JP2007082534A - Heat-stable n-acylamino acid racemase, method for producing the same and application thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a heat-stable N-acylamino acid racemase. <P>SOLUTION: The isolated protein has N-acylamino acid racemase activities, and exhibits (a) thermal stability: having the N-acylamino acid racemase activities after heat treatment for 1 hr at 70°C; and (b) metal ion dependency: exhibiting the N-acylamino acid racemase activities in the presence of Co<SP>2+</SP>, Mn<SP>2+</SP>, Mg<SP>2+</SP>and Fe<SP>2+</SP>. The protein substantially consisting of a polypeptide comprising a specific amino acid sequence, or a polypeptide containing an amino acid sequence having ≥60% identity and ≥80% homology with the amino acid sequence, and having the N-acylamino acid racemase activities after the heat treatment for 1 hr at 70°C. The racemization of the N-acylamino acid by using the protein, and the method for producing an optically active amino acid by combining the protein with a D- or L-aminoacylase are also provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel protein having N-acyl amino acid racemase (hereinafter also referred to as NAAAR) activity, which can be used for the production of optically active α-amino acids. Furthermore, the present invention relates to a method for producing the NAAAR by genetic engineering technology, and a method for producing an optically active α-amino acid using the NAAAR.

  Amino acids can form optical isomers structurally, but most of the α-amino acids that exist in nature and are utilized in living organisms are L-forms. However, the fact that a small amount of D-amino acid is present in the living body has been clarified one after another in recent years, and D-amino acid and its derivatives have also been used for pharmaceuticals and the like due to the usefulness of their physiological activity. ing. Even if they have the same composition, L-amino acids and D-amino acids, which are enantiomers in the three-dimensional structure, have different physiological activities. It is very important to obtain amino acids having the following optical activity.

  Amino acid production methods are roughly divided into a fermentation method for recovering amino acids produced by culturing microorganisms and a chemical synthesis method for artificially synthesizing amino acids under predetermined reaction conditions. In particular, since D-amino acids are currently difficult to be fermented and produced, the production must be dependent on chemical synthesis. However, since amino acids produced by chemical synthesis are in a racemic state in which L and D isomers are optically mixed, there is an operation for separating optical isomers from them and extracting amino acids having desired optical activity. Further required. As this method, a method is known in which D-aminoacylase or L-aminoacylase is allowed to act on an N-acyl-D, L-amino acid to produce an amino acid having a desired optical characteristic and then extracted.

N-acyl amino acid racemase (NAAAR) catalyzes a reversible reaction that replaces N-acyl-D-amino acids and N-acyl-L-amino acids with the corresponding optical isomers, respectively. Therefore, by causing this NAAAR to act on N-acyl-D, L-amino acid together with aminoacylase, a desired optically active amino acid can be obtained in a higher yield than when aminoacylase alone is acted on. it can. Examples of known proteins having N-acylamino acid racemase activity include those derived from, for example, Amycolatopsis sp. TS-1-60 (hereinafter sometimes referred to simply as TS-1-60) (Patent Document 1), those derived from Sebekia benihana (hereinafter sometimes referred to simply as “Benihana”) (Patent Document 2), those derived from Deinococcus radiodurans (Patent Document 3), etc. Can be mentioned. Their gene sequences are also determined (according to Patent Document 3, D. Radiodurans-derived NAAAR has about 46% and 44% identity in amino acid sequence with TS-1-60 and Benihana-derived enzymes, respectively) In addition, expression of recombinant proteins by transformed microorganisms has also been confirmed. In particular, TS-1-60-derived NAAAR has been subjected to crystal structure analysis, and its three-dimensional structure has been determined (Non-patent Document 1).

Usually, if a new enzyme having the same function is to be obtained using the amino acid sequence information of a known enzyme as a clue, a novel gene that is predicted to encode a protein having an amino acid sequence highly homologous to the known sequence is expressed by genetic engineering. Can be easily obtained. Proteins with highly conserved amino acid sequences are considered to be orthologs having the same function. However, in the case of NAAAR, it should be about 40 to about 50% identical to known NAAAR. For example, it is known that the protein does not necessarily have NAAAR activity. For example, the ytfD gene product derived from Bacillus subtilis has an amino acid sequence having about 42% identity with TS-1-60-derived NAAAR and about 43% with safflower-derived NAAR, but lacking NAAAR activity. (Patent Document 3). In addition, the true function of the TS-1-60-derived enzyme, which has been regarded as NAAAR, is o-succinylbenzoate synthase (OSBS), which also acts on N-acyl amino acids due to the sweetness of the substrate specificity to racemize. The hypothesis that a reaction has occurred has been proposed (Non-patent Document 2). According to this document, it is also described that the ytfD gene product also has OSBS activity, and not all enzymes having OSBS activity have NAAAR activity.
That is, a protein having NAAAR activity as a true physiological function has not been known so far, and therefore, a protein having about 50% or less identity in amino acid sequence with a protein having known NAAAR activity. However, whether or not it has NAAAR activity cannot be determined without actually preparing the protein and confirming the activity.

  By the way, in protein molecular design, improving stability is an eternal theme. When industrially using various proteins, deactivation of the protein in storage conditions or usage conditions causes disadvantages such as reduced product quality and high production costs. Forced. In general, the catalytic activity of various enzymes, which are representative examples of proteins, is more efficient at higher temperatures, so the use of highly stable proteins also has the advantage of enabling use under more efficient conditions. .

  One way to achieve protein stabilization is to add stabilizers. In order to improve the stability of the protein in the storage state or the use conditions, it is intended to maintain the function of the protein for a longer period by adding appropriate amounts of various substances. Examples of such chemical substances include saccharides, amino acids, surfactants, proteins such as bovine serum albumin, various preservatives, etc., but any protein can dramatically improve stability. All-purpose stabilizing substances are not known, and the degree of stabilization by these substances is limited. Therefore, it is preferable that the protein itself is excellent in stability.

  Recent advances in genetic engineering techniques have made it easier to introduce mutations into genes encoding proteins. Intensive efforts have been made to improve protein function by substitution, deletion, and addition of amino acid residues, and many examples of obtaining mutant proteins with improved stability have been reported. However, it is not uncommon for mutations that are effective from the viewpoint of improving stability to impair other excellent properties of the protein. Furthermore, genetic engineering protein function modification is based on the performance of the original wild-type protein, so if the original stability is poor, the path to achieving the goal will be far. From the above, when trying to use proteins industrially, it is important to obtain a protein with high stability as a natural property even if it is premised on functional improvement by genetic engineering techniques. .

  The activity maintenance rate in long-term storage at room temperature or higher can be used as an indicator of protein stability, but it is general that proteins with high heat resistance are considered to be excellent in stability. The method of using thermal stability as an index when selecting industrially useful highly stable proteins is a common stone. The thermostable protein is excellent in stability, and is more advantageous in the industry.

The various proteins having the known NAAAR activity described above are also not sufficient in terms of stability, particularly thermal stability, when considering application to industrial D-amino acid production.
JP-A-4-365482 JP 2001-314191 A Japanese Patent Laid-Open No. 2002-238581 Biochemistry (2004) Vol.43, p5716-5727 Biochemistry (1999) Vol.38, p4252-4258

  Accordingly, an object of the present invention is to provide a novel protein having NAAAR activity, which has advantageous properties as compared to conventionally known acylamino acid racemases (hereinafter, unless otherwise specified, has NAAAR activity regardless of true physiological function). The protein is simply referred to as NAAAR), specifically, to provide a heat-resistant NAAAR with improved thermal stability, and optically active amino acids, particularly D-amino acids are industrially produced using the NAAAR Is to provide a method.

As a result of various studies to achieve the above-mentioned object, the present inventors have found that a hypothetical protein having homology with TS-1-60-derived NAAAR that exists in the Chloroflexus aurantiacus genome. The encoding open reading frame (ORF) was isolated and the protein was successfully engineered. The present inventors have found that the protein has NAAAR activity and has excellent thermal stability compared to known NAAAR. Furthermore, the present inventors have desired optical activity by allowing the protein to act on the substrate N-acyl-D, L-amino acid in the presence of L- (or D-) aminoacylase. The present invention was completed by successfully producing α-amino acids.

That is, the present invention has the following configuration.
[1] An isolated protein having N-acylamino acid racemase activity and having the following physicochemical properties.
(a) Thermal stability: N-acylamino acid racemase activity after heating at 70 ° C. for 1 hour
(b) Metal ion dependence: N-acylamino acid racemase activity in the presence of Co ²⁺ , Mn ²⁺ , Mg ²⁺ and Fe ²⁺ [2] Residual N after heating at 70 ° C. for 1 hour -The protein of the above-mentioned [1], wherein the acylamino acid racemase activity is at least 30% or more.
[3] The protein according to [1] or [2], further having the following properties (c) to (e):
(c) Apparent molecular weight by SDS-PAGE: about 39 kDa
(d) pH stability: The residual N-acylamino acid racemase activity after heating at 25 ° C. for 24 hours at a pH of 5 or more is about 80% or more.
(e) Substrate specificity: N-acylamino acid racemase activity against L and D forms of N-acetylated or N-formylated methionine, valine, leucine, phenylalanine and tryptophan [4] Or an isolated protein having N-acylamino acid racemase activity consisting essentially of the polypeptide of (b).
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2
(b) an N-acyl amino acid racemase that includes an amino acid sequence having 60% or more identity and 80% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and that has been heated at 70 ° C. for 1 hour Polypeptide having activity [5] The protein according to [4] above, wherein the remaining N-acylamino acid racemase activity after heating at 70 ° C. for 1 hour is 30% or more.
[6] The protein according to [4] or [5] above, comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2.
[7] A nucleic acid encoding the protein according to any one of the above [1] to [6] (excluding the nucleic acid containing the base sequence represented by SEQ ID NO: 1).
[8] A nucleic acid according to the above [7] for introduction into a heterologous host cell, the nucleic acid comprising a base sequence in which a less frequently used codon is replaced with a more frequently used codon in the host. [9] The nucleic acid according to [8] above, wherein the host is Escherichia coli.
[10] The nucleic acid according to any one of the above [7] to [9], comprising the base sequence represented by SEQ ID NO: 3.
[11] An expression vector in which a nucleic acid encoding the protein according to any one of [1] to [6] is operably linked to a promoter suitable for a host cell into which the nucleic acid is introduced.
[12] The expression vector according to [11] above, wherein the nucleic acid is one according to any of [7] to [10] above.
[13] A transformant obtained by transforming a host cell with the expression vector according to [11] or [12] above.
[14] The transformant according to [13] above, wherein the host cell is Escherichia coli.
[15] The method according to [1] to [6] above, comprising culturing the transformant according to [13] or [14], and collecting a protein having N-acylamino acid racemase activity from the obtained culture. A method for producing the protein according to any one of the above.
[16] Racemized N-acyl amino acid comprising contacting the protein according to any of [1] to [6] above with N-acyl-D-amino acid or N-acyl-L-amino acid Manufacturing method.
[17] The protein according to any one of [1] to [6] above, in the presence of D- or L-aminoacylase, N-acyl-D-amino acid, N-acyl-L-amino acid or a mixture thereof A process for producing a D- or L-amino acid comprising contacting
[18] The method according to [16] or [17] above, wherein the amino acid is selected from the group consisting of methionine, leucine, valine, phenylalanine or tryptophan.

  Since the NAAAR of the present invention is excellent in heat resistance and other stability, it can be used for the production of α-amino acids having optical activity, particularly α-D-amino acids, as compared with conventionally known NAAARs. There is an advantageous effect of bringing about a significant improvement in productivity.

  NAAAR catalyzes a reversible reaction that converts N-acyl-D-amino acids and N-acyl-L-amino acids into the corresponding optical isomers, respectively, while the NAAAR of the present invention exhibits high thermal stability. Is characterized by That is, TS-1-60, Benihana, D.H. Conventionally known NAAARs derived from radiodurance and the like are almost completely deactivated when treated at 70 ° C. for 30 minutes, whereas the NAAAR of the present invention is N− after the heating treatment at 70 ° C. for 1 hour. Acylamino acid racemase activity. Preferably, the NAAAR of the present invention has a residual NAAAR activity (relative activity with 100% of the activity before the heating treatment) after heating at 70 ° C. for 1 hour, more preferably 50% or more. is there. In this specification, the heat stability is determined by performing the above heating treatment in a state where the protein amount is dissolved in 20 mM Tris-HCl buffer (pH 7.5) at a concentration of 6 mg / ml, and before and after the treatment. This can be determined by measuring NAAAR activity and calculating residual NAAAR activity. The conditions for enzyme activity measurement will be described later.

The NAAAR of the present invention exhibits the highest activity in the presence of Co ²⁺ , as in the case of conventionally known NAAARs, but also exhibits activity in the presence of Mn ²⁺ , Mg ²⁺ , and Fe ²⁺ . On the other hand, the enzyme does not show activity in the presence of Cu ²⁺ , Ba ²⁺ and Zn ²⁺ . Comparing such metal ion dependence with conventionally known NAAAR, TS-1-60-derived NAAAR is different from NAAAR of the present invention in that it exhibits a relatively high activity even in the presence of Zn ²⁺ . It differs from the NAAAR of the present invention in that it exhibits a certain activity even in the presence of Ba ²⁺ and Zn ²⁺ . Radiodurance-derived NAAAR exhibits a certain level of activity even in the presence of Zn ²⁺ , while the activity in the presence of Mn ²⁺ , Mg ²⁺ and Fe ²⁺ is lower than that of the NAAAR of the present invention. Different from the NAAAR of the invention. The metal ion dependency in the present specification was determined by substituting a 50 mM cobalt chloride solution with a salt solution of various metal ions having the same concentration under the conditions for enzyme activity measurement described later, and using the cobalt chloride solution. It can be determined by calculating the relative enzyme activity at 100%.

  The NAAAR of the present invention may have different molecular weights due to differences in amino acid composition, glycosylation format depending on host cells, etc., but preferably has an apparent molecular weight of about 39 kDa as determined by SDS-PAGE. Things. The theoretical value calculated from the amino acid composition is, for example, about 40.6 kDa in the case of NAAAR consisting of the amino acid sequence shown in SEQ ID NO: 2 unless modification such as glycosylation by the host cell is taken into consideration. Presumed to have molecular weight.

  The NAAAR of the present invention is stable over a wide pH range of pH 5 or more like the conventionally known NAAAR. That is, the residual N-acylamino acid racemase activity (relative activity with 100% of the activity before the heating treatment) after the heating treatment at 25 ° C. for 24 hours is about 80% or more. In addition, pH stability in this specification is acetic acid buffer (pH 4-5), potassium phosphate buffer (pH 5-7), Tris-HCl buffer instead of HEPES buffer under the conditions of enzyme activity measurement described later. This can be determined by measuring the NAAAR activity before and after the heating treatment using a solution (pH 7-9) or glycine hydrochloride buffer (pH 9-10) and calculating the residual NAAAR activity.

  The NAAAR of the present invention exhibits a substrate specificity similar to the conventionally known NAAAR. That is, it has the highest activity against N-acetyl- or N-formyl-methionine, but also shows activity against N-acetylated or N-formylated valine, leucine, phenylalanine and tryptophan L-form and D-form.

The NAAAR of the present invention is not particularly limited as long as it has the above characteristics, and its origin is not limited. Therefore, in addition to naturally occurring biological origins, all those derived from natural or artificial mutants or transformants obtained by introducing a heterologous (ie, foreign) NAAAR gene are also included. NAAAR derived from a bacterium, preferably a bacterium belonging to the genus Chloroflexus , more preferably a Chloroflexus aurantiacus, is exemplified.

In a preferred embodiment of the present invention, NAAAR is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 60% or more identity and 80% or more homology with the amino acid sequence (hereinafter referred to as A polypeptide having a NAAAR activity even after heating at 70 ° C. for 1 hour (hereinafter, referred to as “substantially the same polypeptide”). An isolated protein consisting essentially of).
As used herein, “identity” and “homology” refer to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is an optimal alignment). The percentage of identical amino acid residues and the percentage of identical and similar amino acid residues with respect to all overlapping amino acid residues in which one may consider the introduction of gaps into one or both of the sequences) (%) Respectively. “Similar amino acids” mean amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn). ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids containing hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Examples of conservative amino acid substitutions are well known in the art and have been described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

  As an algorithm for determining amino acid sequence identity and / or homology, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) The algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48 : 444-453 (1970) [the algorithm is incorporated into the GAP program in the GCG software package], Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm Is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package], described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) Algorithm [The algorithm is a GCG software package. Built into FASTA program in di], and the like, but not limited to.

In addition, here, “being substantially” means, for example, physiological activity possessed by a polypeptide, such as glycosylation by a host cell, N-terminal and / or C-terminal amino acid, or modification of the side chain of an internal amino acid residue That is, it is used in a meaning that includes those that have been modified to the extent that NAAAR activity is not affected. More specifically, the C-terminus may be any of a carboxyl group, a carboxylate, an amide, or an ester (COOR). Here, R in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, or the like, for example, phenyl. C _6-12 aryl groups such as α-naphthyl, for example, C _7- such as phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl ₁₄ aralkyl group, pivaloyloxymethyl group is used. Furthermore, carboxyl groups other than the C terminal may be amidated or esterified. Examples of the ester in this case include the above-mentioned C-terminal ester.
On the other hand, the amino group of the N-terminal amino acid residue may be protected with a protecting group (for example, C _1-6 acyl group such as C _1-6 alkanoyl such as formyl group, acetyl group, etc.) The N-terminal glutamine residue produced by cleavage at the site is pyroglutamine oxidized, or a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) ) May be protected with a suitable protecting group (for example, a C _1-6 acyl group such as a C _1-6 alkanoyl group such as a formyl group or an acetyl group). Alternatively, a complex protein such as a so-called glycoprotein to which a sugar chain is bound is also included.

  The NAAAR of the present invention may be in the form of a salt. Examples of such salts include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts, alkaline earth metal salts). Acceptable acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is C.I. It is encoded by the ORF present in the genome of Aurantiacus, and is presumed to be an enzyme belonging to the L-alanine-D, L-glutamate epimerase and enolase superfamily from the homology of the amino acid sequence with known proteins. In addition to the actual function of the polypeptide, it has not been reported so far that the bacterium actually produces the polypeptide.

The amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 2 is preferably 70% or more, more preferably 80% or more, more preferably the amino acid sequence shown in SEQ ID NO: 2. It has 90% or more identity, particularly preferably 95% or more identity, and most preferably 98% or more identity.
In a preferred embodiment, the amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 2 has one or more amino acids deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2. Amino acid sequence, for example (1) 1 or 2 or more (preferably 1 to 30, more preferably 1 to 10, more preferably 1 to several (2 to 5) in the amino acid sequence shown in SEQ ID NO: 2 ) Amino acid sequence from which amino acids have been deleted, (2) 1 or 2 (preferably 1 to 30, more preferably 1 to 10, more preferably 1 to number (2) in the amino acid sequence shown in SEQ ID NO: 2 To 5) amino acid sequence added, (3) 1 or 2 (preferably 1 to 30, more preferably 1 to 10, more preferably 1 to the amino acid sequence shown in SEQ ID NO: 2) Or an amino acid sequence in which 1 to several (2 to 5) amino acids are inserted, (4) one or more (preferably 1 to 30, more preferably 1) in the amino acid sequence shown in SEQ ID NO: 2. Amino acid sequence in which 10 to 10 amino acids, more preferably 1 to several (2 to 5) amino acids are substituted with other amino acids, or (5) an amino acid sequence in which the mutations (1) to (4) above are combined (In this case, the total number of mutated amino acids is preferably 1-30, more preferably 1-10, and even more preferably 1-number (2-5)).

  A polypeptide substantially identical to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 comprises an amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 2 above, and at 70 ° C. for 1 hour. Although it is not particularly limited as long as it has a detectable NAAAR activity even after heating treatment, it preferably has a residual NAAAR activity of 30% or more, more preferably 50% or more after heating at 70 ° C. for 1 hour. It is a peptide. Here, the definition of the residual NAAAR activity and the method for determining the activity are the same as described above.

  The NAAAR of the present invention is (1) a method of extracting and purifying the tissue or cells producing the enzyme as a raw material, (2) a method of chemically synthesizing, and (3) an operation to express NAAAR by a gene recombination technique. The method can be obtained by appropriately using a method of purifying from the obtained cells, or (4) a method of biochemical synthesis from a nucleic acid encoding NAAAR using a cell-free transcription / translation system.

  For example, NAAAR can be isolated and purified from natural NAAAR-producing cells as follows. NAAAR-producing cells are homogenized in an appropriate buffer solution, and a cell extract is obtained by sonication or surfactant treatment, and then purified by appropriately combining separation techniques conventionally used for protein separation and purification. can do. Such separation techniques include, for example, methods utilizing differences in solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, non-denaturing polyacrylamide electrophoresis (PAGE), sodium dodecyl sulfate-poly A method using molecular weight difference such as acrylamide electrophoresis (SDS-PAGE), a method using charge such as ion exchange chromatography and hydroxyapatite chromatography, a method using specific affinity such as affinity chromatography, and the like Examples include, but are not limited to, a method utilizing a difference in hydrophobicity such as phase high performance liquid chromatography and a method utilizing a difference in isoelectric point such as isoelectric focusing.

The production of NAAAR by chemical synthesis can be performed, for example, by synthesizing all or part of the sequence using a peptide synthesizer based on the amino acid sequence shown in SEQ ID NO: 2. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. When the partial peptide or amino acid capable of constituting the NAAAR of the present invention is condensed with the remaining portion, and the product contains a protecting group, the protecting protein is eliminated, whereby the target protein can be produced. Here, the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the method described in the following (1) and (2).
(1) M. Bodanszkyand MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, NewYork (1965)

The NAAAR of the present invention thus obtained can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
When NAAAR obtained by the above method is a free form, the free form can be converted into an appropriate salt by a known method or a method according thereto, and conversely, when a protein is obtained as a salt. The salt can be converted into a free form or other salt by a known method or a method analogous thereto.

  The NAAAR of the present invention is preferably produced by cloning (or chemically synthesizing) a nucleic acid encoding the protein, and isolating and purifying it from a culture of a transformant containing an expression vector carrying the nucleic acid. be able to.

Cloning of enzyme genes is usually performed by the following method. First, the enzyme is completely or partially purified from a cell or tissue that produces the desired enzyme, and its N-terminal amino acid sequence is determined using the Edman method or mass spectrometry. In addition, the amino acid sequence of an oligopeptide obtained by partially decomposing the enzyme with a protease or chemical substance that cleaves the peptide in a sequence-specific manner is similarly determined by Edman method or mass spectrometry. An oligonucleotide having a base sequence corresponding to the determined partial amino acid sequence is synthesized, and this is used as a probe from a cDNA or genomic DNA library prepared from a cell or tissue producing the enzyme. ) The DNA encoding the enzyme is cloned by a hybridization method.
Alternatively, an antibody against the enzyme is prepared according to a conventional method using all or part of the completely or partially purified enzyme as an antigen, and the antibody is prepared from a cDNA or genomic DNA library prepared from a cell or tissue producing the enzyme. DNA encoding the enzyme can also be cloned by a screening method.
When the gene of an enzyme similar in enzymatic properties to the target enzyme is known, for example, the NCBI BLAST website (http://www.ncbi.nlm.nih.gov/BLAST/) is accessed and the known gene Search for a sequence homologous to the base sequence of the gene, create a probe as described above based on the hit base sequence, and clone the DNA encoding the enzyme by colony (or plaque) hybridization be able to. For example, a C.I. In the case of NAAAR derived from Aurantiacus, it has 51% identity with TS-1-60-derived NAAAR at the amino acid level, and therefore, by homology search with the base sequence of TS-1-60-derived NAAAR gene, C.I. An ORF encoding NAAAR on the Aurantiacus genome can be found.
In addition, based on the hit nucleotide sequence, an appropriate oligonucleotide was synthesized as a primer, and a genomic DNA fraction prepared from cells producing NAAAR, or a total RNA or mRNA fraction was used as a template. , Abbreviated as “PCR method”) or Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”).
The base sequence of the DNA obtained as described above can be determined using a known sequence technique such as Maxam-Gilbert method or dideoxy termination method.

  The nucleic acid encoding the NAAAR of the present invention is preferably a nucleic acid encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a polypeptide substantially identical thereto. The nucleic acid may be DNA, RNA, or a DNA / RNA chimera, preferably DNA. The nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid.

More preferably, the nucleic acid encoding the NAAAR of the present invention includes, for example, a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 (if the nucleic acid is RNA, t is read as u), or SEQ ID NO: 1 A base sequence complementary to the base sequence shown in FIG. 2 and a base sequence that hybridizes under stringent conditions and has the same property as the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 described above, namely 70 Examples thereof include a nucleic acid encoding a polypeptide having the property of having NAAAR activity even after heating at 1 ° C. for 1 hour.
The nucleic acid that hybridizes with the base sequence shown in SEQ ID NO: 1 under stringent conditions is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, with the base sequence shown in SEQ ID NO: 1, Particularly preferred is a nucleic acid containing a base sequence having 90% or more identity, most preferably 95% or more identity.

The identity of the nucleotide sequences in this specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; filtering = ON; match) It can be calculated by score = 1; mismatch score = -3). As another algorithm for determining the homology of a base sequence, the above-mentioned algorithm for calculating homology of amino acid sequences is also preferably exemplified.
Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, Second Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to stringent conditions.

  The stringent conditions include, for example, conditions in which the sodium salt concentration is about 19 to about 40 mM, preferably about 19 to about 20 mM, and the temperature is about 50 to about 70 ° C., preferably about 60 to about 65 ° C. It is done. In particular, it is preferable that the sodium salt concentration is about 19 mM and the temperature is about 65 ° C. A person skilled in the art may appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc. Thus, the desired stringency can be easily adjusted.

  The DNA encoding the NAAAR of the present invention is C.I. It can be obtained from Aurantiacus genomic DNA or RNA (cDNA), but chemically synthesize DNA strands or connect partially synthesized oligo DNA short strands using PCR method By doing so, it is also possible to construct a DNA encoding the full length of NAAAR. The advantage of constructing full-length DNA in combination with chemical synthesis or PCR is that the codons used can be designed over the entire length of the gene in accordance with the host into which the gene is introduced. A plurality of codons encoding the same amino acid are not used uniformly, and the frequency of use varies depending on the species. In general, codons contained in genes that are highly expressed in a given species contain many codons that are frequently used in that species, and conversely, the presence of codons that are used infrequently becomes a bottleneck for genes with low expression levels. There are many examples that prevent high expression. In the expression of a heterologous gene, many examples have been reported so far in which the expression level of the heterologous protein has been increased by replacing the gene sequence with a codon that is frequently used in the host organism. It is expected to be effective in increasing the expression level of heterologous genes.

  For the above reasons, the DNA encoding the NAAAR of the present invention is preferably modified to a codon more suitable for the host cell into which it is introduced (that is, a codon frequently used in the host). The codon usage frequency of each host is defined by the ratio of each codon used in all genes present on the genome sequence of the host organism, and is expressed, for example, by the number of times used per 1000 codons. In addition, the codon usage frequency can be approximately calculated from the sequence of a plurality of typical genes in an organism whose entire genome sequence has not been elucidated. The data on the frequency of codon usage in the host organism to be recombined is, for example, the genetic code usage frequency database published on the website of Kazusa DNA Research Institute (http://www.kazusa.or.jp). Or you can refer to the literature describing the codon usage in each organism, or you can determine the codon usage data for the host organism you are using. By referring to the obtained data and the gene sequence to be introduced and replacing the codon used in the gene sequence that is not frequently used in the host organism with a codon that encodes the same amino acid and is frequently used Good.

The host cell into which the DNA encoding the NAAAR of the present invention is introduced is not particularly limited as long as a recombinant expression system has been established as described later, but preferably bacteria such as Escherichia coli and Bacillus subtilis, actinomycetes And microbial hosts such as gonococci and yeast, insect cells, animal cells, higher plants and the like, more preferably E. coli (for example, K12 strain, B strain, etc.). For example, in the case of the K12 strain, codons frequently used in E. coli are GGT or GGC for Gly, GAA for Glu, GAT for Asp, GTG for Val, GCG for Ala, GCG for Arg, CGT or CGC, Ser for AGC, Lys for AAA, Ile for ATT or ATC, Thr for ACC, Leu for CTG, Gln for CAG, Pro for CCG and the like.
As a DNA encoding NAAAR substituted with a codon frequently used in the host as described above, for example, C.I. Examples include DNA (base sequence shown in SEQ ID NO: 3) in which DNA encoding NAAAR derived from Aurantiacus is substituted with a codon frequently used in E. coli K12, which encodes the same amino acid sequence as NAAAR. .

  The present invention also provides a recombinant vector comprising DNA encoding the NAAAR of the present invention. The recombinant vector of the present invention is not particularly limited as long as it can be replicated and maintained or autonomously propagated in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors and viral vectors. The recombinant vector is simply a known cloning vector or expression vector available in the technical field, and the DNA encoding the NAAAR is converted into an appropriate restriction enzyme and ligase, or, if necessary, a linker or adapter DNA. Can be prepared by ligation using In addition, gene fragments amplified using a DNA polymerase that adds a single base to the amplification end, such as Taq polymerase, can be connected to a vector by TA cloning.

  Examples of the vector include Escherichia coli-derived plasmids such as pBR322, pBR325, pUC18, and pUC19. Examples of yeast-derived plasmids include pSH19 and pSH15. Examples of Bacillus subtilis-derived plasmids include pUB110, pTP5, and pC194. Moreover, bacteriophage such as λ phage, papovirus such as SV40, bovine papilloma virus (BPV), retrovirus such as Moloney murine leukemia virus (MoMuLV), adenovirus (AdV), adeno-associated virus (AAV), Illustrative are animal and insect viruses such as vaccinia virus, baculovirus.

In particular, the present invention provides a NAAAR expression vector in which DNA encoding NAAAR is placed under the control of a promoter functional in the intended host cell. As a vector to be used, a promoter region that functions in various prokaryotic and / or eukaryotic host cells and can control transcription of a gene located downstream thereof (for example, trp promoter when the host is E. coli, When the host is Bacillus subtilis, such as lac promoter, lectA promoter, etc. When the host is a yeast, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. SV40-derived early promoter, MoMuLV-derived long terminal repeat, adenovirus-derived early promoter, and the like, and a transcription termination signal of the gene, that is, a terminator region, The terminator region is not particularly limited as long as it is linked via a sequence containing at least one restriction enzyme recognition site, preferably a unique restriction site that cleaves the vector only at that site. It further contains a selection marker gene for selection of transformants (a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, a gene that complements auxotrophic mutations, etc.) It is preferable. Further, when the DNA encoding NAAAR to be inserted does not contain a start codon and a stop codon, the start codon (ATG or GTG) and the stop codon (TAG, TGA, TAA) are respectively located downstream of the promoter region and the terminator region. A vector contained upstream of is preferably used.
When a bacterium is used as a host cell, the expression vector generally needs to contain a replicable unit capable of autonomous replication in the host cell in addition to the promoter region and terminator region described above. The promoter region includes an operator and a Shine-Dalgarno (SD) sequence in the vicinity of the promoter.
When yeast, animal cells, or insect cells are used as the host, the expression vector preferably further includes an enhancer sequence, 5 'and 3' untranslated regions of NAAAR mRNA, a polyadenylation site, and the like.

  Host organisms into which the prepared recombinant vector is introduced include bacteria such as Escherichia coli and Bacillus subtilis for which recombinant expression systems have been established, microbial hosts such as actinomycetes, bacilli, and yeast, insect cells, animal cells, higher plants, etc. Among them, it is preferable to use Escherichia coli having excellent protein expression ability. As a method for introducing the recombinant plasmid, in addition to introduction by electroporation, introduction by heat shock is possible if the host is made competent by chemical treatment such as calcium chloride. Selection for the presence or absence of transfer of the target recombinant plasmid into the host vector may be performed by searching for a microorganism that simultaneously expresses a marker typified by various drug resistance genes of the vector holding the target DNA and NAAAR activity, For example, a microorganism that grows in a selective medium based on a drug resistance marker and expresses NAAAR may be selected.

  Examples of other vectors, promoters, and host cells that can be applied to the DNA encoding NAAAR of the present invention are preferably those described in Patent Documents 2 and 3, for example.

  The NAAAR of the present invention can be produced by culturing a transformant containing the NAAAR expression vector prepared as described above in a medium and recovering NAAAR from the resulting culture.

  The medium to be used preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large Examples include soybean cake and potato extract. Further, other nutrients [for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.)] may be included as desired. .

Culturing is performed by methods known in the art. Specific media and culture conditions used according to the host cells are exemplified below, but the culture conditions in the present invention are not limited to these.
When the host is a bacterium, actinomycetes, yeast, filamentous fungus or the like, for example, a liquid medium containing the above nutrient source is suitable. Preferably, the medium has a pH of 5-9. When the host is Escherichia coli, preferred media include LB medium, M9 medium [Miller. J., Exp. Mol. Genet, p. 431, Cold Spring Harbor Laboratory, New York (1972)] and the like. Culturing can be performed usually at 14 to 43 ° C. for about 3 to 72 hours with aeration and stirring as necessary. When the host is Bacillus subtilis, it can be carried out usually at 30 to 40 ° C. for about 16 to 96 hours with aeration and stirring as necessary. When the host is yeast, examples of the medium include Burkholder's minimal medium [Bostian. KL et al, Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)], and the pH is 5-8. desirable. The culture is usually carried out at about 20 to 35 ° C. for about 14 to 144 hours, and if necessary, aeration and stirring can be performed.
When the host is an animal cell, examples of the medium include minimal essential medium (MEM) containing about 5 to 20% fetal calf serum [Science, 122, 501 (1952)], Dulbecco's modified Eagle medium (DMEM) [Virology, 8 , 396 (1959)], RPMI 1640 medium [J. Am. Med. Assoc., 199, 519 (1967)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)] and the like. Can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually carried out at about 30 to 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed.
When the host is an insect cell, examples of the medium include Grace's medium containing fetal calf serum [Proc. Natl. Acad. Sci. USA, 82, 8404 (1985)], and the pH is about 5-8. Is preferred. The culture is usually performed at about 20 to 40 ° C. for 15 to 100 hours, and if necessary, aeration and stirring can be performed.

Purification of NAAAR can be performed by appropriately combining various commonly used separation techniques depending on the fraction in which NAAAR activity is present.
NAAAR present in the culture medium is obtained by centrifuging or filtering the culture to obtain a culture supernatant (filtrate), from which, for example, salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration By appropriately selecting a known separation method such as non-denaturing PAGE, SDS-PAGE, ion exchange chromatography, hydroxylapatite chromatography, affinity chromatography, reverse phase high performance liquid chromatography, isoelectric focusing, etc. Obtainable.

  On the other hand, NAAAR present in the cytoplasm collects cells by centrifuging or filtering the culture and suspending it in an appropriate buffer, such as sonication, lysozyme treatment, freeze-thawing, osmotic shock, and / or triton. -After disrupting (dissolving) the cells and the organelle membrane by treatment with a surfactant such as -X100, the debris is removed by centrifugation or filtration to obtain a soluble fraction. It can be isolated and purified by treating with a method.

As a means of obtaining recombinant NAAAR quickly and conveniently, an amino acid sequence (for example, a base such as histidine, arginine, lysine, etc.) that can be adsorbed to a metal ion chelate at a certain part (preferably N or C terminal) of the coding sequence of NAAAR A DNA sequence encoding an amino acid sequence (preferably a sequence consisting of histidine) is added by genetic manipulation and expressed in a host cell, and the metal ion chelate is immobilized from the NAAAR activity fraction of the culture of the cell. A method of separating and recovering NAAAR by affinity with the prepared carrier is preferably exemplified. The DNA sequence encoding the amino acid sequence that can be adsorbed to the metal ion chelate is obtained by, for example, using a hybrid primer in which the DNA sequence is linked to the base sequence encoding the C-terminal amino acid sequence of NAAAR in the process of cloning the DNA encoding NAAAR. The DNA can be introduced into the NAAAR coding sequence by performing PCR amplification or by inserting the NAAAR-encoding DNA in frame into an expression vector containing the DNA sequence before the stop codon. The metal ion chelate adsorbent used for purification contains transition metals such as cobalt, copper, nickel, iron divalent ions, or iron, aluminum trivalent ions, etc., preferably cobalt or nickel divalent ions. The solution is prepared by contacting a ligand, such as an iminodiacetic acid (IDA) group, a nitrilotriacetic acid (NTA) group, a tris (carboxymethyl) ethylenediamine (TED) group, etc., in contact with the ligand. The matrix part of the chelate adsorbent is not particularly limited as long as it is a normal insoluble carrier.
Alternatively, affinity purification can be performed using glutathione-S-transferase (GST), maltose binding protein (MBP), HA, FLAG peptide, or the like as a tag.

  In the purification step, treatment such as membrane concentration, concentration under reduced pressure, addition of an activator and a stabilizer can be performed as necessary. In particular, since the present NAAAR is excellent in heat resistance, heating treatment within a range in which other host cell-derived contaminating proteins can be heat-denatured and can retain NAAAR activity is effective in greatly improving NAAAR purity. The solvent used in these steps is not particularly limited, but various buffer solutions represented by K-phosphate buffer solution, Tris-HCl buffer solution, GOOD buffer solution and the like having a buffer capacity in the range of about pH 6-9 are preferable. .

  When the NAAAR thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein is obtained as a salt, a method known per se Alternatively, the salt can be converted into a free form or other salt by a method analogous thereto.

  The purified enzyme is liquid and can be used for industrial use, but can also be pulverized or further granulated. The liquid enzyme is pulverized by lyophilization by a conventional method.

Furthermore, the NAAAR of the present invention can also be synthesized by in vitro translation using a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc., using RNA corresponding to the DNA encoding it as a template. can do. Whether the RNA encoding the NAAAR of the present invention is purified from the host cell expressing the RNA using the conventional method, as described above in the method for obtaining the cDNA encoding the NAAAR of the present invention. Alternatively, it can be obtained by preparing cRNA using a DNA encoding NAAAR as a template and using a cell-free transcription system containing RNA polymerase. The cell-free protein transcription / translation system may be a commercially available one, or a method known per se, specifically, Escherichia coli extract is obtained from Pratt JM et al., “Transcription and Tranlation”, Hames BD and Higgins SJ eds., IRL Press, Oxford 179-209 (1984). Examples of commercially available cell lysates include E. coli S30 extract system (Promega) and RTS 500 Rapid Tranlation System (Roche), which are derived from E. coli. Rabbit Reticulocyte Lysate System is derived from rabbit reticulocytes. (Promega), and wheat germ-derived ones include PROTEIOS ^™ (TOYOBO). Of these, those using wheat germ lysate are preferred. A method for producing wheat germ lysate is described in, for example, Johnston FB et al., Nature, 179: 160-161 (1957) or Erickson AH et al., Meth. Enzymol., 96: 38-50 (1996). The method can be used.

As a system or apparatus for protein synthesis, there are a batch method [Pratt, JM et al. (1984) mentioned above] and a continuous cell-free protein synthesis system that continuously supplies amino acids and energy sources to a reaction system [Spirin AS et al., Science, 242: 1162-1164 (1988)], dialysis (Kigawa et al., 21st Japan Molecular Biology Society, WID6), or multi-layer method (PROTEIOS ^TM Wheat germ cell-free protein synthesis core kit instruction manual: manufactured by TOYOBO). Furthermore, a method of supplying template RNA, amino acid, energy source and the like to the synthesis reaction system when necessary, and discharging the synthesized product or degradation product when necessary (Japanese Patent Laid-Open No. 2000-333673) can be used.

  The present invention also provides a process for producing a racemized N-acyl amino acid comprising contacting the NAAAR of the present invention with an N-acyl-D-amino acid or N-acyl-L-amino acid.

  The NAAAR of the present invention that can be used for the racemization reaction of N-acylamino acid is not limited to the purified enzyme provided by any of the above-mentioned methods, but also the treated product thereof, the NAAAR-producing cell of the present invention (natural Including both NAAAR-producing cells and transformants produced as described above) and processed products thereof.

  For example, the NAAAR processed product of the present invention includes, for example, NAAAR immobilized on an insoluble carrier by a known method. On the other hand, as a culture of NAAAR-producing cells of the present invention, when the enzyme is secreted extracellularly, the culture supernatant is expressed by intracellular cells when the enzyme is expressed extracellularly. Examples include supernatants obtained by crushing / filtration or centrifugation. Furthermore, the treated product of the culture is specifically the above-mentioned cell whose permeability of the cell membrane has been changed by treatment with an organic solvent such as a surfactant or toluene, or a cell in which bacterial cells have been disrupted by glass beads or enzyme treatment. Extracts and partially purified products are included.

Examples of the substrate N-acylamino acid that can be racemized by the method of the present invention include compounds represented by the following general formula (I).

General formula

(Wherein X represents an acyl derived from a carboxylic acid which may have a substituent, and R represents an alkyl having 1 to 20 carbons which may have a substituent)

Regarding the acyl group (X) of N-acyl-amino acid, carboxylic acids such as alkanoyl, benzoyl, arylalkanoyl and the like can be mentioned, and these acyl groups have substituents (halogen, C _1-3 alkyl, C _1-3 alkoxy, etc.) You may have. Examples of the alkanoyl include acetyl, formyl, chloroacetyl, propionyl and the like. Examples of benzoyl include benzoyl and p-chlorobenzoyl. Examples of the arylalkanoyl include phenylacetyl and phenylpropionyl.

Also, as alkyl represented by R, linear or branched alkyl, hydroxyalkyl, C _1-3 alkylthio, C _1-3 alkyl substituted with thiol, phenyl, hydroxyphenyl or indolyl and amino, carboxy, guanidyl or And C _1-4 alkyl substituted with imidazolyl and the like.

  Preferably, N-acylmethionine, N-acylvaline, N-acylleucine, N-acylphenylalanine, and N-acyltryptophan are applicable as the substrate N-acylamino acid. These N-acylamino acids may be L-form, D-form, or a mixture thereof.

The racemization reaction may be performed under the conditions that allow the NAAAR of the present invention to react. For example, the reaction temperature is 4 to 70 ° C., preferably 20 to 60 ° C., more preferably 30 to 50 ° C., pH 3 to 11, preferably pH 5 to 5. 10. It can be carried out at a substrate concentration of 0.01 to 50%. The substrate can be added all at once at the start of the reaction, but it is desirable to add the substrate continuously or discontinuously so that the substrate concentration in the reaction solution does not become too high. In addition, a divalent metal ion necessary for NAAAR to express its activity, for example, Co ²⁺ , Mn ²⁺ , Mg ²⁺ , Fe ²⁺ is added at 0.1 mM to 10 mM, preferably 1 to 5 mM. To do.

  The present invention further comprises contacting the NAAAR of the present invention with an N-acyl-D-amino acid, N-acyl-L-amino acid or mixture thereof in the presence of D- or L-aminoacylase. A method for producing an L-amino acid is provided. The form of NAAAR of the present invention is the same as in the case of the racemization method.

N-acyl-D-amino acid, N-acyl-L-amino acid or a mixture thereof as a substrate is prepared to an appropriate concentration, and NAAAR, L- (or D-) aminoacylase, and NAAAR are necessary for expressing the activity. Add divalent metal ions, and leave or stir under appropriate temperature and pH conditions. The substrate concentration and temperature or pH conditions are not particularly limited as long as NAAAR and aminoacylase are active, but more preferably the substrate concentration is 0.01% to 50%, and the pH is 5 to 10%. The temperature is 30 ° C. or higher. Further, the divalent metal ion to be added is not particularly limited as long as it functions to activate the NAAAR. Examples of such metal ions include Co ²⁺ , Mn ²⁺ , Mg ²⁺ , Fe ²⁺ may be mentioned. Of these, it is most preferable to use cobalt ions. The concentration at which these metal ions are added is not particularly limited as long as it is a concentration necessary for exhibiting the function as NAAAR, but is preferably 0.1 mM to 10 mM, more preferably 1 mM to 5 mM.

  In this state, if the added aminoacylase is L-aminoacylase, only L-form of N-acyl-D-amino acid and N-acyl-L-amino acid is first deacylated by L-aminoacylase. The desired L-amino acid is produced. NAAAR catalyzes both the reaction of converting the L-form of N-acylamino acid into the D-form and the reaction of transforming the D-form into the L-form, and works to make the ratio almost equal (racemization). Since the racemic state is eliminated when the body is consumed, the reaction of D-form → L-form is further promoted as the reaction of NAAAR. The N-acyl-L-amino acid thus produced is sequentially decomposed into L-amino acids by L-aminoacylase. In principle, all N-acylamino acids can be converted to L-amino acids on the principle. Similarly, if the aminoacylase added is D-aminoacylase, theoretically all N-acylamino acids can be converted to D-amino acids.

The method for producing an optically active amino acid according to the present invention can be carried out in a two-layer mixed system with water or an organic solvent or an aqueous medium that is difficult to dissolve in water.
Further, the reaction can be carried out in a reaction in an aqueous solution, an immobilized enzyme, an immobilized enzyme membrane reactor or the like. The present invention eliminates the need for a chemical racemization method and greatly reduces waste solvents and the like, which is effective in industrial amino acid production such as reduction of environmental burden.

  The N-acylamino acid used as the substrate is not particularly limited as long as the NAAAR can act, but more preferably N-acylmethionine, N-acylvaline, N-acylleucine, N-acylphenylalanine, N-acyltryptophan. Is applicable.

In the present invention, N-acylamino acid racemase activity is measured under the following conditions.
<Reagent>
0.1M HEPES buffer (pH 7.5)
50 mM N-acetyl-L-methionine (pH 7.5)
6.1 mg / ml 4-aminoantipyrine (Daiichi Chemical) solution 32.2 mg / ml TOOS (Dojin Chemical Laboratories) solution 300 U / ml peroxidase (Toyobo PEO-301) solution 20 U / ml D-amino acid oxidase (Biozyme DOX2) solution 1300 U / ml D-aminoacylase (in-house preparation) solution 50 mM cobalt chloride solution 7.5 ml of the above HEPES buffer solution, 2 ml of D-amino acid oxidase solution, 0.1 ml of 4-aminoantipyrine solution, TOOS solution 0 0.1 ml, 0.1 ml of peroxidase solution, 0.2 ml of D-aminoacylase solution, 9.2 ml of N-acetyl-L-methionine solution, and 0.8 ml of cobalt chloride solution are mixed to obtain a reaction reagent.

<Measurement conditions>
Prewarm 2.9 ml of reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 ml of NAAAR solution and mixing gently, the absorbance change at 555 nm is recorded with a spectrophotometer controlled at 37 ° C. with water as a control, and the absorbance change per minute is measured from the linear region. In the blind test, a solvent that dissolves NAAAR is added to the reagent mixture instead of the NAAAR solution, and the change in absorbance per minute is similarly measured. The amount of D-methionine produced per minute is calculated based on a calibration curve prepared in advance using a D-methionine standard solution. The enzyme activity is calculated with the amount of NAAAR enzyme required to produce 1 micromole of D-methionine per minute under the above conditions as one unit.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to a following example.

<Example 1>
Design of N-acylamino acid racemase gene sequence and construction of recombinant plasmid The NAAAR-like protein sequence derived from Chloroflexus aurantiacus is the amino acid sequence of NAAAR protein derived from Amycolatopsis sp. TS-1-60 on the NCBI-BLAST website. Is a sequence of a Hypothetical protein obtained as a result of a search for homologous proteins using as a key, and the homology with the TS-1-60 strain-derived NAAAR is 51%. The amino acid sequence is shown in SEQ ID NO: 2, and the base sequence of the gene encoding it is shown in SEQ ID NO: 1. First, all codons used from the nucleotide sequence of SEQ ID NO: 1 were listed. Next, data on the codon usage of Escherichia coli K12 strain was obtained from the Kazusa DNA Research Institute website, and the usage frequency in Escherichia coli was examined for all the codons in the above base sequence. Then, the less frequently used codons in E. coli were converted to more frequently used codons. The final nucleotide sequence of the gene designed by adjusting the overall codon usage so that there is no bias is as shown in SEQ ID NO: 3. A linear DNA having a sequence to which NdeI and BamHI sites are added upstream and downstream of this sequence is artificially produced by the method described in the cell engineering separate volume “Plant PCR Experiment Protocol” (p84-89, published by Shujunsha). Was synthesized. The DNA molecule thus prepared was sufficiently digested with restriction enzymes NdeI and BamHI, and the digested product was purified using Toyobo's MagExtractor-PCR & Gel Clean Up- to remove the restriction enzyme. The obtained DNA fragment was mixed with pBluescriptII KSN +, which was similarly cleaved with a restriction enzyme and purified, and an equal amount of ligation reagent (Toyobo Ligation High) was added to the mixture and incubated at 16 ° C. for 30 minutes. Ligation was performed. The ligated product was transformed into Escherichia coli DH5α competent cells (Toyobo Competent High DH5α) by heat shock and grown on LB agar medium containing 50 μg / ml of ampicillin. The grown colonies were inoculated with a toothpick in 5 ml of LB liquid medium (containing 50 μg / ml of ampicillin) and cultured with shaking at 37 ° C., and the plasmid was recovered from the grown cells using MagExtractor-Plasmid- (manufactured by Toyobo). . The base sequence downstream of the lac promoter of the recovered plasmid was decoded, and it was confirmed that the inserted fragment had the same sequence as SEQ ID NO: 3 to obtain an expression plasmid pCFNAR.

<Example 2>
Production of transformed microorganism and production of NAAAR The plasmid pCFNAR obtained in Example 1 was transformed into Escherichia coli DH5α strain, and the grown colonies were added to a 100 ml seed medium (0.5% polypeptone) in a 500 ml Sakaguchi flask. , 0.25% yeast extract, 0.5% NaCl, 0.5% glucose, 50 μg / ml ampicillin, pH 7.4), cultured at 30 ° C. for 16 hours at 180 rpm with shaking, and seed culture solution It was. Of the obtained seed culture, 60 ml of 500 ml of production medium (2.4% soybean peptide, 2.4% yeast extract, 1.25% dipotassium hydrogen phosphate, 0.23) in a 2 L Sakaguchi flask % Potassium dihydrogen phosphate, 0.5% glucose, 0.5% maltose, 50 μg / ml ampicillin, pH 7.2), and cultured at 37 ° C. for 48 hours at 180 rpm with shaking. After completion of the culture, the NAAAR activity of the culture was measured and found to be 1.1 U / ml. The culture solution was put into a 500 ml centrifuge tube and centrifuged at 4 ° C. and 8000 rpm for 20 minutes using a high-speed cooling centrifuge (CR21E manufactured by Hitachi, Ltd.). After centrifugation, the supernatant was removed by decantation to obtain bacterial cells. The bacterial cells thus obtained were suspended in 20 mM Tris-HCl buffer (pH 7.5) and crushed using a French press crusher. A 0.7% solution of 3% polyethyleneimine was added to the microbial cell disruption solution, stirred for 30 minutes at room temperature with a magnetic stirrer, and then centrifuged in the same manner as at the time of collection to obtain a supernatant. Further, while stirring this solution with a magnetic stirrer, ammonium sulfate was added and dissolved so as to be saturated to 0.45, and left at room temperature for about 20 minutes. This was centrifuged, and the supernatant was removed by decantation to obtain a precipitate containing the target protein. This precipitate was redissolved in 20 mM Tris-HCl buffer (pH 7.5). This salting-out / re-dissolved solution was desalted by gel filtration using G-25 Sepharose (manufactured by Amersham Biosciences) buffered with 20 mM Tris-HCl buffer (pH 7.5). Further, this solution was adsorbed on DEAE-Sepharose buffered with 20 mM Tris-HCl buffer (pH 7.5), gradient elution with NaCl concentration of 0 to 0.4 M was performed, and finally gel filtration with G-25 Sepharose. The purified protein was obtained by desalting. The specific activity of purified NAAAR (NAAAR activity per unit protein amount) was measured and found to be 0.6 U / mg.

<Example 3>
When SDS-PAGE of molecular weight purified NAAAR was performed using a PhastSystem fully automatic electrophoresis apparatus (manufactured by Amersham Biosciences), a single band was formed by Coomassie blue staining, and the molecular weight was estimated to be about 39,000. . The molecular weight calculated from the amino acid sequence was estimated to be 40,597, which was a result almost in agreement with this.

<Example 4>
Thermostability A solution in which the NAAAR was dissolved in a 20 mM Tris-HCl buffer (pH 7.5) at a protein concentration of 6 mg / ml (active, 3.6 U / ml) was used as a sample. The sample was heated at 37 ° C., 42 ° C., 50 ° C., 60 ° C., and 70 ° C. for 1 hour, and the temperature stability was determined by measuring the NAAAR activity remaining after the treatment. The results are shown in FIG. The enzyme was hardly inactivated even at 60 ° C., and maintained an activity of about 50% in the 70 ° C. treatment as compared with that before the treatment. Known NAAAR has a temperature stability of about ≦ 50 ° C., and it has been confirmed that heat stability is improved by adding magnesium ions in NAAAR derived from the genus Amycolatopsis. Completely deactivated. It was shown that the NAAAR of the present invention has excellent thermal stability compared to known NAAAR.

<Example 5>
pH stability NAAAR solutions were prepared in the pH range of 4 to 10, and the residual activity after incubation at 25 ° C. for 24 hours under each pH condition was examined. The results are shown in FIG. From this result, the stable pH region is 5 or more.

<Example 6>
Metal ion dependent divalent cobalt, copper, iron, manganese, magnesium, barium, zinc NAAAR activity was measured in the presence of 2 mM final concentration. The results are shown in Table 1. Similar to the known NAAAR, the highest NAAAR activity was exhibited in the presence of cobalt ions. NAAAR activity was also exhibited in the presence of iron, manganese and magnesium. On the other hand, NAAAR activity was not detected in the presence of copper, barium and zinc.

<Example 7>
As substrate specificity substrates, N- acetyl -L- methionine and N- acetyl -D- methionine, N- acetyl -L- valine and N- acetyl -D- valine, N- acetyl -L- leucine and N- acetyl - D-leucine, N-acetyl-L-phenylalanine and N-acetyl-D-phenylalanine, N-acetyl-L-tryptophan and N-acetyl-D-tryptophan, and similar N-formyl-L-methionine and N-formyl -D-methionine, N-formyl-L-valine, N-formyl-L-leucine, N-formyl-L-phenylalanine and N-formyl-D-phenylalanine, N-formyl-L-tryptophan and N-formyl-D -For tryptophan, 10 ml of 1% substrate solution (pH 7.5) In, it was added cobalt chloride NAAAR and 1mmol of 1U reacted overnight. It confirmed by measuring optical purity by HPLC method. The optical resolution column was SUMICHIRAL OA-5000 (Sumika Chemical Analysis Service, Ltd.), and 2 mM copper sulfate 10% isopropyl alcohol aqueous solution was used as an eluent, and measurement was performed at a temperature of 50 ° C., a flow rate of 1 ml, and a detection of 254 nm. The results are shown in Table 2. Like the known NAAAR, it has the highest activity against N-acetyl-methionine and N-formyl-methionine, but N-acetyl-valine and N-formyl-valine, N-acetyl-leucine and N-formyl-leucine, N- It also showed activity against acetyl-phenylalanine and N-formyl-phenylalanine, N-acetyl-tryptophan, and L-form and D-form acylamino acids of N-formyl-tryptophan. In addition, the free form L and D-amino acids showed no activity.

<Example 8>
Racemization reaction of N-acyl-amino acid with NAAAR A reaction solution containing NAAAR1U, 1 mM cobalt acetate in 1% N-acetyl-L-methionine and N-acetyl-D-methionine (pH 7.5) at 45 ° C. overnight. Reacted. The reaction solution was confirmed by HPLC using an optically active column. The optical resolution column was SUMICHIRAL OA-5000 (Sumika Chemical Analysis Service, Ltd.), and 2 mM copper sulfate 10% isopropyl alcohol aqueous solution was used as an eluent, and measurement was performed at a temperature of 50 ° C., a flow rate of 1 ml, and a detection of 254 nm. As a result of measuring the optical purity, N-acetyl D, L-methionine was confirmed in the respective reaction liquids of N-acetyl-L-methionine and N-acetyl-D-methionine, and the optical purity was almost 0% ee and the racemate. Met.

<Example 9>
Production method of L-amino acid by NAAAR and L-aminoacylase 100 ml of a reaction solution containing NAUAR100U and L-aminoacylase (Amano Enzyme) 1000 U in 20% N-acetyl-D, L-valine (pH 9.0) , Reacted at 45 ° C. overnight. As the reaction progressed, a supersaturated portion of L-valine formed was precipitated. The precipitated L-valine was separated by filtration, washed with water and dried, and L-valine could be obtained in a yield of 75%. The optical purity of the obtained L-valine was confirmed by HPLC method using an optically active column. The optical resolution column was SUMICHIRAL OA-5000 (Sumika Chemical Analysis Service, Ltd.), and the measurement was performed at a temperature of 50 ° C., a flow rate of 1 ml, and a detection of 254 nm using a 2 mM copper sulfate 5% isopropyl alcohol aqueous solution as an eluent. As a result, the produced L-valine had an optical purity of almost 100% ee.
The NAAAR obtained in the present invention was significant in both yield and quality in industrial L-amino acid production.

<Example 10>
Production method of D-amino acid by NAAAR and D-aminoacylase 100 ml of a reaction solution containing 20% N-acetyl-D, L-methionine (pH 9.0), NAAAR100U and D-aminoacylase (Amano Enzyme) 1000U , Reacted at 45 ° C. overnight. As the reaction proceeded, a supersaturated portion of the produced D-methionine precipitated. The precipitated D-methionine was separated by filtration, washed with water and dried, and D-methionine could be obtained in a yield of 75%. The optical purity of the obtained D-methionine was confirmed by an HPLC method using an optically active column. The optical resolution column was SUMICHIRAL OA-5000 (Sumika Chemical Analysis Service, Ltd.), and the measurement was performed at a temperature of 50 ° C., a flow rate of 1 ml, and a detection of 254 nm using a 2 mM copper sulfate 5% isopropyl alcohol aqueous solution as an eluent. As a result, the produced D-methionine had an optical purity of almost 100% ee.
The NAAAR obtained in the present invention was significant in both yield and quality in industrial D-amino acid production.

  The optically active α-amino acid produced according to the present invention can be used as a food additive or a nutritional supplement component, and can be supplied as an intermediate for producing active ingredients of various pharmaceutical preparations.

C. It is a figure which shows the thermal stability in NAAAR activity of the E. coli recombinant protein which consists of the same amino acid sequence as NAAAR derived from Aurantiacus. The vertical axis represents the residual NAAAR activity (relative activity after 1 hour heating treatment, where the activity before the heating treatment is 100), and the horizontal axis represents the treatment temperature (° C.). C. It is a figure which shows the pH stability in NAAAR activity of the E. coli recombinant protein which consists of the same amino acid sequence as NAAAR derived from Aurantiacus. The vertical axis represents the residual NAAAR activity (relative activity after 24 hours of heat treatment at 25 ° C., assuming that the activity before the heat treatment is 100); the horizontal axis represents the pH of the reaction solution.

Claims (18)

An isolated protein having N-acylamino acid racemase activity and having the following physicochemical properties:
(a) Thermal stability: N-acylamino acid racemase activity after heating at 70 ° C. for 1 hour
(b) Metal ion dependence: N-acylamino acid racemase activity in the presence of Co ²⁺ , Mn ²⁺ , Mg ²⁺ , Fe ²⁺   The protein according to claim 1, wherein the residual N-acylamino acid racemase activity after heating at 70 ° C for 1 hour is at least 30% or more. The protein according to claim 1 or 2, further having the following properties (c) to (e):
(c) Apparent molecular weight by SDS-PAGE: about 39 kDa
(d) pH stability: The residual N-acylamino acid racemase activity after heating at 25 ° C. for 24 hours at a pH of 5 or more is about 80% or more.
(e) Substrate specificity: N-acyl amino acid racemase activity against L and D forms of N-acetylated or N-formylated methionine, valine, leucine, phenylalanine and tryptophan An isolated protein having N-acyl amino acid racemase activity, substantially consisting of the following polypeptide (a) or (b):
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2
(b) an N-acyl amino acid racemase that includes an amino acid sequence having 60% or more identity and 80% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and that has been heated at 70 ° C. for 1 hour Polypeptide having activity   The protein according to claim 4, wherein the residual N-acylamino acid racemase activity after heating at 70 ° C for 1 hour is 30% or more.   6. The protein according to claim 4 or 5, comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2.   A nucleic acid encoding the protein according to any one of claims 1 to 6 (excluding a nucleic acid comprising the base sequence represented by SEQ ID NO: 1).   The nucleic acid according to claim 7 for introduction into a heterologous host cell, wherein the nucleic acid comprises a base sequence in which a less frequently used codon is replaced with a more frequently used codon in the host.   The nucleic acid according to claim 8, wherein the host is Escherichia coli.   The nucleic acid according to any one of claims 7 to 9, comprising the base sequence represented by SEQ ID NO: 3.   An expression vector wherein the nucleic acid encoding the protein according to any one of claims 1 to 6 is operably linked to a promoter suitable for a host cell into which the nucleic acid is introduced.   The expression vector according to claim 11, wherein the nucleic acid is one according to any one of claims 7 to 10.   A transformant obtained by transforming a host cell with the expression vector according to claim 11 or 12.   The transformant according to claim 13, wherein the host cell is Escherichia coli.   The method for producing a protein according to any one of claims 1 to 6, comprising culturing the transformant according to claim 13 or 14, and collecting a protein having N-acylamino acid racemase activity from the obtained culture. .   A method for producing a racemized N-acyl amino acid, which comprises contacting the protein according to any one of claims 1 to 6 with an N-acyl-D-amino acid or an N-acyl-L-amino acid.   Contacting the protein according to any one of claims 1 to 6 with N-acyl-D-amino acid, N-acyl-L-amino acid or a mixture thereof in the presence of D- or L-aminoacylase. A method for producing a D- or L-amino acid.   18. A method according to claim 16 or 17, wherein the amino acid is selected from the group consisting of methionine, leucine, valine, phenylalanine or tryptophan.

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