed to stand at room temperature for 10 minutes. Next, 5 ml of freshly prepared solution II (0.2N NaOH, 1% SDS) was added to the solution and stirred well before it was left at room temperature for 10 minutes. Next, 3.75 ml of solution III (mixed of 60 ml of 5M potassium acetate, 11.5 ml of acetic acid, and 28.5 ml of water) that had been ice-cooled was added to the lysate and stirred well, and the mixture was stirred in ice for 10 minutes. placed. It was centrifuged (16 krpm, 10 min, 4 ° C.) to remove insoluble matters, and the supernatant was transferred to another centrifuge tube. To the supernatant, 0.6 volumes of isopropanol was added and left at room temperature for 10 minutes. The precipitate obtained by centrifugation (16 krpm, 10 min, 20 ° C.) was rinsed with ice-cooled 70% ethanol, air-dried, dissolved by adding 750 μl of TE buffer, and transferred to an Eppendorf tube. To this solution was added 750 μl of 5M LiCl ice-cooled and placed in ice for 10 minutes. Then, the insoluble material was removed by centrifugation (16 krpm, 5 min, 4 ° C.), and the supernatant was transferred to another Eppendorf tube. Of isopropanol was added and left at room temperature for 10 minutes. Next, the precipitate obtained by centrifugation (16 krpm, 5 min, 20 ° C.) was rinsed with ice-cooled 70% ethanol, air-dried, and then dissolved by adding 125 μl of TE buffer. To this solution, 0.5 μl of 10 mg / ml ribonuclease A was added and left at room temperature for 30 minutes. 125 μl of 1.6 M NaCl containing 13% PEG 8000 was added thereto and stirred well, and a precipitate was obtained by centrifugation (16 krpm, 5 min, 4 ° C.). This precipitate was dissolved in 200 μl of TE buffer, extracted with phenol / chloroform, added with 50 μl of 10M ammonium acetate and 500 μl of ethanol, placed at room temperature for 10 minutes, and centrifuged (16 krpm, 5 min, 20 ° C.) to obtain a precipitate. The precipitate was rinsed with ice-cooled 70% ethanol and dissolved in 200 μl of TE buffer.

  Using the plasmid DNA thus prepared, the base sequence of the inserted DNA portion was determined by the dideoxy method. As a result, the base sequence shown in SEQ ID NO: 7 was obtained. The amino acid sequence (SEQ ID NO: 8) encoded by this base sequence was subjected to homology search by FASTA (homology search software) of DDBJ (Japan DNA Data Bank). As a result, this amino acid sequence was amidase derived from various microorganisms. It was found that there is a high homology with the amino acid sequence. The highest homology was derived from Rhodococcus rhodochrous J1, which showed 68.7% homology to the amino acid sequence of SEQ ID NO: 8 of the present application. From the above, in Example 9, the DNA amplified by PCR was considered to be part of the amidase gene.

[Digoxigenin labeling of DNA amplified by PCR]
The DNA amplified by PCR obtained in Example 9 was labeled with digoxigenin as follows using a digoxigenin (DIG) DNA labeling and detection kit (Roche Diagnostics). 15 μl (about 3 μg) of PCR amplified DNA was taken into an Eppendorf tube, heat-denatured in boiling water for 5 minutes, and then rapidly cooled in ice water. To this, 2 μl of a hexanucleotide mixture, 2 μl of dNTP labeling mixture, and 1 μl of Klenow enzyme were added and allowed to react overnight at 37 ° C. 2 μl of 200 mM EDTA (pH 8.0) was added thereto to stop the reaction, and this was stored frozen at −20 ° C.

[Restriction enzyme digestion of genomic DNA and preparation of DNA fragment containing amidase gene]
5 μl (2.5 μg) of the genomic DNA obtained in Example 8 is put into an Eppendorf tube, 5 μl of 10-fold reaction buffer, 5 μl (50 U) of a restriction enzyme (Sac I, Kpn I, Sma I, or BamHI) And 35 μl of water were added and reacted at 37 ° C. (30 ° C. for Sma I) for 2 hours. 1 μl of 5M NaCl was added thereto, phenol / chloroform extraction was performed, 100 μl of ethanol was added, and the mixture was placed on ice for 1 hour or more, and then a precipitate was obtained by centrifugation (16 krpm, 5 min, 4 ° C.). This was rinsed with ice-cooled 70% ethanol, air-dried, and dissolved by adding 20 μl of TE buffer. 5 μl of this DNA solution was subjected to 0.7% agarose gel electrophoresis. As a DNA size marker, Hind III digest (Roche Diagnostics) of lambda phage DNA labeled with digoxigenin was used. The agarose gel after electrophoresis was immersed in 100 ml of denaturing solution (0.5N NaOH, 1.5M NaCl), shaken slowly for 15 minutes, and the operation was repeated once again. Next, the sample was immersed in 100 ml of a neutralization buffer for Southern transfer (0.5 M Tris-HCl buffer [pH 7.5], 3 M NaCl), shaken slowly for 15 minutes, the liquid was changed, and this operation was performed once more. DNA was transferred from the thus treated agarose gel to a nylon membrane (Hybond-N ⁺ ; Amersham Biosciences) using 20 × SSC (3M NaCl, 0.3M sodium citrate) by the capillary method. . Thereafter, the nylon membrane was air-dried, sandwiched between two filter papers, and baked at 80 ° C. for 2 hours to immobilize DNA.

Hybridization and detection with digoxigenin-labeled PCR-amplified DNA was performed using digoxigenin (DIG) DNA labeling and a detection kit (Roche Diagnostics) as follows. The baked membrane obtained above is placed in a hybridization bag and 6 ml of high concentration SDS hybridization buffer (7% SDS, 50% formamide, 5 μg / ml fish sperm DNA (Roche Diagnostics), 5% × SSC, 2% blocking reagent, 50 mM sodium phosphate buffer [pH 7.0], and 0.1% N-lauroyl sarcosine sodium) were added, and prehybridization was performed at 42 ° C. for 1 hour. Next, the solution was discarded, and 1 ml of high-concentration SDS containing 5 μl of digoxigenin-labeled PCR amplified DNA (prepared in Example 11 was heat-denatured in boiling water for 5 minutes and then rapidly cooled in ice water). Hybridization buffer was added and hybridization was performed overnight at 42 ° C. The nylon membrane was removed from the hybridization bag and washed twice with 50 ml of 2 × SSC containing 0.1% SDS at room temperature for 5 minutes. Subsequently, the plate was washed twice at 68 ° C. for 15 minutes using 50 ml of 0.5 × SSC containing 0.1% SDS. The membrane was rinsed with 50 ml of maleic acid buffer (0.15 M NaCl, 0.1 M maleic acid; adjusted to pH 7.5 with 10 N NaOH) containing 0.3% Tween 20 for 1 minute, and 30 ml of blocking solution ( (Maleic acid buffer containing 1% blocking reagent) and gently shaken for 30 minutes. Next, it was immersed in 20 ml of blocking solution containing 4 μl of alkaline phosphatase labeled anti-digoxigenin antibody and gently shaken for 30 minutes. Thereafter, the nylon membrane was washed twice with 50 ml of maleic acid buffer containing 0.3% Tween 20 for 15 minutes, and 50 ml of detection buffer (0.1 M Tris-HCl buffer [pH 9.5], 0.1 M NaCl, 50 mM). Soaked in MgCl ₂ ) for 2 minutes to equilibrate. This nylon membrane was immersed in a freshly prepared chromogenic substrate solution (10 ml of detection buffer containing 200 μl of NBT / BCIP stock solution) and allowed to stand in the dark for 1 hour. After confirming the color development of the band, the nylon membrane was washed with TE buffer and air-dried in the dark. The size of the DNA fragment hybridizing with the PCR amplified DNA obtained in Example 9 was 6010 bp for Sac I digestion, 9810 bp for Kpn I digestion, 3740 bp for Sma I digestion, and 1690 bp for BamH I digestion.

  Take 80 μl (40 μg) of the genomic DNA obtained in Example 8 in an Eppendorf tube, 40 μl of 10 × reaction buffer, 40 μl (400 U) of a restriction enzyme (Sac I, Kpn I, Sma I, or BamH I), and 240 μl of water was added and reacted at 37 ° C. (30 ° C. for Sma I) for 2 hours. 8 μl of 5 M NaCl was added thereto, phenol / chloroform extraction was performed, 800 μl of ethanol was added, and the mixture was placed in ice for 1 hour, and then a precipitate was obtained by centrifugation (16 krpm, 5 min, 4 ° C.). This was rinsed with ice-cooled 70% ethanol, air-dried, and dissolved by adding 100 μl of TE buffer. The total amount of this DNA solution was subjected to 0.7% agarose gel electrophoresis. As a DNA size marker, Hind III digest (Roche Diagnostics) of digoxigenin-labeled λ phage DNA was used. After completion of the electrophoresis, gels containing DNA fragments of 6010 bp for Sac I digestion, 9810 bp for Kpn I digestion, 3740 bp for Sma I digestion, and 3690 bp for BamH I digestion were excised using QIAquick gel extraction kit (Qiagen). DNA was extracted from each gel.

[Cloning of DNA fragment containing amidase gene]
2.7 μg of plasmid pUC18 is taken into an Eppendorf tube, to which 20 μl of 10 × reaction buffer, 5 μl (50 U) of a restriction enzyme (Sac I, Kpn I, Sma I, or BamH I) is added, and water is added. The total volume was 200 μl. This was reacted at 37 ° C. (30 ° C. in the case of Sma I) for 1 hour, and the restriction enzyme was inactivated by heat treatment at 65 ° C. for 15 minutes. To this was added 27 μl of 10 × DP buffer (0.5 M Tris-HCl buffer [pH 8.5], 50 mM MgCl ₂ ), 30 μl (30 U) shrimp alkaline phosphatase (Roche Diagnostics), and 13 μl of water. In addition, the enzyme was inactivated by reacting at 37 ° C. for 30 minutes and heat treatment at 65 ° C. for 15 minutes. 5.4 μl of 5M NaCl was added thereto, phenol / chloroform extraction was performed, 540 μl of ethanol was added, and the mixture was placed on ice for 1 hour, and then a precipitate was obtained by centrifugation (16 krpm, 15 min, 4 ° C.). The precipitate was rinsed with ice-cooled 70% ethanol, air-dried, and dissolved by adding 50 μl of TE buffer.

1 μl of the cloning vector thus prepared and 5 μl of the DNA fragment prepared in Example 12 were mixed, 3 μl of Ligation high (TOYOBO) was added, and the mixture was reacted at 16 ° C. for 1 hour. 5 μl of this was added to 100 μl of E. coli JM109 competent cell (Takara Bio), placed in ice for 30 minutes, and then heat treated at 42 ° C. for 45 seconds. Then, it is ice-cooled, and 900 μl of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO ₄ , 10 mM MgCl ₂ , 20 mM glucose) is added, and slowly at 37 ° C. for 1 hour. Shake. 100 μl of this was applied to an LB agar medium containing 50 μg / ml ampicillin, and the agar medium was kept at 35 ° C. overnight.

The agar medium in which the colonies appeared was placed at 4 ° C. for 30 minutes or more and cooled. On this agar medium, a nylon membrane (Hybond-N ⁺ ; Amersham Biosciences) is placed using tweezers, peeled off after 1 minute, the surface in contact with the colony facing up, and a denaturing solution (0.5 M NaOH, It was placed on two filter papers soaked with 1.5M NaCl). After standing in this state for 15 minutes, the nylon membrane was transferred onto a dry filter paper to absorb excess water. Next, on the two filter papers soaked in the neutralization buffer for colony hybridization (1M Tris-HCl buffer [pH 7.5], 1.5M NaCl), the surface of the nylon membrane that was in contact with the colony was placed. Placed on top. After standing in this state for 15 minutes, the nylon membrane was transferred onto a dry filter paper to absorb excess water. The nylon membrane was then placed on two filter papers soaked with 2 × SSC (30 mM sodium citrate, 0.3 M NaCl). After allowing to stand for 10 minutes in this state, the nylon membrane was air-dried on dry filter paper, and then baked at 80 ° C. for 2 hours to immobilize the DNA on the nylon membrane. Thereafter, the nylon membrane was immersed in 2 × SSC for 5 minutes, and then immersed in a pre-cleaning solution (5 × SSC, 0.5% SDS, 1 mM EDTA) and shaken at 50 ° C. for 30 minutes. Cell debris on the nylon membrane was wiped off using a Kimwipe soaked with pre-cleaning solution, and then the nylon membrane was washed twice with 2 × SSC.

  Place the above nylon membrane in a hybridization bag, 10 ml of formamide-containing standard hybridization buffer (50 ml of formamide [deionized]), 25 ml of 20 containing 50 μg / ml fish sperm DNA (Roche Diagnostics). X SSC, 20 ml of 10% blocking reagent, 1 ml of 10% sodium N-lauroyl sarcosine, 0.2 ml of 10% SDS, and 3.8 ml of water) and pre-high at 42 ° C for 1 hour Hybridization was performed. Next, after discarding this prehybridization solution, 10 μl of digoxigenin-labeled PCR amplified DNA (prepared in Example 11 was heat denatured in boiling water for 5 minutes and then rapidly cooled in ice water. 2 ml of a standard hybridization buffer containing formamide was added, and hybridization was performed overnight at 42 ° C. The nylon membrane was removed from the hybridization bag, and the nylon membrane was washed twice for 5 minutes at room temperature using 2 × SSC containing 0.1% SDS. Subsequently, the nylon membrane was washed twice at 68 ° C. for 15 minutes using 1 × SSC containing 0.1% SDS. The nylon membrane was rinsed with a maleic acid buffer containing 0.3% Tween 20 for 1 minute, then immersed in a blocking solution and gently shaken for 30 minutes. Next, the nylon membrane was immersed in 50 ml of blocking solution containing 10 μl of alkaline phosphatase-labeled anti-digoxigenin antibody and gently shaken for 30 minutes. Thereafter, this nylon membrane was washed twice with a maleic acid buffer containing 0.3% Tween 20 for 15 minutes and then immersed in a detection buffer for 2 minutes to equilibrate. The nylon membrane was placed in a petri dish with the colony side up, and 10 ml of a newly prepared chromogenic substrate solution was added thereto and allowed to stand in the dark for several hours. After confirming the color development of the colonies, the nylon membrane was washed with TE buffer and air-dried in the dark.

  Colonies determined to be positive by the above-described colony hybridization were picked up from the original agar medium, inoculated into 50 ml of LB medium containing 50 μg / ml ampicillin, and cultured with shaking at 37 ° C. overnight. Bacteria were collected from this culture and a plasmid was prepared by the method used in Example 10. The plasmid obtained using Sac I is named pASC2, the plasmid obtained using Kpn I is named pAKP1, the plasmid obtained using Sma I is named pASM3, and the plasmid obtained using BamHI is named pABM11. did. In addition, plasmids with different orientations of the inserted DNA of these plasmids were prepared and named pASC2R, pAKP1R, pASM3R, and pABM11R, respectively.

[Determining the base sequence of the amidase gene]
The various plasmids obtained in Example 13 were digested with various restriction enzymes and analyzed by agarose gel electrophoresis to prepare a restriction enzyme map of the inserted DNA portion. As a result, as shown in FIG. 9, the positional relationship of each cloned DNA fragment was clarified. By comparing the restriction enzyme pattern in the DNA region that is common to all DNA fragments and the restriction enzyme pattern obtained from the base sequence (SEQ ID NO: 7) of the DNA amplified by PCR obtained in Example 10 The position and direction of the amidase gene were estimated as shown in FIG. Therefore, as a result of determining the base sequence in the vicinity, the entire base sequence of the amidase gene shown in SEQ ID NO: 9 was determined. As a result of homology search performed by FASTA (homology search software) of DDBJ (Japan DNA Data Bank) for the amino acid sequence (SEQ ID NO: 10) encoded by this base sequence, the one having the highest homology is Rhodococcus rhodochrous J1 This amidase showed 63.7% homology to the amino acid sequence of SEQ ID NO: 10 of the present application.

  The amino acid sequence of Pseudonocardia thermophila amidase described in International Publication No. 2004/083423 pamphlet showed 85.6% homology to the amino acid sequence of SEQ ID NO: 10 of the present application. However, as shown in FIG. 2, although both amino acid sequences are identical in about 20 amino acid residues on the N-terminal side, the sequences are different over a wide region of about 70 amino acid residues thereafter. It became clear that it was an enzyme. In addition, the N-terminal amino acid sequence determined at the protein level and the internal amino acid sequence were found in the amino acid sequence of SEQ ID NO: 10 of the present application (amino acid sequence subjected to shading in FIG. 2). From this, it was confirmed that the nucleotide sequence of SEQ ID NO: 9 of the present application encodes an amidase protein purified from Pseudocardia thermophila.

[Expression of amidase gene in E. coli]
In order to incorporate the above cloned amidase gene into an expression vector, a PCR forward primer (SEQ ID NO: 11) and reverse primer (SEQ ID NO: 12) were synthesized. PCR was performed as follows. In an ice-cooled 0.2 ml PCR tube, 0.5 μl (2.5 U) Taq DNA polymerase (Takara Bio), 5 μl of 10-fold concentration reaction buffer, 4 μl of dNTP mixture (2.5 mM each), 1.8 ng of plasmid pASM3 (obtained in Example 13), 10 pmol of forward primer, 10 pmol of reverse primer and 2.5 μl of dimethyl sulfoxide were added, and water was added to 50 μl. This was set in a PCR device and the DNA was denatured by treatment at 94 ° C. for 5 minutes, followed by 30 temperature cycles (denaturation step at 94 ° C. for 60 seconds, annealing step at 45 ° C. for 30 seconds, and 68 ° C. The DNA was amplified by a 120 second extension reaction step) and finally the reaction was completed by treatment at 68 ° C. for 2 minutes. After completion of the reaction, the reaction solution was subjected to 1% agarose gel electrophoresis, a gel containing about 1600 bp DNA fragment was excised, and DNA was extracted from the excised gel using a QIAquick gel extraction kit (Qiagen).

The DNA amplified by the above PCR was incorporated into pGEM-T Vector (Promega) as follows. In an Eppendorf tube, place 5 μl of 2 × rapid ligation buffer, 1 μl (50 ng) of pGEM-T Vector, 3 μl of DNA amplified by PCR, and 1 μl (3 U) of T4 DNA ligase at room temperature For 1 hour. 5 μl of this reaction solution was added to 100 μl of E. coli JM109 competent cell (Takara Bio), and placed in ice for 30 minutes, followed by heat treatment at 42 ° C. for 45 seconds. Thereafter, it was ice-cooled, and 900 μl of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO ₄ , 10 mM MgCl ₂ , 20 mM glucose) was added, and then it was added at 37 ° C. Shake slowly for 1 hour. 100 μl of this was applied to a selective agar medium (LB agar medium containing 50 μg / ml ampicillin, 0.5 mM IPTG, and 40 μg / ml X-gal), and this was incubated at 37 ° C. overnight. White colonies that appeared on the selective agar medium were inoculated into 50 ml of LB medium containing 50 μg / ml ampicillin and cultured overnight at 37 ° C. with shaking. Bacteria were collected from this culture and a plasmid was prepared from the cells by the method used in Example 10. The plasmid thus obtained was named pGEM-fPTAM.

Plasmid pGEM-fPTAM was diluted 100-fold with TE buffer (10 mM Tris-HCl buffer [pH 8.0], 1 mM EDTA), 1 μl of this was added to 100 μl of E. coli BL21 (DE3) competent cell (Novagen), and in ice And then heat-treated at 42 ° C. for 45 seconds. Thereafter, the mixture was ice-cooled, 900 μl of SOC medium was added, and the mixture was gently shaken at 37 ° C. for 1 hour. 100 μl of this was applied to an LB agar medium containing 50 μg / ml ampicillin, and this was incubated at 37 ° C. overnight. Colonies that appeared on the agar medium were inoculated into 20 ml of LB medium containing 50 μg / ml ampicillin in a 100 ml baffled Erlenmeyer flask and cultured at 37 ° C. with shaking. When _A600 reached 0.6, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.4 mM, and the mixture was further cultured with shaking at 37 ° C. for 5 hours. The culture was collected by centrifugation (16 krpm, 10 min, 4 ° C.), and the obtained cells were suspended in 100 mM potassium phosphate buffer (pH 7.5).

  An appropriate amount of the above-mentioned bacterial cell suspension was placed in an Eppendorf tube, 100 mM potassium phosphate buffer (pH 7.5) was added to make the total amount 487.5 μl, and the mixture was kept in a 50 ° C. water bath for 5 minutes. To this, 12.5 μl of 20% DL-methionine amide hydrochloride was added and reacted in a 50 ° C. water bath for 20 minutes. After stopping the reaction by adding 100 μl of 2N hydrochloric acid, the supernatant obtained by centrifuging it (16 krpm, 5 min, 20 ° C.) was purified by HPLC (water / acetonitrile / TFA = 950/50/1). analyzed. As a result, 278 U of amidase activity was obtained per 1 g of wet cells. In contrast, the amidase activity per gram of wet cells of the original Pseudonocardia thermophila JCM3095 was 138 U when analyzed under the same conditions. That is, by highly expressing the amidase gene in Escherichia coli, an amidase activity approximately twice that of the original bacterium could be obtained.

  International Publication No. 2004/083423 describes an amidase derived from Pseudonocardia thermophila, which is the same microorganism as the present invention. However, this amidase is considered to be a constitutive enzyme, and its enzyme activity is described as about 2.5 times higher than that of HMB-amide (2-hydroxy-4-methylthiobutyramide) when methionine amide is used as a substrate. Has been. On the other hand, the amidase of the present invention is also derived from Pseudocardia thermophila, but is an inducing enzyme, and its enzyme activity, contrary to the former enzyme, is 2-hydroxy-4-methylthiobutyramide (HMB- It was found that when amide) was used as a substrate, it was about 12 times higher than methionine amide. Therefore, the amidase of the present invention is advantageous for producing 2-hydroxy-4-methylthiobutyric acid (HMBA) from 2-hydroxy-4-methylthiobutyramide.

  Furthermore, as a result of cloning the amidase gene and determining the base sequence, the amino acid sequences of both amidases were identical in about 20 amino acid residues on the N-terminal side, but thereafter a wide range of about 70 amino acid residues. Over time, the sequence was different and was revealed to be another enzyme (FIG. 2).

  To date, various amidases have been found in various microorganisms, and these amidases are strongly inhibited by ammonia produced by hydrolysis of amides (MJ Woods et al., 1979, Biochim. Biophys). Acta 567: 225-237). On the other hand, it has been clarified that the amidase of the present invention has a property that its activity is hardly inhibited by ammonia. Therefore, it can be said that the amidase of the present invention is very advantageous when industrially producing carboxylic acid from amide.

It is a figure which shows the influence of ammonia in the microbial cell reaction in pH7.5. ● is for Pseudonocardia thermophila JCM3095 cells of the present invention, and ○ is for Rhodococcus rhodochrous IFO 15564 cells used as controls. It is the figure which compared the amino acid sequence (Nisso) of the amidase of this invention, and the amino acid sequence (Degussa) of the amidase of international publication 2004/083423 pamphlet. The shaded amino acid sequence is the N-terminal amino acid sequence or internal amino acid sequence determined at the protein level. It is a figure which shows the influence of ammonia in the microbial cell reaction in pH 9.0. ● is for Pseudonocardia thermophila JCM3095 cells of the present invention, and ○ is for Rhodococcus rhodochrous IFO 15564 cells used as controls. It is the figure which analyzed the active fraction in each purification step of amidase by SDS-polyacrylamide gel electrophoresis. Lane 1 is the cell extract, lane 2 is the ammonium sulfate salting out fraction, lane 3 is the phenyl sepharose active fraction, lane 4 is the DEAE sepharose active fraction, lane 5 is the gel filtration active fraction, and lane M is the molecular mass marker. It is. It is a figure which shows the influence of the temperature in the enzyme reaction using purified amidase. It is a figure which shows the thermostability of purified amidase. It is a figure which shows the influence of pH in the enzyme reaction using purified amidase. ◇ is when sodium acetate buffer is used, ● is when potassium phosphate buffer is used, Δ is when Tris-HCl buffer is used, and ■ is when ammonium chloride buffer is used. It is a figure which shows the influence of ammonia in the enzyme reaction using purified amidase. ● represents 30 ° C, ▲ represents 40 ° C, and ■ represents 50 ° C. It is a restriction map of a DNA fragment containing an amidase gene. pAKP1 is a plasmid containing a Kpn I fragment, pASM3 is a Sma I fragment, pASC2R is a Sac I fragment, and pABM11 is a BamH I fragment. Open arrows indicate the position and direction of the amidase gene.

Claims (17)

An amidase characterized by having all the following properties (a) to (f):
(A) Under reaction conditions of pH 9.0, 50% or more amidase activity in the absence of ammonia is exhibited even in the presence of 0.2 M ammonia.
(B) It shows higher specific activity with respect to 2-hydroxy-4-methylthiobutyramide than methionine amide.
(C) The apparent molecular mass on SDS-PAGE is 53 kDa.
(D) The optimum temperature of activity is 60 to 70 ° C.
(E) It is stable at a temperature of 60 ° C. or lower at a neutral pH.
(F) High activity in a wide pH range of pH 5.5 to pH 10.0. The amidase according to claim 1, which is derived from Pseudonocardia thermophila. The protein shown in the following (g) or (h).
(G) A protein consisting of the amino acid sequence represented by SEQ ID NO: 10.
(H) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 10, and having amidase activity. The protein shown in the following (i) or (j).
(I) A protein comprising an amino acid sequence comprising the amino acid sequence at positions 24 to 95 of the amino acid sequence shown in SEQ ID NO: 10 and having amidase activity.
(J) It consists of an amino acid sequence comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of positions 24 to 95 of the amino acid sequence shown in SEQ ID NO: 10, and has amidase activity protein. The protein according to any one of claims 3 to 4, which has the following properties (a) and (b).
(A) Under reaction conditions of pH 9.0, 50% or more amidase activity in the absence of ammonia is exhibited even in the presence of 0.2 M ammonia.
(B) It shows higher specific activity with respect to 2-hydroxy-4-methylthiobutyramide than methionine amide. The protein according to any one of claims 3 to 5, which has all the following properties (c) to (f).
(C) The apparent molecular mass on SDS-PAGE is 53 kDa.
(D) The optimum temperature of activity is 60 to 70 ° C.
(E) It is stable at a temperature of 60 ° C. or lower at a neutral pH.
(F) High activity in a wide pH range of pH 5.5 to pH 10.0. The protein according to any one of claims 3 to 6, which is derived from Pseudocardia thermophila. Amidase gene DNA encoding the protein according to any one of claims 3 to 7. Amidase gene DNA shown in the following (k) or (l).
(K) Amidase gene DNA consisting of the base sequence shown in SEQ ID NO: 9.
(L) Amidase gene DNA comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 9 and encoding a protein having amidase activity. An amidase gene DNA that hybridizes under stringent conditions with a DNA comprising a sequence complementary to the DNA comprising the base sequence shown in SEQ ID NO: 9 and encodes a protein having amidase activity. The amidase gene DNA according to claim 9 or 10, wherein the protein having amidase activity is a protein having the following properties (a) and (b):
(A) Under reaction conditions of pH 9.0, 50% or more amidase activity in the absence of ammonia is exhibited even in the presence of 0.2 M ammonia.
(B) It shows higher specific activity with respect to 2-hydroxy-4-methylthiobutyramide than methionine amide. The amidase gene DNA according to any one of claims 9 to 11, wherein the protein having amidase activity is a protein having all the following properties (c) to (f):
(C) The apparent molecular mass on SDS-PAGE is 53 kDa.
(D) The optimum temperature of activity is 60 to 70 ° C.
(E) It is stable at a temperature of 60 ° C. or lower at a neutral pH.
(F) High activity in a wide pH range of pH 5.5 to pH 10.0. The amidase gene DNA according to any one of claims 9 to 12, wherein the protein having amidase activity is derived from Pseudonocardia thermophila. A recombinant vector in which the amidase gene DNA according to any one of claims 8 to 13 is incorporated. A transformed microorganism, wherein the recombinant vector according to claim 14 is introduced. The amidase according to claim 1 or 2 or a production bacterium thereof or a processed product thereof, or the protein according to any one of claims 3 to 7, or the transformed microorganism according to claim 15 or a processed product thereof are allowed to act on an amide. A process for producing a carboxylic acid characterized by The method for producing a carboxylic acid according to claim 16, wherein the amide is an α-amino acid amide or an α-hydroxy acid amide, and the carboxylic acid is an α-amino acid or an α-hydroxy acid.