JP2015213506A - D-succinylase, and method for producing d-amino acid using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel D-succinylase which has high hydrolytic activity on N-succinyl-D-amino acid and is capable of producing D-amino acid with high yield on an industrial scale and in a period of time suitable for commercial production when combined with succinyl amino acid racemase.SOLUTION: Disclosed is a protein which is represented by any one of the following (a) to (d): (a) a protein that is encoded by a gene consisting of a specific base sequence; (b) a protein consisting of a specific amino acid sequence; (c) a protein encoded by a gene consisting of a base sequence having 90% or more of identity with the specific base sequence, and has D-succinylase activity; and (d) a protein consisting of an amino acid sequence produced by substituting, deleting, inserting and/or adding one or several amino acids in the protein consisting of the specific amino acid sequence, and has D-succinylase activity.

Description

  The present invention relates to a protein having an activity of deacylating N-succinyl-D-amino acid to produce D-amino acid (hereinafter referred to as D-succinylase), which can be used for the production of D-amino acid as a raw material for intermediates such as pharmaceuticals Called). The present invention also relates to a method for efficiently producing D-amino acid from N-succinyl-DL-amino acid by using such D-succinylase in combination with N-succinyl amino acid racemase.

  Optically active amino acids have many demands in the fields of pharmaceuticals, agricultural chemicals, foods and the like, but D-amino acids are difficult to obtain as optically pure amino acids, particularly products (natural amino acids) such as fermentation methods. Therefore, how to efficiently produce D-amino acids is an important industrial issue.

  In response to this problem, a method has been developed in which a racemate of N-acetylamino acid is synthesized and the D form in the racemate is selectively hydrolyzed by an enzymatic method. This method is a method in which N-acetyl-DL-amino acid is hydrolyzed using the specificity of an enzyme such as D-aminoacylase to specifically produce only the necessary D-amino acid, and is excellent in optical purity. Products can be easily obtained (Patent Documents 1, 2, and 3).

  However, in order to improve the yield of D-amino acid by this method, half of the racemate, that is, N-acetyl-L-amino acid remaining in the system without reacting with D-aminoacylase is chemically racemic. It needs to be converted to N-acetyl-DL-amino acid and used again as a raw material. Therefore, an enzyme having a racemase action that catalyzes the racemization of N-acetyl-L-amino acid is useful. For example, D-tryptophan, which is important as a pharmaceutical raw material, can be obtained by hydrolyzing N-acetyl-DL-tryptophan with D-aminoacylase. Then, N-acetyl-L-tryptophan that did not react with D-aminoacylase and remained in the system was racemized again with racemase, and N-acetyl-D-tryptophan in the generated racemate was converted with D-aminoacylase. Hydrolyzes. By continuously performing these two steps as a series of enzymatic reactions, D-tryptophan can be obtained in high yield (Patent Document 4).

  N-acyl amino acid racemase is known as a racemase effective for such use. This enzyme does not act on amino acids that are not N-acyl modified, and is an enzyme that specifically racemates N-acyl amino acids. It is considered that D-amino acid can be obtained in a high yield by reacting a chemically synthesized N-acetylamino acid racemate with D-aminoacylase and N-acylamino acid racemase.

  However, this method has a great obstacle to practical use. This is because the reported reactivity of N-acylamino acid racemase to N-acetylamino acid is extremely low, so that D-amino acid cannot be obtained in high yield on an industrial scale and time.

  In contrast, N-acylamino acid racemase using N-succinylamino acid as a substrate, that is, succinylamino acid racemase, has been identified, and by using this enzyme, efficient N-succinylamino acid on an industrial scale and time can be obtained. It has been reported that racemization has become possible (Patent Documents 5 and 6, Non-Patent Document 1).

  On the other hand, as for the acylase reaction using N-succinylamino acid as a substrate, although it has been known that a conventionally known D-aminoacylase can slightly hydrolyze N-succinyl-D-amino acid (Patent Document 5), N Compared with the case where -acetyl-D-amino acid was used as a substrate, the hydrolysis activity was remarkably low. Therefore, even when a conventionally known D-aminoacylase is combined with succinyl amino acid racemase, it was not possible to obtain D-amino acid in high yield on an industrial scale and time.

JP 2002-320491 A JP 2006-254789 A WO2004 / 055179 JP 2001-46088 A JP 2008-061642 A JP 2008-307006 A

Sakai A.I. et al. , Biochemistry, 2006, 45 (14), 4455-62.

  The present invention was devised in view of the current state of the prior art, and its purpose is to have a high N-succinyl-D-amino acid hydrolyzing activity, and in combination with succinyl amino acid racemase, the industrial scale, time Is to provide a novel D-succinylase capable of producing a D-amino acid in high yield.

In order to achieve the above object, the present inventor screened various soil microorganisms using N-succinyl-D-amino acid hydrolyzing ability as an index. As a result, the present inventor has a novel ability to hydrolyze N-succinyl-D-amino acid. A novel microorganism (Cupriavidus sp. P4-10-C) was found, and the novel microorganism produced a novel D-succinylase. Furthermore, the novel D-succinylase gene was successfully cloned and D-succinylase was also successfully engineered by DNA recombination.
In addition, based on the above knowledge, the present inventors have also eagerly searched for related species of this microorganism deposited in public institutions, and hydrolyzed N-succinyl-D-amino acid from among them. It was found that the substance has an excellent ability to do so, and that the deposited microorganism produces a novel D-succinylase. Furthermore, these novel D-succinylase genes were successfully cloned and D-succinylase was also successfully produced by DNA recombination. And based on these knowledge, the present inventors completed this invention.

That is, according to the present invention, a protein represented by any one of (a) to (d) is provided:
(A) a protein encoded by a gene consisting of the base sequence set forth in SEQ ID NO: 2, 4, 6 or 8;
(B) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 3, 5 or 7;
(C) a protein encoded by a polynucleotide that hybridizes with a base sequence complementary to the base sequence set forth in SEQ ID NO: 2, 4, 6 or 8 under stringent conditions and has D-succinylase activity;
(D) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5 or 7, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and D-succinylase Protein with activity.

In addition, according to the present invention, there is provided a gene characterized by being represented by any one of (a) to (d):
(A) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 2, 4, 6 or 8;
(B) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5 or 7;
(C) a gene that hybridizes with a base sequence complementary to the base sequence set forth in SEQ ID NO: 2, 4, 6 or 8 under a stringent condition and encodes a protein having D-succinylase activity;
(D) consisting of a base sequence corresponding to the amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5 or 7; And a gene encoding a protein having D-succinylase activity.

  Further, according to the present invention, a step of preparing a recombinant vector by inserting the gene into a vector, transforming a host cell with the recombinant vector, preparing a transformant, and culturing the transformant A method for producing the protein is provided.

  Furthermore, according to the present invention, a step of hydrolyzing N-succinyl-D-amino acid in N-succinyl-DL-amino acid using the above protein, and N-succinyl-L-- using N-succinyl amino acid racemase There is provided a method for producing a D-amino acid comprising the step of racemizing an amino acid to produce N-succinyl-D-amino acid.

  Since the D-succinylase of the present invention has a significantly higher hydrolysis activity of N-succinyl-D-amino acid than the conventional enzyme, the efficiency of N-acyl amino acid can be improved by using it in combination with a known succinyl amino acid racemase. Racemization and hydrolysis are possible. Therefore, according to the present invention, it is possible to obtain a D-amino acid useful as a pharmaceutical raw material in a high yield on an industrial scale and time.

FIG. 1 shows Cupriavidus Sp. It is a figure which shows the electrophoresis image by the electrophoresis method of D-succinylase which P4-10-C strain | stump | stock produces. FIG. 2 shows Cupriavidus Sp. It is a figure which shows the residual relative activity at the time of temperature stability measurement of D-succinylase which P4-10-C strain | stump | stock produces. FIG. 3 shows Cupriavidus Sp. It is a figure which shows the relative activity at the time of the optimal temperature measurement of D-succinylase which P4-10-C strain | stump | stock produces. FIG. 4 shows Cupriavidus Sp. It is a figure which shows the relative activity at the time of the optimal pH measurement of D-succinylase which P4-10-C strain | stump | stock produces. FIG. 5 shows the D-succinylase activity of various D-succinylases. FIG. 6 is a graph showing the residual relative activity when measuring the temperature stability of D-succinylase produced by a transformant. FIG. 7 is a diagram showing the relative activity of the D-succinylase produced by the transformant when measuring the optimum temperature. FIG. 8 is a graph showing the residual relative activity when measuring the pH stability of D-succinylase produced by a transformant. FIG. 9 is a graph showing the relative activity of the D-succinylase produced by the transformant when measuring the optimum pH. FIG. 10 is a diagram showing the hydrolysis rate during D-succinylase reaction on N-succinyl-DL-phenylalanine. FIG. 11 is a figure which shows the hydrolysis rate at the time of D-succinylase and succinyl amino acid racemase reaction with respect to N-succinyl-DL-phenylalanine. FIG. 12 is a figure which shows the hydrolysis rate at the time of D-succinylase and succinyl amino acid racemase reaction with respect to N-succinyl-DL-tryptophan. FIG. 13 is a figure which shows the hydrolysis rate at the time of D-succinylase and succinyl amino acid racemase reaction with respect to N-succinyl-DL-biphenylalanine.

  The D-succinylase of the present invention is (a) a protein encoded by a gene consisting of a base sequence described in SEQ ID NO: 2, 4, 6 or 8, or (b) a protein described in SEQ ID NO: 1, 3, 5 or 7. A protein consisting of an amino acid sequence. SEQ ID NO: 2 is a novel soil microorganism Cupriavidus sp. It is the base sequence of D-succinylase of P4-10-C strain, and SEQ ID NO: 1 is its amino acid sequence. SEQ ID NOs: 4, 6, and 8 are Cupriavidus sp. It is the base sequence of D-succinylase of the relative species of P4-10-C strain. Specifically, SEQ ID NO: 4 is from Cupriavidus metallidurans, SEQ ID NO: 6 is from Cupriavidus nekator, and SEQ ID NO: 8 is from Ralstonia picketi Is the base sequence. SEQ ID NOs: 3, 5, and 7 are amino acid sequences corresponding to the base sequences of SEQ ID NOs: 4, 6, and 8, respectively. In addition, some of the above-mentioned bacteria belonging to the genus Cupriavidus are described in the literature as bacteria belonging to the genus Ralstonia, but the genus Cupriavidus has begun to be generally used as an official genus name. The scientific names of these bacteria may be unified or changed due to future reclassification.

  The above proteins (a) and (b) have high hydrolytic activity for N-succinyl-D-amino acid, which is D-form of N-succinylamino acid, and D-amino acid is efficiently generated. It has the feature that can be made. The above proteins (a) and (b) have significantly higher activity against N-succinylamino acids than N-acetylamino acids. From these facts, the proteins (a) and (b) described above are enzymes that catalyze the reaction of efficiently hydrolyzing N-succinyl-D-amino acid to produce D-amino acid and succinic acid, that is, D- It can be said to be a succinylase.

  The physicochemical properties of the D-succinylase of the present invention are as shown in the following (i) to (vi).

(I) Action: Hydrolyzes N-succinyl-D-amino acid to produce D-amino acid.
(Ii) Subunit structure: a heterodimer containing subunits having molecular weights of 50 to 70 kdalton and 20 to 30 kdalton in SDS-polyacrylamide gel electrophoresis.
(Iii) Substrate specificity: acts on any N-succinyl-D-amino acid represented by the following general formula (I).
(In general formula (I), R represents an aryl group having 4 to 20 carbon atoms which may have a substituent, or an aralkyl group having 5 to 20 carbon atoms which may have a substituent.)
Examples of the aryl group having 6 to 20 carbon atoms which may have a substituent in R include a phenyl group and a 4-hydroxyphenyl group. Examples of the substituent include an amino group, hydroxyl group, nitro group, cyano group, carboxyl group, alkyl group, aralkyl group, aryl group, alkanoyl group, alkenyl group, alkynyl group, alkoxyl group, or halogen atom. Moreover, it does not specifically limit as a C7-20 aralkyl group which may have a substituent, For example, a benzyl group, an indolylmethyl group, 4-phenylbenzyl group, 4-hydroxybenzyl group etc. Can be mentioned.
(Iv) Temperature stability: When heat-treated for 60 minutes with enzyme concentration (0.2 mg / mL) and phosphate buffer solution (pH 7.5), the residual activity is 90% or more at 50 ° C.
(V) Optimal temperature: When reacting with an enzyme concentration (0.01 mg / mL) and a phosphate buffer solution (pH 7.5), a temperature of 45 to 50 ° C. is optimal. Optimum means that the enzyme concentration (0.01 mg / mL) and phosphate buffer solution (pH 7.5) are reacted at 30 ° C, 40 ° C, 45 ° C, 50 ° C, and 60 ° C for 5 minutes each. The temperature range in which the relative value is 80% or more when the activity value measured at the temperature at which the activity was highest is 100% is shown.
(Vi) Optimal pH: pH 7-8 is optimal when the reaction is performed at an enzyme concentration (0.01 mg / mL) at 37 ° C. for 60 minutes. Note that “optimum” means enzyme concentration (0.01 mg / mL), phosphate buffer solution (pH 5.5-pH 7.5), Tris-HCl (pH 7.5-pH 8.5), boric acid. Using a buffer solution (pH 8.5-pH 10.0) and reacting at 37 ° C. for 60 minutes, the activity value measured at the pH at which the activity was highest was 100%, and the relative value was 80% or more. Indicates a temperature range.

  The present invention relates to (a) a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 2, 4, 6 or 8, and (b) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5 or 7. Is also included. These are genes corresponding to the above proteins (a) and (b).

  The D-succinylase of the present invention is not limited to those of the above (a) and (b), and (c) a base sequence complementary to the base sequence described in SEQ ID NO: 2, 4, 6 or 8 and stringent 1 or several amino acids in a protein encoded by a polynucleotide that hybridizes under conditions and having D-succinylase activity, or (d) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5 or 7 Includes a protein having an amino acid sequence substituted, deleted, inserted and / or added and having D-succinylase activity. The gene of the present invention hybridizes under stringent conditions with a nucleotide sequence complementary to (c) the nucleotide sequence set forth in SEQ ID NO: 2, 4, 6 or 8, and has D-succinylase activity. A gene encoding a protein, or (d) an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 3, 5 or 7 A gene comprising a corresponding base sequence and encoding a protein having D-succinylase activity is also included. This is often a functionally equivalent protein even if a part of the base sequence of the gene encoding the protein is mutated, and as a result, part of the amino acid sequence of the protein is mutated. Because. In addition, when the D-succinylase gene of the present invention is incorporated into a host organism (such as Escherichia coli) other than the source organism to express the D-succinylase of the present invention, the codon usage of the host organism is used to improve the expression efficiency. This is because the base sequence may be changed together.

  Here, “one or several” is a range that does not significantly impair the three-dimensional structure of the protein of amino acid residues and D-succinylase activity, and specifically 1 to 50, preferably 1 to 30. More preferably, it is 1-20, More preferably, it is 1-10. However, in the case of an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1, 3, 5 or 7 in the sequence listing 50% or more, preferably 80% or more, more preferably 90% or more of the protein having the amino acid sequence described in SEQ ID NO: 1, 3, 5 or 7 in the sequence listing under the conditions of 30 ° C. and pH 7-8 Preferably, 95% or more of D-succinylase activity is retained.

  “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, for example, highly homologous DNAs, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95%. The conditions in which DNAs having the above homology hybridize and DNAs having lower homology do not hybridize to each other (here, homology is the maximum number of bases that match between the sequences to be compared). It is desirable that the values be calculated in such a way that they are arranged in such a manner), or 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C., 0.1, which is a washing condition for normal Southern hybridization X SSC, 0.1% SDS, more preferably at 65 ° C, 0.1 x SSC, 0.1% SDS Conditions, and the like. However, in the case of a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence described in SEQ ID NO: 2, 4, 6 or 8 in the sequence listing, at 30 ° C. and pH 7-8 D-succinylase activity of 50% or more, preferably 80% or more, more preferably 90% or more, and still more preferably 95% or more of the protein having the amino acid sequence described in SEQ ID NO: 1, 3, 5 or 7 in the Sequence Listing It is desirable to hold

  The proteins having the D-succinylase activity and the gene thereof (c) and (d) are, for example, Transformer Mutagenesis Kit; manufactured by Clonetech; EXOIII / Mung Bean Deletion Kit; manufactured by Stratagene; manufactured by QuickChange Kit; -Mutagenesis Kit; can be obtained by modifying the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8 using a commercially available kit such as Toyobo or PCR method. The activity of the protein encoded by the obtained gene is, for example, by introducing the obtained gene into Escherichia coli to produce a transformant, and culturing the transformant to produce an enzyme protein. The cell disruption solution of this transformant or the purified enzyme protein can be added to N-succinyl-DL-amino acid, and the production of D-amino acid can be measured by the method described in the Examples.

  Next, the manufacturing method of D-succinylase of this invention is demonstrated. The D-succinylase of the present invention is produced by inserting a gene into an appropriate vector to prepare a recombinant vector, transforming an appropriate host cell with the recombinant vector, preparing a transformant, This can be done easily by culturing the transformant.

  The vector is not particularly limited as long as it can be retained and autonomously propagated in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors, phage vectors, viral vectors, and the like. Recombinant vectors can be prepared according to conventional methods. For example, the D-succinylase gene of the present invention is added to these vectors using appropriate restriction enzymes and ligases, or, if necessary, linkers or adapter DNAs. It can be easily performed by connecting. 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.

  Moreover, as a host cell, a conventionally known cell can be used and is not particularly limited as long as a recombinant expression system is established. Preferably, microorganisms such as Escherichia coli, Bacillus subtilis, Actinomyces, Neisseria gonorrhoeae and yeast are used. Insect cells, animal cells, higher plants, etc., more preferably microorganisms, and particularly preferably Escherichia coli (for example, K12 strain, B strain, etc.). The transformant may be prepared according to a conventional method.

  When the obtained transformant is cultured for a certain period of time under appropriate culture conditions according to the host cell, the D-succinylase of the present invention is expressed from the incorporated gene and accumulated in the transformant.

  The D-succinylase of the present invention accumulated in the transformant can be used as it is, but may be used after purification. As this purification method, a conventionally known method can be used. For example, a transformed body after culture or a culture thereof is homogenized in an appropriate buffer, and cell extraction is carried out by sonication or surfactant treatment. A liquid can be obtained, and separation techniques conventionally used for protein separation and purification can be appropriately combined therewith. Such separation techniques include salting-out, solvent precipitation methods and other methods utilizing differences in solubility, dialysis, ultrafiltration, gel filtration, non-denaturing polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-polyacrylamide. Method using molecular weight difference such as gel electrophoresis (SDS-PAGE), method using charge such as ion exchange chromatography and hydroxyapatite chromatography, method using specific affinity such as affinity chromatography, reverse Examples include, but are not limited to, a method using a difference in hydrophobicity such as phase high performance liquid chromatography and a method using a difference in isoelectric point such as isoelectric focusing.

  Next, a method for producing a D-amino acid using the D-succinylase of the present invention in combination with a succinyl amino acid racemase will be described. According to the present invention, D-amino acid efficiently hydrolyzes N-succinyl-D-amino acid (D form) in N-succinyl-DL-amino acid (racemic form) using D-succinylase of the present invention. Manufactured by the process.

  Specifically, in this step, the D-succinylase of the present invention and the raw material N-succinyl-DL-amino acid are dissolved in an appropriate solution to prepare a reaction solution, and this reaction solution is set to appropriate conditions. Can be done.

  The solution to be used may be distilled water, but if necessary, a buffering agent such as phosphate or Tris may be used. When using a buffering agent, it is preferable that the density | concentration is 20-200 mM, and it is preferable that pH is 6-8.

  The D-succinylase of the present invention is preferably used at a concentration of 1 to 10,000 mg / L (10 to 100,000 U / L) in the reaction solution.

  The N-succinyl-DL-amino acid to be reacted with the D-succinylase of the present invention can be synthesized by various known methods, for example, Sakai A. et al. et al. , Biochemistry, 2006, 45 (14), 4455-62. The kind of DL-amino acid as a raw material may be appropriately selected according to the kind of D-amino acid to be produced, and may be 20 kinds of naturally occurring amino acids, unnatural amino acids and derivatives thereof. The concentration of N-succinyl-DL-amino acid in the reaction solution is not particularly limited, but is generally 1% by weight to 30% by weight.

  In the method for producing a D-amino acid of the present invention, the temperature at which the reaction proceeds is not particularly limited as long as the D-succinylase of the present invention sufficiently acts. 55 degreeC is more preferable and 30-50 degreeC is further more preferable. Further, the pH during the reaction is not particularly limited as long as the D-succinylase of the present invention sufficiently acts, but generally pH 5 to 10 is preferable, and pH 6 to 9 is more preferable. The reaction time is not particularly limited, but is generally about 1 to 120 hours, preferably about 1 to 72 hours, and more preferably about 1 to 24 hours. The reaction time can be appropriately selected experimentally in consideration of the type of D-amino acid to be produced and the desired yield, yield, amount of enzyme or substrate used, quantity ratio, reaction temperature, reaction pH, and the like.

  The D-succinylase used in the method for producing a D-amino acid of the present invention is not limited to a purified one or an unpurified one (crude enzyme solution, etc.), and is contained in a transformant. It may be. In this case, the transformant is added to the reaction system, and the reaction is allowed to proceed simultaneously while culturing the transformant, or the transformant cultured in advance may be added to the reaction system.

  The method for producing a D-amino acid according to the present invention further includes a step of generating N-succinyl-D-amino acid by racemizing N-succinyl-L-amino acid using N-succinyl amino acid racemase. Since the D-succinylase of the present invention mainly hydrolyzes N-succinyl-D-amino acid in N-succinyl-DL-amino acid (racemate), N-succinyl-L-amino acid in the racemate as it is. Many of them are wasted. Therefore, if N-succinyl-L-amino acid is racemized using N-succinyl amino acid racemase to produce N-succinyl-D-amino acid, the remaining N-succinyl-L-amino acid is also converted to D-amino acid. can do.

  N-succinylamino acid racemase is an enzyme that catalyzes both the reaction of converting the L-form of N-succinylamino acid into the D-form and the reaction of converting the D-form into the L-form, and the ratios thereof are almost equal (racemization). . The N-succinyl amino acid racemase used in the production method of the present invention is not particularly limited as long as the N-succinyl amino acid can be racemized, and the N-acyl amino acid racemase described in JP-A-2007-82534 and JP-A Conventionally known ones such as N-acylamino acid racemase described in 2008-61642 can be used.

The reaction of racemizing N-succinyl-D-amino acid using N-succinyl amino acid racemase is performed by, for example, mixing in a reaction solution containing N-succinyl amino acid, N-succinyl amino acid racemase and a buffer under the following conditions. Do. The reaction temperature is not particularly limited as long as the N-succinyl amino acid racemase to be used is sufficiently effective, but generally 25 to 70 ° C is preferable, and 37 to 60 ° C is more preferable. The pH during the reaction is not particularly limited as long as the N-succinyl amino acid racemase to be used is sufficiently effective, but in general, pH 5 to 9 is preferable, and pH 6 to 8 is more preferable. N-succinyl amino acid racemase is preferably used at a concentration of 50 to 15000 mg / L (5000 to 1500,000 U / L) in the reaction solution. The activity of N-succinyl amino acid racemase is significantly improved by adding a divalent metal ion at a final concentration of 0.1 mM to 1 M (preferably 0.1 to 1 mM). Examples of the divalent metal ion to be added include Mn ²⁺ , Co ²⁺ , Mg ²⁺ , Fe ²⁺ and Ni ²⁺ . As the buffer used for the reaction of N-succinylamino acid racemase, the same buffer as that used for the reaction of D-succinylase can be used. The N-acylamino acid racemase described in JP-A-2007-82534 has been found from the subsequent studies to be an N-succinyl amino acid racemase using N-succinylamino acid as a more suitable substrate. Therefore, the N-acyl amino acid racemase described in JP-A-2007-82534 can be used in combination with the D-succinylase of the present invention.

  The racemization reaction with the N-succinyl amino acid racemase and the hydrolysis reaction with D-succinylase can be performed separately, but are preferably performed simultaneously. When performed simultaneously, when viewed microscopically, the D-form of N-succinyl-DL-amino acid is first hydrolyzed (desuccinylated) by the D-succinylase of the present invention to produce the desired D-amino acid. Since the racemic state is eliminated when the D-form of the substrate is consumed, N-succinyl amino acid racemase further promotes the reaction of converting the L-form to the D-form. N-succinyl-D-amino acid produced by N-succinyl amino acid racemase is sequentially decomposed into D-amino acids by the D-succinylase of the present invention. This repetition can theoretically convert almost all N-succinyl-DL-amino acids to D-amino acids. The reaction conditions when the racemization reaction and the hydrolysis reaction are performed simultaneously are not particularly limited as long as the N-succinyl amino acid racemase and the D-succinylase of the present invention exhibit the activity, but the substrate concentration is 0.01. It is preferable to carry out at a weight% to 30 weight%, pH 5-9, and temperature 40-50 degreeC. The time required for the racemization reaction and the hydrolysis reaction is not particularly limited as long as it is a time until the N-succinyl-DL-amino acid used as a raw material can be converted into a desired amount of D-amino acid. Generally, it is about 1 to 7 days.

The acylase activity of D-succinylase used in the present invention can be measured, for example, by the following D-amino acid oxidase method.
<Reagent>
10 mM N-succinyl-D-valine 1.0 (U / mL) peroxidase (Toyobo PEO-301)
1.0 mM 4-aminoantipyrine (Daiichi Chemical)
1.0 mM TOOS (Doujin Chemical Laboratory)
6.0 (U / mL) D-amino acid oxidase (Biozyme DOX2)
A 25 mM phosphate buffer solution containing
In addition, the synthesis | combination of N-succinyl-D-valine can be performed as follows, for example. D-valine (Nacalai Tesque, 4.7 g) and succinic anhydride (Nacalai Tesque, 4.0 g) are dissolved in 40 mL of acetic acid (Nacalai Tesque). Next, the solution is heated to 50 to 60 ° C. to volatilize the solvent and crystallize. The crystallized white precipitate is then collected and recrystallized in a mixture of 20 mL ethyl acetate and 20 mL methanol. The precipitate is crushed with a mortar and dried to obtain N-succinyl-D-valine. Details are described in Non-Patent Document 1.
<Measurement conditions>
Prewarm 2.7 mL of reaction reagent at 37 ° C. for 5 minutes. After adding 0.3 mL of the enzyme solution and mixing gently, the absorbance change at 555 nm was recorded for 5 minutes with a spectrophotometer controlled at 37 ° C. using water as a control, and the absorbance change per minute (ΔOD _TEST) ). In the blind test, a solution dissolving the enzyme is added to the reagent mixture instead of the enzyme solution, and the change in absorbance per minute (ΔOD _BLANK ) is measured in the same manner. From these values, D-succinylase activity is determined according to the following formula. Here, 1 unit (U) in D-succinylase activity is defined as the amount of enzyme that produces 1 micromole of D-amino acid per minute under the above conditions.

(Equation 1)
Activity (U / mL) =
{(ΔOD _TEST −ΔOD _BLANK ) × 3.0 × dilution factor} /
{31.0 × 0.3 × 1/2 × 1.0}

In the formula, 3.0 is the amount of the reaction reagent + enzyme solution (mL), 31.0 is the molar molecular extinction coefficient (cm ² / micromol) under this activity measurement condition, and 1/2 is generated by the enzyme reaction. The coefficient due to the quinoneimine dye formed from one molecule of H ₂ O ₂ being ½ molecule, 0.3 is the volume of the enzyme solution (mL), 1.0 is the optical path length (cm) of the cell .

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not limited to the following Example.

[Example 1] Screening for D-succinylase The bacterial strain of the present invention was isolated by screening from the soil by the following method. As a medium, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.2%, disodium hydrogen phosphate 0.3%, magnesium sulfate heptahydrate 0.05%, yeast extract 0.01%, polypeptone 0. A small amount of soil was added to a medium of pH 7.5 containing 01% and the inducer N-succinyl-D-phenylalanine 0.2%, and the culture was shaken in a test tube at 35 ° C., and the accumulation culture was performed several times from the soil. . A petri dish medium containing 2% agar was prepared with the same medium, and the culture solution subjected to enrichment culture was plated out to obtain an isolated colony. The isolated colony was cultured again in a test tube in the above-mentioned medium, and a strain having D-succinylase activity was selected by the following method.

Selection of producing bacteria by HPLC analysis: In order to select a strain having a strong activity against N-succinyl-D-phenylalanine in a colony-isolated strain culture solution, the following analysis by HPLC was performed.
Column: Inertsil ODS-2 (5 μm, 4.6 mmφ × 150 mm) (GL Sciences Inc.), mobile phase: pH 2.3 aqueous phosphoric acid solution: acetonitrile = 80: 20, temperature: 40 ° C., flow rate: 1.0 mL / min , Detection: N-succinyl-D-phenylalanine and D-phenylalanine elute from decomposition of N-succinyl-D-phenylalanine and production of D-phenylalanine in the culture supernatant centrifuged after culturing at 210 nm. It was analyzed from the peak area of the time to be.
The strain obtained through such a method had the following mycological properties. This strain was designated as Cupriavidus Sp. It was named P4-10-C (Cupriavidus sp. P4-10-C).

(Bacteriological test I)
1. Cell morphology: Neisseria gonorrhoeae (0.7-0.8 × 1.2-2.0 μm)
2. 2. Gram stainability: negative Presence or absence of spores: negative 4. 4. Mobility: Yes Colony state Medium: Nutrient agar
Incubation time: 24 hours
Diameter: 2.0-3.0mm
Color: Light yellow
Shape: Circular
Raised state: lenticular
Perimeter: all edges
Surface shape: smooth
Transparency: opaque
Viscosity: butter shape Growth temperature test: 37 ° C
Growth at 45 ° C 7. Catalase reaction: positive 8. 8. Oxidase reaction: positive Acid / gas production from glucose (acid production / gas production): negative / negative O / F test (oxidation / fermentation): negative / negative

(Bacteriological examination III)
1. Use of citric acid (Simmons method): positive 2. Starch hydrolysis: negative 3. Lipase activity (Tween 20): negative 4. Acid production from D-ribose: negative Acid production from inositol: negative 6. 6. Acid production from mannitol: negative Acid production from L-arabinose: negative

As a result of the above biochemical and mycological properties, it is a gram-negative bacillus having motility, does not oxidize glucose, and is positive for catalase and oxidase, which is consistent with the general properties of Cupriavidus.
As a result of the API test, C.I. There are many similarities to the properties of gillardii, except that citric acid is used and inositol is not oxidized.

Furthermore, as a result of a simple molecular system using 16S rDNA of the upper strain of homology search for Apollon DB-BA 4.0 using BLAST, C.I. It formed a cluster with giliadi's 16S rDNA and showed that it was closely related.
However, there are 21 base differences between the sequences. Cupriavidus sp. closely related to gillardii. It was reasonable to say.

  For this reason, microorganisms newly discovered from nature were classified as bacteria belonging to the genus Cupriavidus. In addition, the strain discovered by this invention is Cupriavidus sp. As P4-10-C (Cupriavidus sp. P4-10-C), 1-1-1 Higashi 1-chome, Tsukuba, Ibaraki, Japan Center No. 6 FERM BP- It is deposited internationally as 11387 (international deposit date: June 28, 2011).

[Example 2] Cupriavidus SP. Purification of D-succinylase produced by the P4-10-C strain Cupriavidus SP. P4-10-C strain, normal bouillon "Eiken" 1.8%, powdered yeast extract D-3 0.2% (Wako Pure Chemical Industries, Ltd., Code: 390-00531), casamino acid "Digo" In a 500 mL flask, 0.1% (Wako Pure Chemical Industries, Ltd., Code: 392-00655), pH 7.5 medium supplemented with 0.3% dipotassium hydrogen phosphate and 0.2% glucose The culture was carried out for 2 days at 35 ° C., 150 r / min, with rotary stirring using 27 mediums that were sterilized by adding 200 mL medium and autoclaved. The turbidity (ABS 660 nm) at the end of the culture was 2.7 and pH 8.6.
After culturing, the cells were collected by centrifuging at 8000 r / min for 30 minutes using a cooling centrifuge (manufactured by Hitachi Koki Co., Ltd.). The collected cells were washed with a 20 mmol / L HEPES-NaOH (pH 7.5) buffer and then centrifuged again to obtain 36 g of cells.
After suspending in 100 mL of 20 mM HEPES-NaOH (pH 7.5) buffer, which is about 3 times the amount of cells obtained, using an ultrasonic cell disruption apparatus BD-1 (manufactured by Toago Denki Co., Ltd.) under ice cooling, Ultrasonic crushing was performed 10 times in a 5-minute cycle. After crushing, the mixture was centrifuged at 8000 r / min and 4 ° C. for 60 minutes with a high-speed cooling centrifuge (manufactured by Hitachi Koki Co., Ltd.) to obtain 178 mL of the supernatant. This was used as a crude enzyme solution.
In addition, this strain produced D-succinylase without adding N-succinyl-D-phenylalanine at the time of culture.

  The crude enzyme solution is packed in a dialysis tube, poured into a buffer solution containing 20 mM HEPES-NaOH (pH 7.5) 1.2 M ammonium sulfate, and the buffer solution is changed several times while stirring in a low temperature chamber (4 ° C.). Dialysis was performed. After completion of dialysis, the mixture was centrifuged at 4000 r / min and 4 ° C. for 30 minutes with a high-speed cooling centrifuge (manufactured by Hitachi Koki Co., Ltd.) to obtain 100 mL of the supernatant. The supernatant was applied to a Butyl-TOYOPEARL 650M column (manufactured by Tosoh Corporation) (3.2 cmφ × 20 cm) previously equilibrated with a buffer containing 20 mM HEPES-NaOH (pH 7.5) 1.2 M ammonium sulfate. Enzyme was adsorbed, and 20 mM HEPES-NaOH (pH 7.5) 1.2 M ammonium sulfate-containing buffer and 20 mM HEPES-NaOH (pH 7.5) buffer were used in a total volume of 2000 mL, and the enzyme was eluted by a linear concentration gradient method. A fraction having succinylase activity was collected. The active fraction obtained with Butyl-TOYOPEARL was concentrated with 70% ammonium sulfate saturation, and the precipitate was collected by centrifugation at 10,000 r / min, 4 ° C. for 60 minutes. The collected precipitate was dialyzed against 5 mM phosphate buffer (pH 7.2). 33 mL of the dialyzed enzyme solution was adsorbed on a Macro-Prep CHT Type I (manufactured by BIO-RAD) hydroxyapatite column (1.6 cmφ × 20 cm) previously equilibrated with 5 mM phosphate buffer (pH 7.2). Next, the enzyme is eluted by a linear concentration gradient method using a total amount of 150 mL of 5 mM phosphate buffer (pH 7.2) and 300 mmol / L phosphate buffer (pH 7.2), and a fraction having D-succinylase activity is obtained. It was collected. An active fraction (130 mL) obtained by Macro-Prep CHT Type I was concentrated to 20 mL using an ultrafiltration membrane with a molecular weight cut off of 10,000 from Vivaspin 20 (manufactured by Sartorius Co., Ltd.). The collected concentrated solution was dialyzed against 20 mM HEPES-NaOH (pH 7.5) acid buffer. This dialysate was previously equilibrated with 20 mM HEPES-NaOH (pH 7.5) buffer solution. F. Column 5 mL (manufactured by GE Healthcare Bioscience), followed by linear concentration using 20 mM HEPES-NaOH (pH 7.5) and 20 mM HEPES-NaOH (pH 7.5) 0.3 M sodium chloride buffer solution in a total amount of 200 mL The enzyme was eluted by a gradient method, and a fraction having D-aminoacylase activity was recovered.

  A small amount of the fraction in which the D-succinylase activity was confirmed was subjected to a conventional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the fraction free of impure protein was collected to obtain a novel D-succinylase. The result of electrophoresis after purification is shown in FIG.

[Example 3] Cupriavidus SP. Enzymatic properties of D-succinylase produced by strain P4-10-C The enzymatic properties of D-succinylase obtained in Example 2 were measured by the following method.

1. Molecular weight Measurement was performed by SDS-polyacrylamide gel electrophoresis (SuperSep Ace 10-20%, manufactured by Wako Pure Chemical Industries, Ltd.) as a standard method. Protein molecular weight marker (manufactured by Sekisui Medical Co., Ltd., protein molecular weight marker “Daiichi” III) (this marker is phosphorylase b (97,400 dalton), bovine serum albumin (66,267 dalton)), aldolase (42 , 400 dalton), carbonic anhydrase (30,000 dalton), trypsin inhibitor (20,100 dalton), and lysozyme (14,400 dalton)). The molecular weight was a heterodimer containing subunits of 50-70 kDaltons and 20-30 kDaltons (FIG. 1).

2. Substrate specificity The above-mentioned D-amino acid oxidase method (however, when measuring the activity value for N-succinyl-D-tryptophan, N-succinyl-D-phenylalanine, N-succinyl-D-serine, 10 mM N as a reaction substrate) -Instead of succinyl-D-valine, 10 mM N-succinyl-D-tryptophan, 10 mM N-succinyl-D-phenylalanine, 10 mM N-succinyl-D-serine are measured, respectively) and the HPLC described below According to the analysis method, the following N-succinyl-D-amino acids were reacted to confirm.
For N-succinyl-D-aspartic acid, N-succinyl-D-aspartic acid, and N-succinyl-D-glutamic acid, to which amino acid oxidase is difficult to act, the column: Inertsil ODS-2 (5 μm, 4.6 mmφ) was measured by HPLC measurement. × 150 mm) (GL Sciences Inc.), mobile phase: pH 2.3 phosphoric acid aqueous solution: acetonitrile = 90: 10, temperature: 40 ° C., flow rate: 0.5 mL / min, detection: 210 nm under the conditions of D-amino acid A calibration curve was prepared, the reaction solution was analyzed, and the D-amino acid concentration was measured.
N-succinyl-D-glutamic acid having a carboxy group, N-succinyl-D-aspartic acid, N-succinyl-D- in which the carboxy group is amidated in order to examine the difference in activity depending on the type of substrate side chain Asparagine, N-succinyl-D-phenylalanine having a hydrophobic aromatic ring, N-succinyl-D-tryptophan, N-succinyl-D-valine having a hydrophobic aliphatic hydrocarbon group, having a hydrophilic hydroxy group Experiments were performed on N-succinyl-D-serine.
The results are shown in Table 4. As is clear from Table 4, it was confirmed that the D-succinylase obtained in Example 2 exhibited enzyme activity against various types of amino acids, although there were differences due to side chains.

Furthermore, regarding the ability to hydrolyze 10 mM of various N-succinyl-D-amino acids into D-amino acids, the D-succinylase obtained in Example 2 and the known D-aminoacylase Amano (Wako Pure Chemical Industries, Ltd. Code: 329) -61063, Japanese Patent Application Laid-Open No. 2001-000185, derived from Alkalinegenes xylose oxydans subspecies xylose oxydans A-6).
The results are shown in Table 5. As is apparent from Table 5, the D-succinylase obtained in Example 2 was superior to the known D-aminoacylase Amano by about 1000 times or more, although it varied depending on the substrate.
Regarding the D-succinylase obtained in Example 2, the measurement method was performed according to the above-mentioned D-amino acid oxidase and HPLC analysis method. Moreover, about the well-known D-aminoacylase amano, it was made to react in the solution containing 0.1 mM cobalt chloride, and all were performed according to the HPLC analysis method.
The hydrolysis activity of N-succinyl-D-amino acid to D-amino acid of D-succinylase obtained in Example 2 is 12 U / mg or more.

3. Temperature stability and optimum temperature Using the D-succinylase enzyme solution obtained in Example 2, the temperature stability and optimum temperature were examined by the D-amino acid oxidase method. For temperature stability, the enzyme solution (0.2 mg / mL) was treated in a phosphate buffer solution at pH 7.5 at 4 ° C, 40 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, or 70 ° C for 60 minutes. The enzyme activity was determined. The optimum temperature was determined by changing the reaction temperature from 30 ° C. to 60 ° C. in a phosphate buffer solution having a pH of 7.5 with an enzyme solution (0.01 mg / mL). Evaluation of temperature stability was shown by the residual relative activity which made 100% the case where it processed at 4 degreeC. Moreover, the evaluation of the optimum temperature was shown by the relative activity with the enzyme activity at 50 ° C. being 100%. The results are shown in FIG. 2 (temperature stability) and FIG. 3 (optimum temperature). As is apparent from FIG. 2, it was found that the enzyme activity maintained 90% or more of the enzyme activity when treated at 4 ° C. even by heat treatment at 50 ° C. Further, as is clear from FIG. 3, the optimum temperature of the present enzyme is about 45 ° C. to about 50 ° C. In addition, the optimum temperature was reacted at 30 ° C., 40 ° C., 45 ° C., 50 ° C., and 60 ° C. for 5 minutes in enzyme concentration (0.01 mg / mL) and phosphate buffer solution (pH 7.5). A temperature range in which the relative activity is 80% or more when the highest temperature is 100% is shown.

4). PH optimum
Using the D-succinylase enzyme solution (0.01 mg / mL) obtained in Example 2, the optimum pH was examined by the HPLC analysis method described above. As the buffer solution, phosphate buffer solution (pH 5.5-pH 7.5), Tris-HCl (pH 7.5-pH 8.5), borate buffer solution (pH 8.5-pH 10.0) are used, and each pH is adjusted. And then reacted at 37 ° C. for 60 minutes, and the enzyme activity was determined. The evaluation of pH stability was shown by relative activity with the pH = 7.45 as 100. The results are shown in FIG. As is clear from FIG. 4, this enzyme showed an activity of 80% or more when treated at pH = 6.99 to 8.40 and at pH = 7.45.

5. Effect of Metal Ion Using the D-succinylase enzyme solution (0.01 mg / mL) obtained in Example 2, various divalent metal ions shown in Table 6 below were each 37 ° C. in the presence of 1 mM final concentration. The D-succinylase activity was measured by the HPLC analysis method described above, and indicated as a relative activity with the additive-free enzyme activity as 100. The results are shown in Table 6. As is apparent from Table 6, this enzyme activity was not significantly affected by metal ions.

[Example 4] Cupriavidus SP. 1. Isolation of D-succinylase gene from P4-10-C strain Acquisition of partial sequence After SDS-PAGE, a band corresponding to the enzyme was transferred from an acrylamide gel to a PVDF membrane (Bio-Rad: SK blot PVDF membrane), and N-terminal amino acid sequence analysis and internal amino acid sequence analysis were performed. The internal amino acid sequence from this band; SNNWVIAGSTRSTGR, the N-terminal amino acid sequence from the band of about 23 kDa; APPTDRYAAPGLEKP and the internal amino acid sequence; MARDFGPAYVDGDRR were obtained. A degenerate primer was synthesized based on these identified amino acid sequences (SEQ ID NOs: 9 and 10 in Table 8), and a DNA polymerase was extracted using genomic DNA extracted from Cupriavidas SP P4-10-C by a known method as a template. PCR was performed under the recommended conditions using KOD-Plus (Toyobo). Using the cloning kit Target Clone-Plus (manufactured by Toyobo), the PCR product amplified by the PCR was operated according to the protocol, cloned into the vector pBluescript, and Escherichia coli DH5α strain competent cell (Toyobo). To obtain a transformant. The transformant was cultured in LB medium, the plasmid was extracted, the gene sequence was confirmed by BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), and a partial sequence of 575 bp was obtained.

2. Cupriavidas SP. Acquisition of genomic DNA from P4-10-C strain 50 mL of the culture solution was subjected to centrifugation, and the cells were purified with a genome extraction kit (Toyobo: Genomic DNA purification Kit) according to the protocol. . However, in the genomic DNA purified with this kit, a long-chain DNA fragment could not be amplified in the PCR reaction for cloning of the full-length sequence. Since no improvement was seen even when the PCR conditions were examined, it was considered that there was a problem with the purity of the genomic DNA. Based on the known method described in “current protocols in molecular biology”, the genomic DNA was again analyzed. Extraction was performed. First, after culturing in a 50 mL medium, the collected cells are suspended and washed in TE (50 mM Tris-HCl (pH 8.0), 20 mM EDTA), and after collecting the cells by centrifugation, the cells are collected again. This cell was suspended in 11.3 mL of TE. Further, 0.06 mL of 20 mg / mL proteinase K (manufactured by Nacalai Tesque) and 0.6 mL of 10% SDS solution were added to this suspension, followed by incubation at 37 ° C. for 1 hour. After incubation, 2 mL of 5 M NaCl solution was added and stirred thoroughly. Thereafter, 1.6 mL of CTAB / NaCl solution (10% CTAB / 0.7 M NaCl) was mixed and incubated at 65 ° C. for 10 minutes. After incubation, deproteinization was performed with an equal volume of 25: 24: 1 phenol / chloroform / isoamyl alcohol solution. In the case of normal bacteria, turbidity was not observed in the aqueous layer in this step, but in the case of this bacterium, turbidity remained. Therefore, deproteinization was performed once again with an equal amount of 25: 24: 1 phenol / chloroform / isoamyl alcohol solution. As a result, the turbidity was completely removed. Then, an equal amount of 24: 1 chloroform / isoamyl alcohol was added to the separated aqueous layer and mixed to recover the aqueous layer. An equivalent amount of isopropanol was added to the aqueous layer to precipitate and collect DNA. The precipitated DNA was dissolved in 0.5 mL of TE, 5 μl of 10 mg / mL RNase was added, and the mixture was reacted at 37 ° C. overnight. After the reaction, deproteinization was performed with an equal amount of 25: 24: 1 phenol / chloroform / isoamyl alcohol solution, 24: 1 chloroform / isoamyl alcohol was added and stirred, and the aqueous layer was recovered. 3M sodium acetate solution (pH 5.2) was added to the aqueous layer obtained after performing this operation twice more so that the final concentration was 0.4M, and further 2 volumes of ethanol was added. The DNA produced as a precipitate was collected, washed with 70% ethanol, dried, and dissolved in 1 mL of TE.

3. Acquisition of full length sequence Next, in order to acquire a full length sequence using the genomic DNA obtained as mentioned above, the following operation was performed. TAKARA LA PCR Using In Vitro Cloning Kit (Takara Bio), following the protocol, we succeeded in amplifying a DNA fragment from the known sequence toward the C-terminal, from the N-terminal including the start codon to the known partial gene sequence. Was determined. However, since the above-mentioned kit was unable to determine the base sequence on the C-terminal side, two types of primers (SEQ ID NOS: 11 and 12 in Table 8) directed to the outside of the gene were newly added based on the known base sequence. Designed. Using this primer, an inverse PCR method was performed using the previously obtained DNA as a template. Thereby, a DNA fragment containing a gene part outside the already obtained partial gene was obtained, and the C-terminal sequence was determined. Subsequently, a DNA primer (SEQ ID NO: 34 in Table 8) having a sequence obtained by binding a NdeI cleavage site to a portion presumed to be upstream from the N-terminus of the enzyme, and EcoRI cleavage to a portion presumed to be downstream from the C-terminus Using a DNA primer (SEQ ID NO: 35 in Table 8) having a sequence to which the sites are bound, the DNA between these sequences is amplified by PCR using the previously obtained DNA as a template, whereby the D-succinylase gene A DNA fragment containing the full length of was obtained. The base sequence of the obtained DNA fragment was analyzed, it was confirmed that the full length of the D-succinylase gene was included, and the amino acid sequence was estimated.

  Cupriavidas sp. Obtained in the present invention. When the D-succinylase gene derived from the P4-10-C strain was searched by NCBI-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), Peptidase selseSonse C45 of Peptidase sella seinse S45, No. .YP — 587763) and 76% homology with the gene registered. The penicillin acylase family to which this penicillin amidase belongs is an enzyme that hydrolyzes penicillin G, cephalosporin C, etc., and it is estimated that such an enzyme hydrolyzes N-succinyl-D-amino acid. It is difficult, and it is almost impossible to obtain this gene by homology search predicted from the function. Moreover, the enzyme which hydrolyzes N-succinyl-D-amino acid is not known until now, and it can be said that D-succinylase in this invention is a novel enzyme. Further, in the comparison of amino acid homology in the same genus Cupriavidus Metalidurans, Cupriavidus Neketa, and the closely related genus Ralstonia picketi, in which the cloning and confirmation of the hydrolytic ability of N-succinyl-D-amino acids were performed in the present invention, From 50% to about 80%, it was found that the amino acid sequence was greatly different even in closely related genera (Table 7).

[Example 5] Isolation of the gene for D-succinylase derived from Cupriavidas metareduran A strain of Cupriavidas metareduran (NBRC No. 101272) was obtained from National Institute of Technology and Evaluation, and cultured in a liquid medium. A genome was extracted from the cells obtained by the culture using a genome extraction kit (Toyobo: Genomic DNA purification Kit). Gene-specific primers were synthesized (SEQ ID NOs: 13 and 14 in Table 8), and the gene was obtained by PCR using DNA polymerase KOD-Plus (manufactured by Toyobo) using the obtained genome as a template. Using cloning kit Target Clone-Plus (manufactured by Toyobo Co., Ltd.), the operation was performed according to the protocol, and cloning was performed in vector pBluescript to obtain recombinant expression plasmid pDSACm. pDSACm was transformed into Escherichia coli DH5α strain competent cell (manufactured by Toyobo Co., Ltd.) to obtain a transformant. The transformant was cultured in LB medium, the plasmid was extracted, the gene sequence was confirmed by BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), and the amino acid sequence was deduced. The primers used for the sequencing are shown in Table 8 (SEQ ID NOS: 19, 20, 21, 22, 23, 24 in Table 8).

[Example 6] Isolation of D-succinylase gene derived from Cupriavidus neketa A strain of Cupriavidus neketer (NBRC No. 102504) was obtained from National Institute of Technology and Evaluation, and cultured in a liquid medium. From the cells obtained by culturing, the genome was extracted with a genome extraction kit (Toyobo: Genomic DNA purification Kit), and gene-specific primers were synthesized (SEQ ID NOS: 15 and 16 in Table 8). In the same manner as described, the full length of the gene sequence was obtained and the amino acid sequence was estimated. The primers used for the sequencing are shown in Table 8 (SEQ ID NOs: 25, 26, 27, 28, and 29 in Table 8).

[Example 7] Isolation of Ralstonia pickety-derived D-succinylase gene A strain of Ralstonia pickety (NBRC No. 102503) was obtained from National Institute of Technology and Evaluation, and cultured in a liquid medium. From the cells obtained by culturing, the genome was extracted with a genome extraction kit (Toyobo: Genomic DNA purification Kit), and gene-specific primers were synthesized (SEQ ID NOs: 17 and 18 in Table 8). In the same manner as described, the full length of the gene sequence was obtained and the amino acid sequence was estimated. The primers used for the sequencing are shown in Table 8 (SEQ ID NOs: 30, 31, 32, and 33 in Table 8).

[Example 8] Preparation of recombinant plasmids expressing various D-succinylase genes Cupriavidas SP. Recombinant plasmid for the D-succinylase gene derived from the P4-10-C strain A sequence obtained by binding the restriction enzyme NdeI and EcoRI cleavage sites to the N-terminal and C-terminal parts of the D-succinylase gene obtained in Example 4, respectively. A DNA fragment containing an open reading frame was obtained by amplifying the DNA during this period by PCR using the genomic DNA obtained in Example 4 as a template using the primers (SEQ ID NOs: 34 and 35 in Table 8). . Ligation was performed by cleaving this DNA fragment with the restriction enzymes NdeI and EcoRI, mixing with the vector plasmid pBSK cleaved with the enzymes, and adding the same amount of ligation reagent (Toyobo Ligation High) as the mixture. did. Thus, a recombinant plasmid pBSKDSAP4-10-C designed to express a large amount of the D-succinylase gene was obtained.

2. Recombinant plasmid of D-succinylase gene derived from Cupriavidas metalidurans pDSACm obtained in Example 5 was treated with restriction enzymes NdeI and BamHI, the DSA gene was excised, mixed with vector plasmid pBSK cleaved with the enzyme, and mixed Ligation was carried out by adding an equal amount of ligation reagent (Toyobo Ligation High) to the solution and incubating. Thus, a recombinant plasmid pBSKDSACm designed to express a large amount of the D-succinylase gene was obtained.

3. Recombinant plasmid of the D-succinylase gene derived from Cupriavidas neketer The pDSACn obtained in Example 6 was treated with restriction enzymes NdeI and PstI, the DSA gene was excised, mixed with the vector plasmid pBSK cleaved with the enzyme, and mixed solution Ligation was carried out by adding an equal amount of ligation reagent (Toyobo Ligation High) and incubating. Thus, a recombinant plasmid pBSKDSACn designed to express a large amount of the D-succinylase gene was obtained.

4). Recombinant plasmid of D-succinylase gene derived from Ralstonia picketi pDSARp obtained in Example 7 was treated with restriction enzymes NdeI and BamHI, the DSA gene was excised, mixed with vector plasmid pBSK cleaved with the enzyme, and mixed solution Ligation was carried out by adding an equal amount of ligation reagent (Toyobo Ligation High) and incubating. Thus, a recombinant plasmid pBSKDSARp designed to express a large amount of the D-succinylase gene was obtained.

[Example 9] E. coli of various D-succinylase genes. Expression in E. coli Plasmids constructed in Example 8: pBSKDSAP4-10-C, pBSKDSACm, pBSKDSACn, and pBSKDSSARp, respectively, were transferred to Escherichia coli DH5α strain competent cells (Toyobo Competent High DH5α) according to the protocol attached to this product. Transformation was performed to obtain a transformant. The obtained colonies of each transformant were inoculated into 5 mL of LB liquid medium (containing 50 μg / mL ampicillin) sterilized in a test tube, and then aerobically cultured by shaking at 37 ° C. for 20 hours. . The bacterial cells are collected from the obtained culture broth by centrifugation, suspended in a 25 mM phosphate buffer solution (pH 7.5), disrupted by ultrasonic waves, and centrifuged to remove insoluble matter derived from the bacterial cells. The D-succinylase crude enzyme solution in the transformants of various origins was obtained. To 100 μl of these crude enzyme solutions, 100 μl of 150 mM N-succinyl-D-amino acid and 800 μl of 25 mM phosphate buffer (pH 7.5) were added and reacted at 37 ° C. for 16 hours. A part of the reaction solution is applied to a TLC plate (Merck), developed with a developing solvent consisting of butanol: acetic acid: water (3: 1: 1, v / v), and various D-amino acids are obtained by ninhydrin spray. Detected. The results are shown in FIG. Trp, Phe, Val, Ser, Asp, Asn, and Glu in FIG. 5 represent tryptophan, phenylalanine, valine, serine, aspartic acid, asparagine, and glutamic acid, respectively. Moreover, a color shows the color when it develops with a ninhydrin spray. As a result, strong D-succinylase activity can be confirmed for D. succinylase derived from Cupriavidas sp. P4-10-C and D. succinylase derived from Cupriavidas metalidurans, and certain D-succinylase derived from Cupriavidas neketter and Ralstonia picketi D-succinylase activity could be confirmed.

[Example 10] Preparation of Cupriavidus sp. P4-10-C-derived D-succinylase from transformant A colony of a transformant transformed with pBSKP4-10-C obtained in Example 9 A 60 mL LB medium (containing 50 μg / mL ampicillin) in a Sakaguchi flask was inoculated, and cultured at 30 ° C. for 16 hours at 180 rpm with shaking as a seed culture solution. This seed culture was inoculated into 6 L of LB medium (containing 50 μg / mL of ampicillin) contained in a 10 L jar fermenter, and cultured for 21 hours at 30 ° C. with a shaking speed of 420 rpm. Turbidity (Abs 660 nm) at the end of the culture was 32.7. The bacterial cells thus obtained were collected by centrifugation, suspended in a 25 mM phosphate buffer solution (pH 7.5), and crushed using a French press crusher. The obtained crude enzyme solution was subjected to an ammonium sulfate fractionation treatment, and then separated and purified with 5 mL of HiTrap Octyl FF column (GE Healthcare Bioscience) and 5 mL of HiTrap CM FF column (GE Healthcare Bioscience). When the D-succinylase obtained by this method was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, it was confirmed that almost no impure protein was present. Using this, the following examples were used. Evaluation of enzyme properties and production of D-amino acids were performed.

[Example 11] Enzymatic properties of D-succinylase produced by a transformant Optimum temperature and thermostability of D-succinylase produced by the transformant Using the D-succinylase enzyme solution produced by the transformant obtained in Example 10, the temperature stability and the optimum are achieved by the D-amino acid oxidase method. The appropriate temperature was examined. For temperature stability, the enzyme solution was heated at a predetermined temperature for 3 hours and then ice-cooled to determine the remaining activity. The optimum temperature was determined by changing the reaction temperature to 30 ° C. or 65 ° C. and determining the enzyme activity. Evaluation of temperature stability was shown by the residual relative activity which made 100% the case where it processed at 4 degreeC. In addition, the optimum temperature was evaluated based on the relative activity with the enzyme activity at 50 ° C. being 100%. The results are shown in FIG. 6 (temperature stability) and FIG. 7 (optimum temperature). As is apparent from FIGS. 6 and 7, even when the enzyme was treated at 50 ° C., it maintained 100% activity when treated at 4 ° C., and the optimum reaction temperature was 45 ° C. to 50 ° C.

2. Optimum pH and pH stability of D-succinylase produced by the transformant Using the D-succinylase enzyme solution produced by the transformant obtained in Example 10, the pH was measured by the D-amino acid oxidase method and the HPLC analysis method. Stability and optimum pH were investigated. Buffer solutions include acetate buffer solution (pH 4.5 to pH 5.5), phosphate buffer solution (pH 5.5 to pH 7.5), Tris-HCl (pH 7.5 to pH 8.5), or borate buffer solution ( pH stability was determined by measuring enzyme activity after treatment at 25 ° C. for 20 hours, and optimum pH was determined by determining enzyme activity after reacting at each pH for 60 minutes. It was. Enzyme activity was expressed as relative activity with each of pH 7.5 (pH stability) and pH 7.0 (optimum pH) as 100. The results are shown in FIG. 8 (pH stability) and FIG. 9 (optimum pH). As is apparent from FIGS. 8 and 9, this enzyme exhibits high activity at pH 6 to 8, and exhibits 80% or more activity when treated with phosphate buffer solution pH 7.5 in the range of pH 4 to pH 10, with a wide range. Very stable at a pH range.

4). Effect of metal ion of D-succinylase produced by transformant Using the D-succinylase enzyme solution produced by the transformant obtained in Example 10, various divalent metal ions shown in Table 9 below were used. Each was reacted at 37 ° C. for 60 minutes in the presence of a final concentration of 1 mM, the D-succinylase activity was measured by HPLC analysis, and the relative activity with no added enzyme activity as 100 was shown. The results are shown in Table 9. As is clear from Table 9, this enzyme activity was not significantly affected by metal ions.

5. Effects of inhibitors of D-succinylase produced by transformants Using the D-succinylase enzyme solution produced by the transformant obtained in Example 10, various inhibitors shown in Table 10 below were each at final concentrations. The reaction was carried out at 37 ° C. for 60 minutes in the presence of 1 mM, and the D-succinylase activity was measured by HPLC analysis. The results are shown in Table 10. As is apparent from Table 10, this enzyme is not inhibited by DTT, EDTA, and N-ethylmaleimide, but is inhibited by cysteine modifiers such as PCMB, iodoacetamide, and iodoacetic acid. It was found to be involved in enzyme stability.

[Example 12] Synthesis of N-succinyl-DL-phenylalanine, N-succinyl-DL-tryptophan and N-succinyl-DL-biphenylalanine 10 g DL-phenylalanine (Code: 168-01312 manufactured by Wako Pure Chemical Industries, Ltd.) and water Sodium oxide 7.3 g (Wako Pure Chemical Industries, Code: 198-13765) was dissolved in 150 mL of water, and succinic anhydride 18.7 g (Wako Pure Chemical Industries, Code: 198-04355) and hydroxylation were added thereto. The reaction was conducted at 50 ° C. or lower while adding 7.3 g of sodium. This was concentrated under reduced pressure and neutralized with hydrochloric acid (Wako Pure Chemical Industries, Code: 087-01076) to pH 2 or lower to obtain crude crystals. Next, the crude crystal was recrystallized with n-hexane (Wako Pure Chemical Industries, Code: 085-00416), the precipitate was collected by filtration, and the precipitate was pulverized and dried to obtain 45 g of N-succinyl-DL-phenylalanine.
N-succinyl-DL-tryptophan and N-succinyl-DL-biphenylalanine were obtained in the same procedure except that DL-phenylalanine was changed to DL-tryptophan or DL-biphenylalanine.

[Example 13] Synthesis of D-phenylalanine from N-succinyl-DL-phenylalanine using D-succinylase Transformation using 10 mL of 5 wt% N-succinyl-DL-phenylalanine aqueous solution (pH 7.5) as a substrate The hydrolysis rate and optical activity of phenylalanine from N-succinyl-DL-phenylalanine when 0.05 mg D-succinylase enzyme solution was added and reacted at 45 ° C. for 3 days were calculated by the following HPLC measurement method did. The production rate of phenylalanine by hydrolysis with an enzyme is as follows. Column: Inertsil ODS-2 (5 μm, 4.6 mmφ × 150 mm) (GL Sciences Inc.), mobile phase: pH 2.3 phosphoric acid aqueous solution: acetonitrile = 80: 20, The peak area value of phenylalanine was measured under the conditions of temperature: 40 ° C., flow rate: 1.0 mL / min, detection: 210 nm. The optically active substance ratio of the generated phenylalanine is as follows: Column: CROWNPAK CR (+) (5 μm, 4.0 mmφ × 150 mm) (Daicel Chemical Industries, Ltd.), mobile phase: perchloric acid aqueous solution (pH 2.0), temperature: Measurement was performed under the conditions of 15 ° C., flow rate: 1.0 mL / min, detection: 210 nm. The results are shown in FIG. As is apparent from FIG. 10, in the reaction system in which D-succinylase of the transformant is added to the N-succinyl-DL-phenylalanine aqueous solution, 50% of N-succinyl-DL-phenylalanine in the reaction solution on the third day of reaction. The above was hydrolyzed, and 90% of D-phenylalanine and 10% of L-phenylalanine were hydrolyzed. From this, it is clear that the present D-succinylase acts also on L-phenylalanine but acts more strongly on D-phenylalanine.

[Example 14] Synthesis of D-phenylalanine from N-succinyl-DL-phenylalanine using D-succinylase and succinyl amino acid racemase Using 10 mL of 5 wt% N-succinyl-DL-phenylalanine aqueous solution (pH 7.5) as a substrate N-succinyl obtained by adding 0.05 mg of a D-succinylase enzyme solution of the transformant and 50 mg of a succinyl amino acid racemase (SEQ ID NO: 36) solution derived from Chloroflexus aurantiacus and reacting at 45 ° C. for 4 days The hydrolysis rate of D-phenylalanine from -DL-phenylalanine and the ratio of optically active substances were calculated by the following HPLC measurement method. Hydrolysis rate: column: Inertsil ODS-2 (5 μm, 4.6 mmφ × 150 mm) (GL Sciences Inc.), mobile phase: pH 2.3 phosphoric acid aqueous solution: acetonitrile = 80: 20, temperature: 40 ° C., flow rate : 1.0 mL / min, detection: measured at 210 nm. The optically active substance ratio is as follows: Column: CROWNPAK CR (+) (5 μm, 4.0 mmφ × 150 mm) (Daicel Chemical Industries, Ltd.), mobile phase: perchloric acid aqueous solution (pH 2.0), temperature: 15 ° C., flow rate : 1.0 mL / min, detection: measured at 210 nm. The hydrolysis rate was calculated from the peak area value of the substrate before and after the enzyme reaction. Furthermore, the optically active substance ratio of the generated phenylalanine was calculated. The results are shown in FIG. As is clear from FIG. 11, in the reaction system in which the present enzyme D-succinylase and succinyl amino acid racemase are added to an aqueous solution of N-succinyl-DL-phenylalanine, N-succinyl-DL-phenylalanine in the reaction solution is obtained on the 4th day of reaction. More than 80% of this was converted to D-phenylalanine. In addition, by coexisting succinyl amino acid racemase in the reaction system, conversion of N-succinyl-L-phenylalanine remaining in large amounts to N-succinyl-DL-phenylalanine and hydrolysis reaction to D-phenylalanine are preferential. And succeeded in suppressing the production of L-phenylalanine. The optical purity on the 4th day of the reaction was 93.6% ee.

Example 15 Synthesis of D-tryptophan from N-succinyl-DL-tryptophan using D-succinylase and succinyl amino acid racemase 10 mL of 5 wt% N-succinyl-DL-tryptophan aqueous solution (pH 7.5) as a substrate When 0.05 mg of a transformant D-succinylase enzyme solution and 50 mg of a succinyl amino acid racemase (SEQ ID NO: 36) solution derived from Chloroflexus aurantiacus were added and reacted at 45 ° C. for 4 days, N-succinyl The hydrolysis rate of D-tryptophan and the optically active substance ratio from -DL-tryptophan were calculated by the following HPLC measurement method. Hydrolysis rate: column: Inertsil ODS-2 (5 μm, 4.6 mmφ × 150 mm) (GL Sciences Inc.), mobile phase: pH 2.3 phosphoric acid aqueous solution: acetonitrile = 80: 20, temperature: 40 ° C., flow rate : 1.0 mL / min, detection: measured at 210 nm. The optically active substance ratio is as follows. Column: CROWNPAK CR (+) (5 μm, 4.0 mmφ × 150 mm) (Daicel Chemical Industries, Ltd.), mobile phase: perchloric acid aqueous solution (pH 2.0) / methanol = 90/10, Measurement was performed under the conditions of temperature: 15 ° C., flow rate: 0.7 mL / min, detection: 210 nm. The hydrolysis rate was calculated from the peak area value of the substrate before and after the enzyme reaction. Furthermore, the optically active substance ratio of the produced tryptophan was calculated. The results are shown in FIG. As is clear from FIG. 12, this enzyme D-succinylase and succinyl amino acid racemase are added to an aqueous solution of N-succinyl-DL-tryptophan, and N-succinyl-DL-tryptophan in the reaction solution on the 4th day of reaction. More than 80% of the product was hydrolyzed. The optical purity on the 4th day of the reaction was 96.1% ee.

[Example 16] Synthesis of D-biphenylalanine from N-succinyl-DL-biphenylalanine using D-succinylase and succinyl amino acid racemase 10 mL of 5 wt% N-succinyl-DL-biphenylalanine aqueous solution (pH 7.5) As a substrate, 0.05 mg of a transformant D-succinylase enzyme solution and 50 mg of a succinyl amino acid racemase (SEQ ID NO: 36) solution derived from Chloroflexus aurantiacus were added and reacted at 45 ° C. for 3 days. The hydrolysis rate of D-biphenylalanine from N-succinyl-DL-biphenylalanine and the optically active substance ratio were calculated by the following HPLC measurement method. Hydrolysis rate: column: Inertsil ODS-2 (5 μm, 4.6 mmφ × 150 mm) (GL Sciences Inc.), mobile phase: pH 2.3 phosphoric acid aqueous solution: acetonitrile = 80: 20, temperature: 50 ° C., flow rate : 1.5 mL / min, detection: measured at 210 nm. The optically active substance ratio is as follows. Column: CROWNPAK CR (+) (5 μm, 4.0 mmφ × 150 mm) (Daicel Chemical Industries, Ltd.), mobile phase: perchloric acid aqueous solution (pH 2.0) / methanol = 85/15, Measurement was performed under the conditions of temperature: 50 ° C., flow rate: 1.5 mL / min, detection: 210 nm. The hydrolysis rate was calculated from the peak area value of the substrate before and after the enzyme reaction. Furthermore, the optically active substance ratio of the produced biphenylalanine was calculated. The results are shown in FIG. As is apparent from FIG. 13, in the reaction system in which the present enzyme D-succinylase and succinyl amino acid racemase are added to an aqueous solution of N-succinyl-DL-biphenylalanine, N-succinyl-DL- More than 99% of biphenylalanine was hydrolyzed. The optical purity on the 4th day of the reaction was 88.3% ee.

  Since the D-succinylase of the present invention has a significantly higher hydrolysis activity of N-succinyl-D-amino acid than the conventional enzyme, the efficiency of N-acyl amino acid can be improved by using it in combination with a known succinyl amino acid racemase. Racemization and hydrolysis are possible. Therefore, the D-succinylase of the present invention is useful for obtaining a D-amino acid useful as a pharmaceutical raw material and the like at a high yield on an industrial scale and time.

Claims (5)

A protein represented by any one of the following (a) to (d):
(A) a protein encoded by a gene consisting of the base sequence set forth in SEQ ID NO: 6;
(B) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5;
(C) a protein encoded by a gene consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence set forth in SEQ ID NO: 6 and having D-succinylase activity;
(D) A protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the protein comprising the amino acid sequence set forth in SEQ ID NO: 5, and having D-succinylase activity. A gene represented by any one of the following (a) to (d):
(A) a gene consisting of the base sequence set forth in SEQ ID NO: 6;
(B) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5;
(C) a gene comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence set forth in SEQ ID NO: 6 and encoding a protein having D-succinylase activity;
(D) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 consisting of a base sequence corresponding to an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and D-succinylase activity A gene encoding a protein having   A step of preparing a recombinant vector by inserting the gene of claim 2 into a vector, preparing a transformant by transforming a host cell with the recombinant vector, and culturing the transformant; The method for producing a protein according to claim 1.   The step of hydrolyzing N-succinyl-D-amino acid in N-succinyl-DL-amino acid using the protein according to claim 1 and the racemic N-succinyl-L-amino acid using N-succinyl amino acid racemase A method for producing a D-amino acid, comprising the step of producing an N-succinyl-D-amino acid by conversion into a non-amino acid.   The step of hydrolyzing N-succinyl-D-amino acid in N-succinyl-DL-amino acid using the protein according to claim 1 and the racemic N-succinyl-L-amino acid using N-succinyl amino acid racemase The method according to claim 4, wherein the step of converting to N-succinyl-D-amino acid is performed simultaneously.