JP2001314191A - Method for racemizing N-acyl-amino acids and method for producing optically active amino acids - Google Patents

Abstract

[PROBLEMS] To provide a method for racemizing N-acyl-amino acids using a racemase having racemizing activity for a wide range of substrates, and a method for producing optically active amino acids. A racemization method using N-acylamino acid racemase (NAAR) derived from Sebekia benihana and a method for producing an optically active amino acid based on the method are provided. N
-Acylalanine, N-acyl-aspartic acid, N-
Acyl amino acids such as acyl-leucine or N-acyl-valine can also be used as a substrate to catalyze efficient racemization. Furthermore, when applied to the production of optically active amino acids, optically active amino acids useful as raw materials for pharmaceuticals and the like can be efficiently obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for racemizing N-acyl-amino acids by racemase, and further to a method for producing an optically active amino acid corresponding to each by reacting with L-aminoacylase or D-aminoacylase. .

[0002]

2. Description of the Related Art N-acyl-amino acid racemase (hereinafter referred to as N-acyl-amino acid racemase)
NAAR) is an enzyme that does not act on amino acids and specifically racemizes N-acyl-amino acids. Stre
An enzyme having NAAR activity has been found in actinomycetes such as the genus ptomyces, the genus Amycolatopsis, and the genus Sebekia (Japanese Patent No. 2712331). Amycolatopsis species TS- is also known as an acylamino acid racemase producing bacterium.
1-60 and a manufacturing method using the same (JP-A-6-2056)
68), a DNA fragment encoding an acylamino acid racemase derived from the strain (JP-A-4-365482)
It has been known. However, NAAR that has been isolated and purified and whose substrate specificity has been revealed is a Streptomyces
Only two enzymes derived from Species Y-53 and Amycolatopsis species TS-1-60. In all of these NAARs whose substrate specificity has been clarified, acyl amino acids that act substantially are limited. The activity of NAAR derived from Streptomyces species Y-53 is 50 or more with respect to N-acylleucine, N-acylphenylalanine, and N-acylvaline when the activity with respect to N-acylmethionine is 100, but N-
Less than 50 for acyltryptophan, N-acylalanine, N-acylaspartic acid. Also derived from Amycolatopsis species TS-1-60
The activity of NAAR indicates that the activity on N-acylmethionine is 1
00, N-acylphenylalanine, N-
It is 50 or more for acylvaline, but less than 50 for N-acyltryptophan, N-acylalanine, N-acylaspartic acid and N-acylleucine.
Furthermore, the present inventors have already found that Sebekia benihana
enihana) has been successfully isolated and expressed in recombinant form (Abstracts of the Annual Meeting of the Society of Biotechnology, 1999, p.166). However, although it is known that S. benihana has N-acyl-amino acid racemase activity on N-acyl-L-methionine, there is no specific knowledge on the substrate specificity of the enzyme.

[0003] Racemization of acyl amino acids is an important step in the production of optically active amino acids. Enzymes not only have a high catalytic function, but also exhibit stereospecificity as well as substrate specificity and reaction specificity. The stereospecificity of the enzyme is almost absolute, with some exceptions. By the way, with the refinement of research in recent years, medicines, pesticides,
The importance of handling optically active substances in fields such as feeds and flavors is increasing. Since optical isomers may have completely different physiological activities, techniques for specifically obtaining them are important. For example, in thalidomide, the D (R) form has no teratogenicity, but the L (S) form has strong teratogenicity.
The practical use of the racemate caused thalidomide to cause phytotoxicity. Furthermore, when only one of the enantiomers exhibits an effective bioactivity, the coexistence of both causes not only the problem that the other isomer does not have any activity, but also a competitive inhibition on the effective enantiomer. . As a result, the biological activity of the racemate may be drastically reduced to less than 1/2 that of the effective enantiomer. Therefore, obtaining (synthesizing or resolving) an optically pure enantiomer is an important industrial issue.

[0004] To solve this problem, a method of synthesizing a racemic body and then effectively resolving it has been widely used. However, in the method based on the division after the synthesis, an enantiomer that is not intended is always synthesized as a by-product, so that there remains a problem in terms of effective utilization of the raw materials. Even if the recovered by-product is recycled as a raw material, the synthesis of a fixed amount of by-product is always repeated. Therefore, optical resolution by an enzymatic method that does not generate by-products and a large amount of waste liquid has attracted attention. The optical resolution by the enzymatic method is a method for specifically generating a necessary enantiomer using the specificity of an enzyme. Since synthesis of unnecessary enantiomers can be suppressed to a low level, a product having excellent optical purity can be easily obtained. It is also advantageous in terms of effective utilization of raw materials. Regardless of whether optical resolution is used or a specific enantiomer is specifically synthesized enzymatically, the action of racemase is useful for synthesizing a racemate as a substrate. That is, NAAR is required to catalyze the racemization of acyl amino acids.

[0005] For example, D-tryptophan is one of the important D-amino acids as a raw material for pharmaceuticals. D-tryptophan can be obtained by deacylation of N-acyl-DL-tryptophan. However, a racemase that can racemize N-acyltryptophan to efficiently catalyze N-acyl-DL-tryptophan is not known. Similarly, in the case of acyl amino acids such as N-acylalanine, N-acyl-aspartic acid, N-acyl-leucine, or N-acyl-valine, a racemase that can catalyze efficient racemization using these as a substrate is unknown.

[0006]

SUMMARY OF THE INVENTION The present invention relates to a method for racemizing N-acyl-amino acids using a racemase having racemizing activity on a wide range of substrates, and a method for producing optically active amino acids using this method. Providing is an issue.

[0007]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have conducted a search for a racemase which is effective for a wide range of substrates. And NAAR derived from Sebekia benihana reported earlier by the present inventors
Have a substrate specificity that can be used for industrial racemization of N-acyl-amino acids, and completed the present invention. That is, the present invention relates to a method for racemizing an N-acyl-amino acid with NAAR having the following specificity of a substrate, and a method for producing an optically active amino acid based on the racemization method. [1] In a method for racemizing an N-acyl-amino acid, comprising the step of allowing a racemase or a processed product thereof to act on an optically active N-acyl-amino acid and racemizing the N-acyl-amino acid, the racemase comprises the following ( A racemization method comprising an N-acyl-amino acid racemase comprising the protein according to any one of (a) to (c). (A) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (B) In the amino acid sequence of SEQ ID NO: 2, 1
Or several amino acids are substituted, deleted, inserted, and / or
Or a protein comprising an added amino acid sequence and having the physicochemical properties described in (1) and (2) below. (C) a protein encoded by a polynucleotide that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and a physicochemical property described in the following (1) and (2) N with
A protein having an acyl-amino acid racemase activity. (1) Action: acts on N-acyl-amino acid to cause racemization. (2) Substrate specificity: among N-acyl-amino acids, especially N-acylalanine, N-acyl-aspartic acid,
N-acyl-leucine, N-acyl-valine, and N-acyl-tryptophan have at least 50 or more relative activities to N-acyl-amino acids of N-acyl-methionine, respectively. [2] A racemase-producing bacterium or a processed product thereof is treated with an optically active N
A method of racemizing an N-acyl-amino acid, the method comprising the step of acting on an acyl-amino acid to racemize the N-acyl-amino acid, wherein the racemase-producing bacterium is one of the following (a) to (d): A racemization method, characterized in that the transformant is a transformant that retains a protein encoded by the polynucleotide described in an expressible manner. (A) a polynucleotide comprising the protein coding region in the nucleotide sequence of SEQ ID NO: 1; (B) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2; (C) a polynucleotide which hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and which has the physicochemical properties described in (1) and (2) below. -A polynucleotide encoding a protein having amino acid racemase activity. (D) consists of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2, and has the physicochemical properties described in (1) and (2) below. A polynucleotide encoding a protein having a property of N-acyl-amino acid racemase activity. (1) Action: acts on N-acyl-amino acid to cause racemization. (2) Substrate specificity: among N-acyl-amino acids, especially N-acylalanine, N-acyl-aspartic acid,
N-acyl-leucine, N-acyl-valine, and N-acyl-tryptophan have at least 50 or more relative activities to N-acyl-amino acids of N-acyl-methionine, respectively. [3] N-acyl-amino acid is N-acylalanine,
-Acyl-aspartic acid, N-acyl-leucine, N
The method for racemizing an N-acyl-amino acid according to [1] or [2], which is at least one N-acyl-amino acid selected from the group consisting of -acyl-valine and N-acyl-tryptophan. [4] In the presence of D- or L-aminoacylase, N
D), wherein the racemization method described in [1] or [2] is performed using an acyl-DL-amino acid as a substrate.
Or a method for producing an L-amino acid.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides a method for racemizing N-acyl-amino acids by NAAR comprising a protein described in any of the following (a) to (c). (A) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (B) In the amino acid sequence of SEQ ID NO: 2, 1
Or several amino acids are substituted, deleted, inserted, and / or
Or a protein comprising an added amino acid sequence and having the physicochemical properties described in (1) and (2) below. (C) a protein encoded by a polynucleotide that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and a physicochemical property described in the following (1) and (2) N with
A protein having an acyl-amino acid racemase activity. (1) Action: acts on N-acyl-amino acid to cause racemization. (2) Substrate specificity: among N-acyl-amino acids, especially N-acylalanine, N-acyl-aspartic acid,
N-acyl-leucine, N-acyl-valine, and N-acyl-tryptophan have at least 50 or more relative activities to N-acyl-amino acids of N-acyl-methionine, respectively.

The NAAR having the amino acid sequence of SEQ ID NO: 2 can be isolated from Sebekia benihana. S.benihana is, for example, IFO 14
Available as 309. More specifically, Sb
enihana is cultured by a known method, and N
AAR can be purified. For example, after crushing the cells,
Protamine sulfate precipitation is performed, and the centrifuged supernatant is salted out using ammonium sulfate, and further purified by a combination of anion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration and the like. S.benihana IFO 143
The NAAR purified from 09 has the following physicochemical and enzymatic properties (3) to (6). (3) Molecular weight: about 440 molecular weight by SDS-PAGE
00, molecular weight about 340000 by gel filtration (4) Inhibitor: Inhibited by PCMB. (5) Range of optimal temperature for operation: The optimal temperature is 40-60 ° C. (6) Stable pH range: stable in the pH range of 7.5 to 10.

[0010] As the NAAR in the present invention, a recombinant obtained by expressing a polynucleotide encoding the NAAR can also be used. The NAAR in the present invention includes a NAAR consisting of the protein described as (b) or (c) above. NA purified from S.benihana IFO 14309
A protein consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and / or added to the amino acid sequence of AR (SEQ ID NO: 2) is a NAAR consisting of the amino acid sequence of SEQ ID NO: 2. It is likely to show an enzyme activity close to. Therefore, those skilled in the art routinely select a NAAR mutant that can be used in the present invention from a protein having such an amino acid sequence.

[0011] Furthermore, a protein encoded by a polynucleotide that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, wherein the protein is a physicochemical compound described in (1) or (2) above. A protein having specific properties can be used as NAAR in the present invention. A protein encoded by a polynucleotide capable of hybridizing under stringent conditions to a DNA encoding NAAR isolated from S. benihana IFO 14309 includes a protein having an enzyme activity similar to that of the NAAR. Are included. NA that can be used in the method of the present invention from among these enzymes
Selecting AR mutants is a routine matter for those skilled in the art.

In selecting the NAAR mutant of the present invention, N-
Racemase activity for acyl-amino acids can be measured as follows. Tris-HCL buffer (pH 7.5
-50 mM), a reaction solution containing cobalt chloride-1.0 mM, N-acyl-amino acid-20 mM and an enzyme was reacted at 30 ° C. for 5 minutes,
The reaction was stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, 3U
Of L-aminoacylase was added and reacted at 30 ° C. again for 1 hour. One hour later, the reaction was stopped by heating at 100 ° C. for 3 minutes. After stopping the reaction, centrifuge (15000 rpm, 10 minutes, 4 ° C)
Then, the amino acids generated by the TNBS method were measured. 1U
Is the amount of enzyme capable of racemizing 1 μmol of N-acyl-amino acid per minute. The protein is quantified by a dye binding method using a Bio-Rad protein assay kit. The TNBS method was performed as follows. First, add 0.5 ml of 100 mM Na ₂ B to a sample containing 0.5 ml of amino acids.
₄ O ₇ was added, and 20 μl of 110 mM TNBS (Trinitrobenz
enesulfonic acid) solution and stirred quickly. After 5 minutes, a 100 mM NaHPO ₄ containing 1.5mM of Na ₂ SO ₃ was added 2 ml, to stop the color reaction and the absorbance was measured at 420 nm.

The polynucleotide which can be hybridized under stringent conditions for obtaining the mutant of the NAAR of the present invention is defined as a probe DNA, for example, ECL
direct nucleic acid labeling and detection system
(Amersham Pharmaica Biotech) using the conditions described in the manual (wash: 42 ° C, primar containing 0.5x SSC)
ywash buffer). As the probe DNA, a DNA obtained by selecting one or a plurality of any at least 20, preferably at least 30, for example, 40, 60 or 100 continuous sequences in the base sequence described in SEQ ID NO: 1 Can be used.

The nucleotide sequence shown in SEQ ID NO: 1 is S. beniha
na This is the nucleotide sequence of DNA encoding NAAR isolated from IFO 14309. Such DNA can be obtained by PCR using a genomic library or cDNA library of S. benihana as a template. Primers required for PCR can be designed by those skilled in the art based on the nucleotide sequence shown in SEQ ID NO: 1. Alternatively, it can be obtained by screening a genomic library or a cDNA library with a DNA probe having a base sequence selected from this base sequence. Methods for cloning a gene by PCR or hybridization of a probe are known.

That is, a microorganism belonging to the genus Sevecchia and capable of producing N-acyl-amino acid racemase is cultured,
The spheroplasts are converted to spheroplasts by a cell wall degrading enzyme and used in a usual method (for example, J. Biol. Chem. 268, 26212-26219 (1993), Me.
th. Cell. Biol. 29, 39-44 (1975))
Is prepared. As the microorganism, S. benihana IFO 14309 can be used. The prepared chromosomal DNA is completely or partially digested with a suitable restriction enzyme (HindIII, EcoRI, BamHI, Sau3AI, etc.) to obtain a DNA fragment of about 2 to 8 kb. This genomic DNA is subjected to Southern hybridization using a probe designed based on SEQ ID NO: 1, to form a hybrid with a target DNA fragment. The plurality of hybridized DNA fragments are recovered and introduced into an E. coli expression vector. Escherichia coli (such as Escherichia coli JM109 strain) is transformed with the obtained recombinant plasmid to prepare a genomic library. Expression vectors include pUC18 (Takara Shuzo), pKK223-3
(Manufactured by Pharmacia), pET derivatives (such as Takara Shuzo), or pMAL-p2 (manufactured by NEB) can be used. Positive colonies are obtained from the genomic library by colony hybridization using the same probe.
Plasmids are extracted from positive colonies and cut with restriction enzymes to obtain insert DNA. The target gene can be confirmed by performing Southern hybridization with the same probe and forming a hybrid between the insert DNA and the probe. Further, by determining the base sequence of the insert and comparing it with the base sequence shown in SEQ ID NO: 1, it can be confirmed that the target gene has been isolated. The obtained DNA is integrated into an appropriate expression vector, and transformed into a host cell, whereby a highly expressing strain of NAAR encoded by the DNA can be obtained. Such a highly expressed strain can be used for producing an optically active amino acid according to the present invention.

[0016] NAAR can be purified from a strain highly expressing NAAR. That is, the high expression strain is cultured under conditions that induce NAAR expression, and the enzyme gene is expressed. Cultured cells can be collected and crushed, and the resulting supernatant can be used as a crude enzyme solution for the enzyme reaction. Alternatively, purified NAAR can be obtained by the method described above. NA of crude enzyme solution
AR activity can be determined as follows. That is, L along with the crude enzyme solution and the substrate N-acetyl-D-methionine.
-N-acyl-amino acid racemase activity is measured by reacting at 30 ° C. for 5 to 60 hours in the presence of -aminoacylase and measuring the amount of L-methionine produced. 1 μmole per minute
The amount of the enzyme required to produce L-methionine is 1 U. Methionine formation was determined by thin-layer chromatography (TL
It can also be qualitatively confirmed by C).

The microorganism having N-acyl-amino acid racemase-producing ability used as a gene source at the time of cloning includes all strains, mutants, and strains belonging to the genus Seveccia having N-acyl-amino acid racemase-producing ability. Including variants. Among them, particularly preferred strains are Sebekia benihana.
Seeds and the like. In addition, an expression vector in E. coli, for example, pUC18, pKK223-3, pET, pMAL-p2, etc., using an open reading frame downstream of the promoter in the forward direction, N-acyl-amino acid racemase derived from the genus Seveccia It can be expressed directly or as a fusion protein.

The present invention is a method for racemizing N-acyl-amino acids using the above NAAR. The target enzyme reaction can be carried out by bringing the culture containing the NAAR or its highly expressing strain or the treated product thereof into contact with a reaction solution containing an N-acyl-amino acid. That is, the reaction can be carried out in an organic solvent hardly soluble in water or water, for example, an organic solvent such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, or a two-phase mixed system with an aqueous medium. The racemization method according to the present invention can be carried out using an immobilized enzyme, a membrane reactor, or the like. The contact form between the enzyme and the reaction solution is not limited to these specific examples.
The reaction solution is a solution obtained by dissolving a substrate in an appropriate solvent that provides a desired environment for the expression of enzyme activity. The processed product of the microorganism containing NAAR in the present invention is, specifically, a microorganism in which the permeability of the cell membrane is changed by treatment with an organic solvent such as a surfactant or toluene, or by crushing cells by treatment with glass beads or an enzyme. Includes cell-free extracts and partially purified ones. On the other hand, the processed material of NAAR is NA
It includes those in which AR is immobilized on an insoluble carrier by a known method. Note that NAAR in the present invention is not limited to a purified enzyme, and can be used as a crudely purified product or the like.

The N-acyl-amino acid in the method for racemizing an N-acyl-amino acid according to the present invention includes, for example, compounds represented by the following general formula (1). General formula (1) (In the formula, X represents an acyl derived from a carboxylic acid which may have a substituent, and R represents an alkyl having 1 to 20 carbon atoms which may have a substituent.)

Regarding the acyl group (X) of the N-acyl-amino acid, these acyl groups may have a substituent (halogen, alkyl, alkoxy, etc.), and include alkanoyl such as formyl, acetyl, chloroacetyl, benzoyl, Benzoyl such as p-chlorobenzoyl; acyl carboxylate such as arylalkanoyl such as phenylacetyl and phenylpropionyl. Also, R
Linear or branched alkyl, hydroxylalkyl, C1-3alkylthio,
C1-4 alkyl substituted with thiol, phenyl, hydroxyphenyl or indolyl and C1-4 alkyl substituted with amino, carboxyl, guanidyl or imidazolyl and the like can be mentioned. More specifically, N-acylalanine, N-acyl-aspartic acid, N-acyl-leucine, N-acyl-valine, and N-acyl-tryptophan, which are particularly reactive, are preferably used.

The racemization reaction of the present invention is carried out at a reaction temperature of 4-6.
The reaction can be carried out at 0 ° C., preferably 10-40 ° C., pH 3-11, preferably pH 6-9, at a substrate concentration of 0.01-90%, preferably 0.1-30%. Reaction yield is 50
It often progresses at -100%. The substrate can be added all at once at the start of the reaction, but it is preferable to add the substrate continuously or discontinuously so that the substrate concentration in the reaction solution does not become too high.

[0022] In addition, the present invention relates to a method for producing an optically active amino acid by combining the above NAAR with L-aminoacylase or D-aminoacylase. That is, the above-described NAAR, or a microorganism producing the enzyme or a processed product thereof is allowed to act on an N-acyl-amino acid, and then L-aminoacylase or D-aminoacylase is acted on, thereby obtaining an optically active reaction product. Amino acids can be produced. The method for producing an optically active amino acid according to the present invention comprises an organic solvent that is hardly soluble in water or water, for example, ethyl acetate, butyl acetate, toluene,
The reaction can be carried out in an organic solvent such as chloroform or n-hexane or in a two-phase mixed system with an aqueous medium.
The reaction of the present invention can be carried out using an immobilized enzyme, a membrane reactor, or the like. The racemization method according to the present invention is advantageous in industrial use because, for example, a residual raw material discarded in the production of an optically active amino acid using acylase can be reused.

The method for producing an optically active amino acid according to the present invention comprises a reaction temperature of 4 to 60 ° C., preferably 10 to 40 ° C., p.
H3-11, preferably pH 6-9, substrate concentration 0.01
It can be carried out at -90%, preferably 0.1-30%. The reaction yield often proceeds at 50-100%.
The substrate can be added all at once at the start of the reaction, but it is preferable to add the substrate continuously or discontinuously so that the substrate concentration in the reaction solution does not become too high. The resulting optically active amino acid can be purified by appropriately combining bacterial cells, protein, centrifugation, separation by membrane treatment, solvent extraction, crystallization, and the like. For example, in the case of D-tryptophan, a reaction solution containing microbial cells is centrifuged to remove the microbial cells, proteins are removed by ultrafiltration, the filtrate is dehydrated and concentrated, and the precipitate is separated by filtration. Can be easily purified.

[0024]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto. [Example 1] Cloning of N-acyl-amino acid racemase An oligonucleotide probe was prepared based on the base sequence of the N-acyl-amino acid racemase gene derived from Amycolatopsis sp. TS-1-60. Then, when Southern hybridization was performed on the chromosomal DNA of Sebekia benihana IFO14309 partially cut with five types of restriction enzymes using the prepared probe, a Sma I fragment of about 5 kbp and about 4.5 kbp were obtained.
It was confirmed that a hybrid was formed with the kbp SphI fragment. Then, a Sma I fragment of about 5 kbp was recovered and ligated to the Sma I site of plasmid vector pUC18.
The strain was transformed into 5α strain to prepare a genomic library.
As a result of performing colony hybridization on the genomic library using the same probe, one positive colony out of about 1000 colonies was obtained. As a result of extracting a plasmid from this colony and cutting with a restriction enzyme SmaI, an insert DNA of about 5 kbp was confirmed. When Southern hybridization was performed with the same probe, it was confirmed that the insert DNA of about 5 kbp and the probe formed a hybrid.

Next, the N-acyl-amino acid racemase gene was specified. First, the entire nucleotide sequence of the approximately 5 kbp Sma I chromosomal DNA fragment was determined by the dideoxy method. The actinomycete gene has a GC content of about 70%, which is extremely high among all species, and the characteristic that the GC content of the third letter of the codon is close to 100% makes it possible to determine the codon in the determined base sequence. The position of the ORF could be estimated by examining the region where the GC content of the third letter of the above became close to 100%. As a result of examining the GC content, three ORFs / ORFs were found in this fragment.
It was presumed that 1, ORF2 and ORF3 were present. One of them
A sequence showing homology to the oligonucleotide probe was found in two ORFs, ORF2, and ORF2 was found to be Amycolatopsis
It showed homology with the N-acyl-amino acid racemase gene from sp. TS-1-60. This ORF2 consists of 1104 bp, 36
It encoded eight amino acids. The initiation codon was ATG, and a sequence presumed to be an SD sequence was found upstream of the initiation codon. First, a primer having a restriction enzyme Nde I site at the start codon and a restriction enzyme Bgl II site downstream of the stop codon was prepared, and ORF2 was amplified by PCR. The obtained PCR-amplified fragment was digested with restriction enzymes Nde I and Bgl II, and ligated downstream of the T7 promoter of the expression vector pET-3c to prepare an expression vector pET-NR.

The prepared pET-NR was transformed into Escherichia coli BL21 (DE3) strain, and expression of ORF2 in the transformant was attempted. To avoid insolubilization of the expressed protein, the transformant was cultured at 28 ° C. under the induction of IPTG at a final concentration of 0.01 mM. The cultured cells were collected and disrupted, and the obtained supernatant was used as a crude enzyme solution for the enzyme reaction. For enzyme reaction, crude enzyme solution, substrate N-acetyl
L-aminoacylase coexists with -D-methionine,
The reaction was performed at 0 ° C. for 5 to 60 hours. In this reaction system, NAAR activity was measured by measuring the amount of L-methionine produced. The enzyme activity was defined as 1 U when 1 μmole of L-methionine was produced in 1 minute. In addition, in the case of only confirming the production of methionine, TLC was used. The amount of the generated amino acids was determined by the TNBS method. The TNBS method adds 0.5 ml of 100 mM Na ₂ B ₄ O ₇ to a sample containing 0.5 ml of amino acids,
To this, add 20 μl of 110 mM TNBS (Trinitrobenzenesulfonic
acid) solution and stirred quickly. After 5 minutes, 1.5 mM
2 ml of 100 mM NaHPO ₄ containing Na ₂ SO ₃ was added to stop the color reaction. The absorbance at 420 nm was measured.

Example 2 Purification of N-acyl-amino acid racemase from transformants <Method> Preparation of crude enzyme solution: E. coli transformants were incubated at 30 ° C. for 24 hours (final concentration 0.01 mM at 4 hours after the start of culture). (IPTG added). 50 g of the wet bacterial cells obtained by collecting the culture solution are mixed with 160 ml of 50 mM Tris-HC.
l (pH 7.5) and sonicated (190 W, 40 min) (200 ml). Centrifuge the cell lysate (18,000 rpm, 4
C., 30 mim), and 190 ml of the obtained supernatant was used as a crude enzyme solution. -Ammonium sulfate fractionation 1st: 80% saturated ammonium sulfate solution (in 50
mM Tris-HCl (pH 7.5)) and a final concentration of 25% saturation (13% W /
After V), it was left still at 4 ° C. for 16 hours. After 16h, centrifuge (8,
500 rpm, 4 ° C., 30 mim) to obtain 250 ml of the supernatant.

Butyl-Toyopearl 650M-1st time: 250 ml of the supernatant obtained from the ammonium sulfate fraction is equilibrated in advance with a 25% saturated (13% W / V) ammonium sulfate solution (in 50 mM Tris-HCl (pH 7.5)). 1
After passing through an 80 ml Butyl-Toyopearl 650M column and washing,
5 while adding ammonium sulfate concentration gradient (25% saturation → 0%)
The adsorbed protein was eluted with twice the volume (900 ml) of 50 mM Tris-HCl (pH 7.5). The fraction in which N-acyl-amino acid racemase activity was confirmed was collected (280 ml). -Ammonium sulfate fractionation-second time: The sample collected in the first time of Butyl-Toyopearl 650M was dialyzed to obtain a 385 ml sample.
80% saturated ammonium sulfate solution (in 50mM Tri
After adding s-HCl / pH7.5) to a final concentration of 25% saturation (13% W / V), the mixture was allowed to stand at 4 ° C. for 16 hours. After 16h, centrifuge (8,500rp
m, 4 ° C., 30 mim) to obtain 545 ml of supernatant. -Butyl-Toyopearl 650M 2nd time: Pass 545 ml of the supernatant obtained from the ammonium sulfate fractionation through a 180 ml Butyl-Toyopearl 650M column pre-equilibrated with a 25% saturated (13% W / V) ammonium sulfate solution.
After washing, apply a gradient (25% saturation → 0%)
The adsorbed protein was eluted with twice the volume (900 ml) of 50 mM Tris-HCl (pH 7.5). The fraction in which N-acyl-amino acid racemase activity was confirmed was collected (200 ml).

Superose 12HR Butyl-Toyopearl 650M The sample collected in the second round was dialyzed to obtain a 280 ml sample. Equilibrated in advance with 50 mM Tris-HCl (pH 7.5) containing 0.15 M sodium chloride
200 μl of this sample was passed through a Superose 12HR (flow rate 0.5 mim / ml). A 1.9 ml sample having N-acyl-amino acid racemase activity confirmed was obtained. Total activity and total protein content were converted to values for the total volume of 280 ml. Table 1 shows the outline of the above NAAR purification, and FIG. 1 and FIG. 2 (chromatograms by Butyl-Toyopearl 650M) showing the results of the purification.
And FIG. 3 (SDS-PAGE before and after purification). Butyl-To
After two purifications with yopearl 650M and a purification with Superose12HR, it was confirmed that NAAR could be finally purified as almost pure protein.

[0030]

[Table 1]

Example 3 Measurement of molecular weight of N-acyl-amino acid racemase 1. Measurement of molecular weight The molecular weight of NAAR was determined by (1) gel filtration and (2) SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Was measured. (1) Gel filtration method The solution was applied to a TSKgel G3000SWXL column (Tosoh, 7.8 mmID x 30 cm) equilibrated with a phosphate buffer (50 mM, pH 7.0) containing 0.2 M sodium chloride in advance, and 20 ml of the same buffer was used. Was eluted at a flow rate of 0.5 ml / min. The molecular weight markers include MW-Marker proteins (HPLC) (manufactured by Oriental Yeast): glutamate dehydrogenase (290,000), lactate dehydrogenase (142,000), enolase (6
7,000), myokinase (32,000), cytochrome C (1
2,400). As a result, the molecular weight of this enzyme was about 34
It was suggested to be 0,000 (FIG. 4).

(2) SDS-polyacrylamide gel electrophoresis The SDS-polyacrylamide gel electrophoresis is based on a Laemmli method (Laemmli, UK: Nature, 227, pp680) using a mini gel slab electrophoresis apparatus (manufactured by Nippon Aido). make use of,
Electrophoresis was performed. As the gel, a polyacrylamide gel (12%) was used. The sample was prepared in the same amount as the enzyme solution (4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2-ME, 0.005% BPB (Bromo phenol Blu
e) containing 125 mM Tris · HCl (pH 6.8) buffer), and heat at 100 ° C. for about 5 minutes using a block heater.
10 μl of the solution cooled to room temperature was subjected to electrophoresis. Detection was performed by a staining method using CBB-R. SDS-PAGE Molecular Weight Standards, Low Range
(Phosphorylase b: 97,400, albumin: 66,200, ovalbumin: 45,000, carbonic anhydrase:
31,000, trypsin inhibitor: 21,500, lysozyme: 14,400, manufactured by BIO RAD). As a result, NAAR
Was suggested to have a molecular weight of about 44,000 (FIG. 5). The molecular weight determined by gel filtration was about 340,000, suggesting that NAAR may be present as an octamer.

[Example 4] Method for measuring enzyme activity of N-acylamino acid racemase The activity of N-acylamino acid racemase was measured by the method described below. First, the following reaction solutions were prepared. N-acetyl-D-amino acid (100 mM) 100 µl (20 mM) Cobalt chloride (100 mM) 5 µl (1 mM) Tris-HCl (0.5 M / pH7.5) 50 µl (50 mM) Sterile distilled water 295 µl Total 450 µl 50 µl in this reaction solution Add 500 μl of enzyme solution to 30 ℃
5 minutes (several tens of minutes if the activity is low)
The reaction was stopped by heating at 3 ° C. for 3 minutes. After stopping the reaction, 3U L
-Aminoacylase was added, and the reaction was performed again at 30 ° C. for 1 hour. One hour later, the reaction was stopped by heating at 100 ° C. for 3 minutes.
After stopping the reaction, the generated L-methionine was measured by the TNBS method. The enzyme activity was defined as 1 unit (U) when 1 μmole of N-acetyl-L-methionine was generated from N-acetyl-D-methionine per minute.

Example 5 Optimum temperature and thermostability of N-acylamino acid racemase The optimum temperature for the action was 25 ° C. in the enzyme activity measurement method.
The temperature was changed to 7070 ° C., and L-methionine produced after the reaction was measured and determined. The thermostability of this enzyme was determined by heating the enzyme solution at a predetermined temperature for 30 minutes, immediately cooling it with ice, and measuring the remaining activity according to the enzyme activity measurement method. Enzyme activity is shown as relative activity with 100 at the case of 30 ° C for both the optimum temperature and the thermostability. The results are shown in FIG. 6 (optimal temperature) and FIG. 7 (thermal stability). The optimal temperature for NAAR was 40-60 ° C.

[Example 6] Optimum pH and pH stability of N-acylamino acid racemase The optimum pH for the action was determined as a buffer solution in the enzyme activity measurement method.
Using a bistris hydrochloride buffer solution (pH 5.0 to 7.0) and a Tris hydrochloride buffer solution (pH 7.0 to 10.0), the reaction was performed at 30 ° C. for 30 minutes to determine the activity. The pH stability is determined by using a buffer solution of the specified pH (optimal pH
Add the enzyme solution (20 times dilution) and leave it overnight
The enzyme activity was measured and determined according to the enzyme activity measurement method. Enzyme activities were shown as relative activities, with the case of pH 7.5 as 100. The results are shown in FIG. 8 (optimal pH) and FIG. 9 (pH stability). NAAR was stable in the pH range of 7.5 to 10.

[Example 7] Substrate specificity of N-acylamino acid racemase The substrate specificity was determined by changing the substrate in the reaction solution and measuring according to the enzyme activity measurement method. The enzymatic activity was shown as a relative activity with N-acetyl-D-methionine as 100. It was confirmed that NAAR acts on a wide range of N-acetyl-amino acids to have racemizing activity. In particular, N-acetylalanine, N-acetyl-aspartic acid, N-acetyl-leucine, N-acetyl-valine, N-acetyl-
It was confirmed that the relative activity of tryptophan with respect to each N-acetyl-amino acid was at least 50 when N-acetyl-methionine was defined as 100. Based on this substrate specificity, an enzymatic reaction as described below was constructed.

[Example 8] Influence of metal ions on N-acylamino acid racemase In the enzyme activity measurement method, various metal salts were added to the final concentrations except for cobalt chloride (final concentration 1 mM) added to the reaction solution.
The reaction was carried out at 30 ° C. for 5 minutes, and the produced L-methionine was measured to examine the influence of various metal ions on N-acylamino acid racemase. The enzyme activity was 100 when Co was added.
And expressed as relative activity. The results are shown in Table 2. Co had an activating effect.

[0038]

[Table 2]

[Example 9] Influence of N-acylamino acid racemase inhibitor In the enzyme activity measurement method, various enzyme inhibitors were added so that the final concentrations in the reaction solution became 1 mM, respectively, and the case where no enzyme inhibitor was added was controlled. At 30 ° C. for 10 minutes to examine the effect of the inhibitor on the enzyme. In addition, L- used during the reaction
Aminoacylase was added in excess so as not to be affected by the inhibitor. Enzyme activity was shown as a relative activity with the case of no addition as 100. For EDTA, the final concentration in the reaction solution is
5 mM was also examined. The results are shown in Table 3. NAAR
Was inhibited by Monoiodoacetic acid, PCMB and EDTA.

[0040]

[Table 3]

Example 10 Racemization of N-acyl-amino acid with N-acyl-amino acid racemase 100 mM potassium phosphate buffer (pH 6.5), 1 U of N-acyl-amino acid racemase, 0.5% N-acetyl −
The reaction was carried out at 30 ° C. overnight in a reaction solution containing L-tryptophan or N-acetyl-D-tryptophan. Racemization was confirmed by measuring the optical purity of tryptophan in the reaction solution by HPLC. The optical purity of N-acyl-tryptophan was determined as follows. N-acyl-tryptophan was extracted from the reaction solution with methyl ethyl ketone, desolvated, and analyzed by liquid chromatography using an optical resolution column. The optical resolution column is CHIRALPAKW manufactured by Daicel Chemical Industries, Ltd.
H was measured at room temperature using a 0.25 mM copper sulfate aqueous solution as an eluent. (Flow rate: 1 ml / min, detection: 254 nm) and the quantification and optical purity were measured.
-Acetyl-tryptophan is made of N-acetyl-L
In the case of -tryptophan and N-acetyl-D-tryptophan, the optical purity after the completion of the reaction was almost 0% ee and a racemic form.

Example 11 Production of D-amino acid using N-acyl-amino acid racemase and D-aminoacylase 100 mM potassium phosphate buffer (pH 6.5), 1 U of N-acyl-amino acid racemase, 1 U of D-aminoacylase 1
The reaction was carried out overnight at 30 ° C. in 100 ml of a reaction solution containing U and 15% N-acetyl-DL-tryptophan. As the reaction proceeded, the produced D-tryptophan saturated the aqueous layer, and the supersaturated portion was deposited in the aqueous layer, but the reaction continued. D-tryptophan precipitated in the aqueous layer was separated by filtration, washed with water and dried. (Yield 70%) The optical purity of the produced D-tryptophan was determined as follows. Using CHIRALPAKWH manufactured by Daicel Chemical Industries, Ltd., measurement was performed at room temperature using a 0.25 mM copper sulfate aqueous solution as an eluent. (Flow rate: 1m
1 / min, detection: 254 nm) As a result, D-tryptophan produced according to the present invention had an optical purity of almost 100% ee.

Example 12 Production of L-amino acid using N-acyl-amino acid racemase and L-aminoacylase 100 mM potassium phosphate buffer (pH 6.5), 1 U of N-acyl-amino acid racemase, L-aminoacylase (Sigma) The reaction was carried out overnight at 30 ° C. in 100 ml of a reaction solution containing 1 U of 15% N-acetyl-DL-tryptophan. As the reaction proceeded, the produced L-tryptophan saturated the aqueous layer, and the supersaturated portion precipitated in the aqueous layer, but the reaction continued. L-tryptophan precipitated in the aqueous layer was separated by filtration, washed with water and dried. (Yield 70%) The optical purity of the produced L-tryptophan was determined as follows. Using CHIRALPAKWH manufactured by Daicel Chemical Industries, Ltd., measurement was performed at room temperature using a 0.25 mM copper sulfate aqueous solution as an eluent. (Flow rate: 1 ml / min, detection: 254 nm) As a result, the optical purity of L-tryptophan produced by the present invention was almost 100% ee.

[0044]

The N-acyl-amino acid racemase which is advantageous for industrial production has been shown. By using this enzyme, a method for efficiently producing an optically active amino acid having high optical purity was provided by combining a method for racemizing an N-acyl-amino acid with D-aminoacylase or L-aminoacylase. Optically active amino acids are useful as intermediates in the manufacture of pharmaceuticals.

[0045]

[Sequence List] SEQUENCE LISTING <110> DAICEL Chemical Industries LTD. <120> Methods for Racemization of N-acyl-amino acid, and manufacturing of optical active amino acid. <130> D1-A0003Y1 <140> <141> <150 > JP 2000-60358 <151> 2000-03-01 <160> 2 <170> PatentIn Ver. 2.0 <210> 1 <211> 1104 <212> DNA <213> Sebekia benihana <220> <221> CDS <222 > (1) .. (1104) <400> 1 atg aaa atc acc gga gtg gag ctg cgc cgg atc gcg atg ccg ctg gtc 48 Met Lys Ile Thr Gly Val Glu Leu Arg Arg Ile Ala Met Pro Leu Val 1 5 10 15 gcg ccg ttc cgc acg tcg ttc ggc acc gag cac gac agg gac gtc ctc 96 Ala Pro Phe Arg Thr Ser Phe Gly Thr Glu His Asp Arg Asp Val Leu 20 25 30 ctg gtc aga gtg gtc acc ccc gat gcc gag ggg tgg tgc gtg 144 Leu Val Arg Val Val Thr Pro Asp Ala Glu Gly Trp Gly Glu Cys Val 35 40 45 gcg atg tcg gag ccg ctc tac tcc tcc gag tac gtc gac gga gcc gcc 192 Ala Met Ser Glu Pro Leu Tyr Ser Ser Glu Tyr Val Asp Gly Ala Ala 50 55 60 gcg gtg atc cgc cgc ttc ctg ctg ccc gcg ctg ccc gac aac gtc gac 240 Ala V al Ile Arg Arg Phe Leu Leu Pro Ala Leu Pro Asp Asn Val Asp 65 70 75 80 gcg tac ggc gtg ggc cac gcc ctc gag ccg atc aag ggc cac cgc atg 288 Ala Tyr Gly Val Gly His Ala Leu Glu Pro Ile Lys Gly His Arg Met 85 90 95 gcc aag gcg gcg ctg gag acg gcc gtg ctc gac gcc cag ctg agg gcc 336 Ala Lys Ala Ala Leu Glu Thr Ala Val Leu Asp Ala Gln Leu Arg Ala 100 105 110 tcg ggc gag tcg ttc ggc cac ggc gcc acg agg gac agg gtg 384 Ser Gly Glu Ser Phe Gly Gln Phe Leu Gly Ala Thr Arg Asp Arg Val 115 120 125 ccg tgc ggg gtg tcg gtc ggc atc atg gac tcc atc ccg cag ctg ctc 432 Pro Cys Gly Gly Ile Met Asp Ser Ile Pro Gln Leu Leu 130 135 140 gag gcg gtg gag ggc tat ctg gac gag ggc tac gtc agg atc aag ctg 480 Glu Ala Val Glu Gly Tyr Leu Asp Glu Gly Tyr Val Arg Ile Lys Leu 145 150 155 aag atc gag ccc ggc tgg gac gtc gag ccg gtg cgc gcg gtc agg gag 528 Lys Ile Glu Pro Gly Trp Asp Val Glu Pro Val Arg Ala Val Arg Glu 165 170 175 cgg ttc ggc gac gag gtg gg gcc tac 576 Arg Phe Gly Asp Glu Val Leu Leu Gln Val Asp Ala Asn Ala Ala Tyr 180 185 190 acc ctg gtc gac gcc cag cag ctg gcc agg ctc gac gac ttc ggc ctg 624 Thr Leu Val Asp Ala Gln Gln Leu Ala Asg Leu As Phe Gly Leu 195 200 205 ctg ctg atc gag cag ccg ctg gcg aac gac gac ctg gtg cag cac gcc 672 Leu Leu Ile Glu Gln Pro Leu Ala Asn Asp Asp Leu Val Gln His Ala 210 215 220 gag ctg gcc ag cgc acc ccg atc tgc ctg gat gag tcg atc 720 Glu Leu Ala Lys Arg Leu Arg Thr Pro Ile Cys Leu Asp Glu Ser Ile 225 230 235 240 gag tcg gcc gag cac gcg gcg gcc gcc atc tcg ctc aag gcc tgc Acg Glu Gc G768 Gc His Ala Ala Ala Ala Ile Ser Leu Lys Ala Cys Ser 245 250 255 atc gtc aac atc aag ccg ggc agg atc ggc gga tac ctg gag gcg cgg 816 Ile Val Asn Ile Lys Pro Gly Arg Ile Gly Gly Tyr Leu Glu Ala Arg 260 265 270 cgc atc cac gac ctg tgc agg gcg cac ggc atc gcg gtg tgg tgc ggg 864 Arg Ile His Asp Leu Cys Arg Ala His Gly Ile Ala Val Trp Cys Gly 275 280 285 ggc atg ctg gag acg ggc gc gc cc gc gc gc gc gc cccgcg ctg gcc 912 Gly Met Leu Glu Thr Gly Leu Gly Arg Ala Ala Asn Val Ala Leu Ala 290 295 300 gcg ctg ccc ggc ttc acc ctg ccg ggc gac acc tca ggc tcg cgc cgt 960 Ala Leu Pro Gly Phe Thr Thr Thr Ser Gly Ser Arg Arg 305 310 315 320 tac tac gcc acc gac atc acc gag ccc ttc gag ctc gac ggc ggc cac 1008 Tyr Tyr Ala Thr Asp Ile Thr Glu Pro Phe Glu Leu Asp Gly Gly His 325 330 335 335 ctc acc gtg ccg tcg ggc ccc ggc ctg ggc gtc gat ccc gtc aag gag 1056 Leu Thr Val Pro Ser Gly Pro Gly Leu Gly Val Asp Pro Val Lys Glu 340 345 350 atc ctc gag gag gtc acc acctcc acg gag tgg atc ccg ctc ggc tga Leu Glu Glu Val Thr Thr Ser Thr Glu Trp Ile Pro Leu Gly 355 360 365 <210> 2 <211> 367 <212> PRT <213> Sebekia benihana <400> 2 Met Lys Ile Thr Gly Val Glu Leu Arg Arg Ile Ala Met Pro Leu Val 1 5 10 15 Ala Pro Phe Arg Thr Ser Phe Gly Thr Glu His Asp Arg Asp Val Leu 20 25 30 Leu Val Arg Val Val Thr Pro Asp Ala Glu Gly Trp Gly Glu Cys Val 35 40 45 Ala Met Ser Glu Pro Leu Tyr Ser Ser Glu Tyr Val A sp Gly Ala Ala 50 55 60 Ala Val Ile Arg Arg Phe Leu Leu Pro Ala Leu Pro Asp Asn Val Asp 65 70 75 80 Ala Tyr Gly Val Gly His Ala Leu Glu Pro Ile Lys Gly His Arg Met 85 90 95 Ala Lys Ala Ala Leu Glu Thr Ala Val Leu Asp Ala Gln Leu Arg Ala 100 105 110 Ser Gly Glu Ser Phe Gly Gln Phe Leu Gly Ala Thr Arg Asp Arg Val 115 120 125 Pro Cys Gly Val Ser Val Gly Ile Met Asp Ser Ile Pro Gln Leu Leu 130 135 140 Glu Ala Val Glu Gly Tyr Leu Asp Glu Gly Tyr Val Arg Ile Lys Leu 145 150 155 160 Lys Ile Glu Pro Gly Trp Asp Val Glu Pro Val Arg Ala Val Arg Glu 165 170 175 Arg Phe Gly Asp Glu Val Leu Leu Gln Val Asp Ala Asn Ala Ala Tyr 180 185 190 Thr Leu Val Asp Ala Gln Gln Leu Ala Arg Leu Asp Asp Phe Gly Leu 195 200 205 Leu Leu Ile Glu Gln Pro Leu Ala Asn Asp Asp Leu Val Gln His Ala 210 215 220 Glu Leu Ala Lys Arg Leu Arg Thr Pro Ile Cys Leu Asp Glu Ser Ile 225 230 235 240 Glu Ser Ala Glu His Ala Ala Ala Ala Ile Ser Leu Lys Ala Cys Ser 245 250 255 Ile Val Asn Ile Lys Pro Gly Arg Ile Gly Gly Tyr Leu Glu Ala Arg 260 265 270 Arg Ile His Asp Leu Cys Arg Ala His Gly Ile Ala Val Trp Cys Gly 275 280 285 Gly Met Leu Glu Thr Gly Leu Gly Arg Ala Ala Asn Val Ala Leu Ala 290 295 300 Ala Leu Pro Gly Phe Thr Leu Pro Gly Asp Thr Ser Gly Ser Arg Arg 305 310 315 320 Tyr Tyr Ala Thr Asp Ile Thr Glu Pro Phe Glu Leu Asp Gly Gly His 325 330 335 Leu Thr Val Pro Ser Gly Pro Gly Leu Gly Val Asp Pro Val Lys Glu 340 345 350 Ile Leu Glu Glu Val Thr Thr Ser Thr Glu Trp Ile Pro Leu Gly 355 360 365

[Brief description of the drawings]

FIG. 1 is a first chromatogram of Butyl-Toyopearl 650M of a crude enzyme solution obtained from Escherichia coli transformed with a NAAR expression vector. The vertical axis (left) shows the absorbance at 280 nm, the vertical axis (right) shows the (NH ₄ ) ₂ SO ₄ concentration (%), and the horizontal axis shows the fraction number.

FIG. 2 is a second chromatogram of crude enzyme solution obtained from Escherichia coli transformed with a NAAR expression vector using Butyl-Toyopearl 650M. The vertical axis (left) shows the absorbance at 280 nm, the vertical axis (right) shows the (NH ₄ ) ₂ SO ₄ concentration (%), and the horizontal axis shows the fraction number.

FIG. 3 is a photograph showing a result of SDS-PAGE in a purification process of a crude enzyme solution obtained from Escherichia coli transformed with a NAAR expression vector. Lane 1: Crude enzyme solution (supernatant of cell lysate), Lane 2: First time of Butyl-Toyopearl 650M, Lane 3: Second time of Butyl-Toyopearl 650M, Lane 4: Supe
rrose 12HR, M: molecular weight marker

FIG. 4 is a graph showing the results of molecular weight measurement by gel filtration. The vertical axis indicates molecular weight (kDa), and the horizontal axis indicates elution volume (ml).

FIG. 5 is a graph showing the results of molecular weight measurement by SDS-PAGE. The vertical axis indicates the molecular weight (kDa), and the horizontal axis indicates the relative mobility.

FIG. 6 is a graph showing an analysis result of an optimum temperature of NAAR. The vertical axis indicates relative activity (%), and the horizontal axis indicates temperature.

FIG. 7 is a graph showing an analysis result of thermal stability of NAAR. The vertical axis indicates relative activity (%), and the horizontal axis indicates temperature.

FIG. 8 is a graph showing the results of analyzing the optimum pH of NAAR.
The vertical axis indicates relative activity (%), and the horizontal axis indicates pH.

FIG. 9 is a graph showing an analysis result of pH stability of NAAR. The vertical axis indicates relative activity (%), and the horizontal axis indicates pH.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:19) C12N 15/00 ZNAA F-term (Reference) 4B024 AA03 BA71 BA75 CA03 CA09 DA06 GA11 4B050 CC03 DD02 EE10 LL01 LL05 4B064 AE03 AE04 AE05 AE06 AE17 AE34 CA02 CA21 CB30 CC03 CD12 DA01 DA16

Claims (4)

[Claims] (1) A method for producing racemase or a processed product thereof using an optically active N
A method of racemizing an N-acyl-amino acid, the method comprising the step of acting on an acyl-amino acid to racemize the N-acyl-amino acid;
A racemization method comprising an N-acyl-amino acid racemase comprising the protein according to any one of (c). (A) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (B) In the amino acid sequence of SEQ ID NO: 2, 1
Or several amino acids are substituted, deleted, inserted, and / or
Or a protein comprising an added amino acid sequence and having the physicochemical properties described in (1) and (2) below. (C) a protein encoded by a polynucleotide that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and a physicochemical property described in the following (1) and (2) N with
A protein having an acyl-amino acid racemase activity. (1) Action: acts on N-acyl-amino acid to cause racemization. (2) Substrate specificity: among N-acyl-amino acids, especially N-acylalanine, N-acyl-aspartic acid,
N-acyl-leucine, N-acyl-valine, and N-acyl-tryptophan have at least 50 or more relative activities to N-acyl-amino acids of N-acyl-methionine, respectively. 2. A racemase-producing bacterium or a treated product thereof is allowed to act on an optically active N-acyl-amino acid to produce the N-acyl-amino acid.
In a method for racemizing an N-acyl-amino acid comprising a step of racemizing an amino acid, the racemase-producing bacterium holds a protein encoded by the polynucleotide according to any one of the following (a) to (d) so that it can be expressed: Racemization method, characterized in that it is a transformed transformant. (A) a polynucleotide comprising the protein coding region in the nucleotide sequence of SEQ ID NO: 1; (B) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2; (C) a polynucleotide which hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and which has the physicochemical properties described in (1) and (2) below. -A polynucleotide encoding a protein having amino acid racemase activity. (D) consists of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2, and has the physicochemical properties described in (1) and (2) below. A polynucleotide encoding a protein having a property of N-acyl-amino acid racemase activity. (1) Action: acts on N-acyl-amino acid to cause racemization. (2) Substrate specificity: among N-acyl-amino acids, especially N-acylalanine, N-acyl-aspartic acid,
N-acyl-leucine, N-acyl-valine, and N-acyl-tryptophan have at least 50 or more relative activities to N-acyl-amino acids of N-acyl-methionine, respectively. 3. An at least one N-acyl-amino acid selected from the group consisting of N-acylalanine, N-acyl-aspartic acid, N-acyl-leucine, N-acyl-valine, and N-acyl-tryptophan. N-
The N- according to claim 1 or 2, which is an acyl-amino acid.
A racemization method for acyl-amino acids. 4. The method according to claim 1, wherein an N-acyl-DL-amino acid is used as a substrate in the presence of D- or L-aminoacylase.
Or a method for producing a D- or L-amino acid, which comprises performing the racemization method described in 2.