WO2012002450A1 - D-succinylase et procédé de production d'un acide d-aminé l'utilisant - Google Patents

D-succinylase et procédé de production d'un acide d-aminé l'utilisant Download PDF

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WO2012002450A1
WO2012002450A1 PCT/JP2011/064943 JP2011064943W WO2012002450A1 WO 2012002450 A1 WO2012002450 A1 WO 2012002450A1 JP 2011064943 W JP2011064943 W JP 2011064943W WO 2012002450 A1 WO2012002450 A1 WO 2012002450A1
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amino acid
succinyl
succinylase
protein
seq
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PCT/JP2011/064943
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Japanese (ja)
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伸弥 熊谷
西矢　芳昭
幸夫 岩井
洋輔 角田
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積水メディカル株式会社
東洋紡績株式会社
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Priority to JP2012522670A priority Critical patent/JP5806664B2/ja
Publication of WO2012002450A1 publication Critical patent/WO2012002450A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C12P41/007—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures by reactions involving acyl derivatives of racemic amines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)

Definitions

  • 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).
  • D-succinylase D-succinylase
  • 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, etc., but D-amino acids are difficult to obtain as optically pure amino acids, particularly products such as fermentation methods (natural amino acids). Therefore, how to efficiently produce D-amino acids is an important industrial issue.
  • N-acetyl-L-amino acid remaining in the system without reacting with D-aminoacylase is chemically racemic. It is necessary to convert it to N-acetyl-DL-amino acid and use it again as a raw material. Therefore, an enzyme having a racemase action that catalyzes the racemization of N-acetyl-L-amino acid is useful.
  • D-tryptophan which is important as a pharmaceutical raw material, can be obtained by hydrolyzing N-acetyl-DL-tryptophan with D-aminoacylase.
  • 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 resulting racemate was converted with D-aminoacylase. Hydrolyzes.
  • D-tryptophan can be obtained in high yield (Patent Document 4).
  • N-acylamino acid racemase is known as a racemase effective for such use. This enzyme does not act on amino acids not modified with N-acyl and is an enzyme that specifically racemates N-acyl amino acids. It is considered that D-amino acid can be obtained in high yield by reacting D-aminoacylase with N-acylamino acid racemase using a chemically synthesized racemic N-acetylamino acid as a raw material.
  • N-acylamino acid racemase using N-succinylamino acid as a substrate that is, succinylamino acid racemase
  • succinylamino acid racemase succinylamino acid racemase
  • the present invention was devised in view of the current state of the prior art.
  • the purpose of the present invention is to increase the hydrolysis activity of N-succinyl-D-amino acid, and to combine it with succinyl amino acid racemase to achieve industrial scale, time. It is an object of the present invention to provide a novel D-succinylase capable of producing a D-amino acid in high yield.
  • the present inventor screened various soil microorganisms using N-succinyl-D-amino acid hydrolyzing ability as an index.
  • a novel microorganism Cupriavidus sp. P4-10-C
  • the novel microorganism produced a novel D-succinylase.
  • the novel D-succinylase gene was successfully cloned and D-succinylase was also successfully engineered by DNA recombination.
  • 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.
  • a protein characterized by being represented by any one of (a) to (d): (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 under stringent conditions with a base sequence complementary to the base sequence set forth in SEQ ID NO: 2, 4, 6 or 8 and having 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.
  • 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.
  • 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.
  • 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.
  • the D-succinylase of the present invention has a much 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, a D-amino acid useful as a pharmaceutical raw material can be obtained in high yield on an industrial scale and time.
  • FIG. 1 shows Cupriavidus Sp.
  • FIG. 3 is a diagram showing an electrophoresis image of D-succinylase produced by a P4-10-C strain by electrophoresis.
  • FIG. 2 shows Cupriavidus Sp.
  • FIG. 4 is a graph showing the residual relative activity when measuring the temperature stability of D-succinylase produced by the P4-10-C strain.
  • FIG. 3 shows Cupriavidus Sp.
  • FIG. 3 is a graph showing the relative activity of D-succinylase produced by the P4-10-C strain when measuring the optimum temperature.
  • FIG. 4 shows Cupriavidus Sp.
  • FIG. 4 is a graph showing the relative activity of D-succinylase produced by the P4-10-C strain when measuring the optimum pH.
  • FIG. 4 shows Cupriavidus Sp.
  • FIG. 4 is a graph showing the relative activity of D-succinylase produced by the P4-10-C strain when measuring the optimum pH.
  • 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 the transformant.
  • FIG. 7 is a graph 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 D-succinylase produced by the transformant when measuring the optimum pH.
  • FIG. 10 is a graph showing the hydrolysis rate during the D-succinylase reaction on N-succinyl-DL-phenylalanine.
  • FIG. 10 is a graph showing the hydrolysis rate during the D-succinylase reaction on N-succinyl-DL-phenylalanine.
  • FIG. 11 is a graph showing the hydrolysis rate during the reaction between D-succinylase and succinyl amino acid racemase for N-succinyl-DL-phenylalanine.
  • FIG. 12 is a graph showing the hydrolysis rate during the reaction of D-succinylase and succinyl amino acid racemase for N-succinyl-DL-tryptophan.
  • FIG. 13 is a diagram showing the hydrolysis rate during the reaction between D-succinylase and succinyl amino acid racemase for 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.
  • SEQ ID NO: 2 is a novel soil microorganism Cupriavidus sp. This is the base sequence of D-succinylase of the P4-10-C strain, and SEQ ID NO: 1 is its amino acid sequence.
  • SEQ ID NOs: 4, 6, and 8 are Cupriavidus sp. This is the base sequence of D-succinylase of a closely related species of the P4-10-C strain.
  • SEQ ID NO: 4 is for Cupriavidus metallidurans
  • SEQ ID NO: 6 is for Cupriavidus nekator
  • SEQ ID NO: 8 is for 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.
  • 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 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).
  • N-succinyl-D-amino acid is hydrolyzed to yield D-amino acid.
  • Subunit structure a heterodimer containing subunits having molecular weights of 50 to 70 kDalton and 20 to 30 kDalton in SDS-polyacrylamide gel electrophoresis.
  • Substrate specificity acts on any N-succinyl-D-amino acid represented by the following 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.
  • the aryl group having 6 to 20 carbon atoms which may have a substituent for R include a phenyl group and a 4-hydroxyphenyl group.
  • 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.
  • the aralkyl group having 7 to 20 carbon atoms which may have a substituent is not particularly limited, and examples thereof include a benzyl group, an indolylmethyl group, a 4-phenylbenzyl group, and a 4-hydroxybenzyl group. Can be mentioned.
  • 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.
  • Optimum 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.
  • 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 the 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 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 a 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 (c) hybridizes with a base sequence complementary to the base sequence described in SEQ ID NO: 2, 4, 6 or 8 under stringent conditions and has D-succinylase activity.
  • a gene encoding a protein having a corresponding base sequence and having D-succinylase activity is also included.
  • “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, specifically 1 to 50, preferably 1 to 30.
  • the number is preferably 1 to 20, more preferably 1 to 10.
  • 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%.
  • highly homologous DNAs for example, 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95%.
  • homology is the maximum number of bases that match between the sequences to be compared).
  • 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 at a salt concentration corresponding to SDS 'S conditions, and the like.
  • the conditions are 30 ° C. and pH 7-8.
  • D-succinylase activity 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 set forth 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 (c) and (d) and the gene thereof are, for example, Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Selection Kit; manufactured by Stratagene, QuickChangeSiteDistrate; -Mutagenesis Kit: It can be obtained by modifying the base sequence described in 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 confirmed by the method described in the Examples.
  • 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 replicated and autonomously propagated in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors, phage vectors, virus vectors and the like.
  • Recombinant vectors may be prepared according to conventional methods.
  • the gene of the D-succinylase of the present invention may be added to these vectors using appropriate restriction enzymes and ligases, or, if necessary, linkers or adapter DNAs. It can be easily performed by connecting.
  • 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.
  • a conventionally known cell can be used and is not particularly limited as long as a recombinant expression system is established.
  • 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.
  • 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 it may be used after purification.
  • a conventionally known method can be used.
  • 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.
  • separation techniques include methods utilizing the difference in solubility such as salting out, solvent precipitation, 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, and 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.
  • 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.
  • D form N-succinyl-D-amino acid
  • racemic form N-succinyl-DL-amino acid
  • 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 buffer such as phosphate or Tris may be used.
  • a buffering agent such as phosphate or Tris
  • the concentration is preferably 20 to 200 mM
  • the pH is preferably 6 to 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.
  • N-succinyl-DL-amino acid to be reacted with D-succinylase of the present invention can be synthesized by various known methods, for example, Sakai A.I. 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.
  • the temperature at which the reaction proceeds is not particularly limited as long as the D-succinylase of the present invention acts sufficiently, but it is generally preferably 5 to 60 ° C. 55 ° C. is more preferable, and 30 to 50 ° C. is more preferable.
  • the pH during the reaction is not particularly limited as long as the D-succinylase of the present invention acts sufficiently, but in general, 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 and reaction pH.
  • the D-succinylase used in the D-amino acid production method of the present invention is not limited to purified or unpurified one (crude enzyme solution, etc.), and is contained in the transformant. It may be.
  • 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 D-amino acid according to the present invention further includes a step of racemizing N-succinyl-L-amino acid with N-succinyl amino acid racemase to produce N-succinyl-D-amino acid. 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 among the racemates remains as it is. Many of them are wasted.
  • 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-succinyl amino acid racemase is an enzyme that catalyzes both the reaction of converting the L-form of N-succinyl amino acid into the D-form and the reaction of converting the D-form into the L-form, and the ratios 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 is generally preferably 25 to 70 ° C, more preferably 37 to 60 ° C.
  • the pH during the reaction is not particularly limited as long as the N-succinyl amino acid racemase to be used acts sufficiently, 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).
  • the divalent metal ion to be added include Mn 2+ , Co 2+ , Mg 2+ , Fe 2+ and Ni 2+ .
  • the buffer used for the reaction of N-succinyl amino acid racemase the same buffer as that used for the reaction of D-succinylase can be used.
  • N-acylamino acid racemase described in Japanese Patent Application Laid-Open No. 2007-82534 has been found to be an N-succinyl amino acid racemase using N-succinylamino acid as a more suitable substrate through subsequent studies. 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 using N-succinylamino acid racemase and the hydrolysis reaction using D-succinylase can be performed separately, but are preferably performed simultaneously.
  • 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, the 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.
  • the reaction conditions when the racemization reaction and the hydrolysis reaction are carried out 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 preferably carried out at a weight percentage of 30 to 30 weight percent, a pH of 5 to 9, and a temperature of 40 to 50 ° C.
  • the time required for the racemization reaction and 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, and depends on the amount charged. 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.
  • a 25 mM phosphate buffer solution containing The synthesis of N-succinyl-D-valine can be performed, for example, as follows.
  • D-succinylase activity is determined according to the following formula.
  • one 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.
  • 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.
  • Example 1 Screening for D-succinylase The strain of the present invention was isolated from soil by screening in the following manner. 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 an inducer N-succinyl-D-phenylalanine 0.2%, and agitation culture was performed several times from the soil by shaking in a test tube at 35 ° C. .
  • 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 medium, and a strain having D-succinylase activity was selected by the following method.
  • the strain discovered by this invention is Cupriavidus sp.
  • P4-10-C Cupriavidus sp. P4-10-C
  • 1-1-1 Higashi 1-chome Tsukuba City, Ibaraki, Japan 6th National Institute of Advanced Industrial Science and Technology (AIST), Patent Biodeposition Center, FERM BP- It is deposited internationally as 11387 (international deposit date: June 28, 2011).
  • Cupriavidus SP Purification of D-succinylase produced by P4-10-C strain Cupriavidus SP.
  • the P4-10-C strain was obtained by adding 1.8% of ordinary bouillon “Eiken”, 0.2% of powdered yeast extract D-3 (Code: 390-00531) manufactured by Wako Pure Chemical Industries, Ltd. In a 500 mL flask, 0.1% (Wako Pure Chemical Industries, Ltd., Code: 392-00655), pH 7.5 medium supplemented with dipotassium hydrogen phosphate 0.3% and glucose 0.2% 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.
  • the crude enzyme solution is packed in a dialysis tube, poured into a buffer 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.) overnight. 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 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 cm ⁇ ⁇ 20 cm) equilibrated in advance with a 5 mM phosphate buffer (pH 7.2).
  • the enzyme was 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 was 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 (Vivapin 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. Apply to 5 mL of column (GE Healthcare Bioscience), then use 20 mM HEPES-NaOH (pH 7.5) and 20 mM HEPES-NaOH (pH 7.5) 0.3 M sodium chloride buffer to obtain a linear concentration.
  • the enzyme was eluted by a gradient method, and a fraction having D-aminoacylase activity was collected.
  • 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.
  • 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).
  • N-succinyl-D-aspartic acid N-succinyl-D-aspartic acid, N-succinyl-D-glutamic acid to which amino acid oxidase does not act easily
  • the column: Inertsil ODS-2 (5 ⁇ m, 4.6 mm ⁇ ) 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 210 nm 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.
  • 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.
  • 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, Alkaligenes xylose oxydans subspecies xylose oxydans A-6 strain).
  • 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.
  • the measurement method was performed according to the above-mentioned D-amino acid oxidase and HPLC analysis method. Further, the known D-aminoacylase amano was reacted in a 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.
  • Example 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.
  • 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).
  • 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: KK 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.
  • degenerate primers were synthesized (SEQ ID NOs: 9 and 10 in Table 8), and DNA polymerase was extracted from genomic DNA extracted by a known method from Cupriavidas SP P4-10-C as a template. PCR was performed under recommended conditions using KOD-Plus (Toyobo). The PCR product amplified by the PCR was operated in accordance with its protocol using a cloning kit Target Clone-Plus (manufactured by Toyobo), cloned into the vector pBluescript, and Escherichia coli DH5 ⁇ strain competent cell (Toyobo). To obtain a transformant.
  • Target Clone-Plus manufactured by Toyobo
  • Escherichia coli DH5 ⁇ strain competent cell Toyobo
  • 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.
  • the collected cells are suspended in TE (50 mM Tris-HCl (pH 8.0), 20 mM EDTA) and washed, and after collecting the cells by centrifugation, the cells are collected again.
  • This cell was suspended in 11.3 mL of TE.
  • 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.
  • 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.
  • a DNA fragment containing a gene part outside the already obtained partial gene was obtained, and the C-terminal sequence was determined.
  • 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, so that the D-succinylase gene A DNA fragment containing the full length of was obtained.
  • the nucleotide sequence of the obtained DNA fragment was analyzed to confirm 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.
  • D4 succinylase gene derived from the P4-10-C strain was searched by NCBI-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), Peptidase SSI, No. Peptidase SSI, 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 assumed that such an enzyme hydrolyzes N-succinyl-D-amino acid.
  • Example 5 Isolation of a gene of D-succinylase derived from Cupriavidas metalidurance
  • a strain of Cupriavidus metalidurance (NBRC No. 101272) was obtained from the 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.
  • cloning kit Target Clone-Plus manufactured by Toyobo Co., Ltd.
  • the operation was performed according to the protocol, and the product was cloned into the vector pBluescript to obtain a recombinant expression plasmid pDSACm.
  • pDSACm was transformed into Escherichia coli DH5 ⁇ 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 Cupriavidas neketa
  • a strain of Cupriavidas neketter (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 D-succinylase gene derived from Ralstonia picketi
  • a strain of Ralstonia picketi (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 in which the cleavage sites of the restriction enzymes NdeI and EcoRI were respectively linked to the N-terminal and C-terminal parts of the D-succinylase gene obtained in Example 4
  • 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. In this way, a recombinant plasmid pBSKDSAP4-10-C designed to express a large amount of the D-succinylase gene was obtained.
  • Recombinant plasmid of D-succinylase gene derived from Cupriavidas metalidurance 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.
  • ligation reagent Toyobo Ligation High
  • Recombinant plasmid of the D-succinylase gene derived from Cupriavidas neketer The pDSACn obtained in Example 6 was treated with the 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.
  • 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. In this way, a recombinant plasmid pBSKDSARp designed to express a large amount of the D-succinylase gene was obtained.
  • 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 DH5 ⁇ competent cell (Toyobo Competent High DH5 ⁇ ) 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 ⁇ g / mL ampicillin) sterilized in a test tube, and then aerobically cultured by shaking at 37 ° C. for 20 hours. .
  • LB liquid medium containing 50 ⁇ g / mL ampicillin
  • 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. After removal, a crude D-succinylase enzyme solution was obtained from transformants of various origins. To 100 ⁇ l of these crude enzyme solutions, 100 ⁇ l of 150 mM N-succinyl-D-amino acid and 800 ⁇ l of 25 mM phosphate buffer (pH 7.5) were added and reacted at 37 ° C. for 16 hours.
  • 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 ⁇ g / 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 ⁇ g / 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.
  • LB medium containing 50 ⁇ g / mL of ampicillin
  • 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).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • 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 D-amino acid oxidase method was used to achieve temperature stability and high temperature stability. 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.
  • 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.
  • 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 is obtained from enzyme activity after treatment at 25 ° C. for 20 hours, and optimum pH is obtained from enzyme activity after reaction 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, the present 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.
  • Example 12 Synthesis of N-succinyl-DL-phenylalanine, N-succinyl-DL-tryptophan and N-succinyl-DL-biphenylalanine 10 g of 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.
  • 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 added with 0.05 mg of D-succinylase enzyme solution 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.
  • the optically active substance ratio of the generated phenylalanine is as follows: Column: CROWNPAK CR (+) (5 ⁇ m, 4.0 mm ⁇ ⁇ 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.
  • Example 14 Synthesis of D-phenylalanine from N-succinyl-DL-phenylalanine using D-succinylase and succinyl amino acid racemase 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 transformant D-succinylase enzyme solution and 50 mg of a succinyl amino acid racemase (SEQ ID NO: 36) solution derived from Chloroflexus aurantiacus and reacting at 45 ° C.
  • SEQ ID NO: 36 succinyl amino acid racemase
  • the optically active substance ratio is as follows: Column: CROWNPAK CR (+) (5 ⁇ m, 4.0 mm ⁇ ⁇ 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.
  • the optically active substance ratio of the generated phenylalanine was calculated. The results are shown in FIG. As is clear from FIG.
  • N-succinyl-DL-phenylalanine in the reaction solution is obtained on the fourth day of the reaction. More than 80% of the product was converted to D-phenylalanine.
  • succinyl amino acid racemase in the reaction system gives priority to conversion of a large amount of remaining N-succinyl-L-phenylalanine to N-succinyl-DL-phenylalanine and hydrolysis to D-phenylalanine. 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
  • D-succinylase and succinyl amino acid racemase 10 mL of 5 wt% N-succinyl-DL-tryptophan aqueous solution (pH 7.5) as a substrate
  • SEQ ID NO: 36 succinyl amino acid racemase
  • 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 is obtained 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.
  • SEQ ID NO: 36 succinyl amino acid racemase
  • the hydrolysis rate of D-biphenylalanine from N-succinyl-DL-biphenylalanine and the ratio of optically active substances were calculated by the following HPLC measurement method.
  • Hydrolysis rate: column: Inertsil ODS-2 (5 ⁇ m, 4.6 mm ⁇ ⁇ 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.
  • N-succinyl-DL-biphenylalanine aqueous solution N-succinyl-DL- More than 99% of biphenylalanine was hydrolyzed.
  • the optical purity on the 4th day of the reaction was 88.3% ee.
  • the D-succinylase of the present invention Since the D-succinylase of the present invention has a much 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 or the like in a high yield on an industrial scale and time.

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Abstract

La présente invention concerne une D-succinylase inédite présentant une forte activité hydrolytique sur l'acide N-succinyl-D-aminé et se montrant capable de produire de l'acide D-aminé avec un bon rendement à l'échelle industrielle et dans des délais adaptés à une production commerciale lorsqu'elle est combinée au racémase d'un acide aminé succinylique. L'invention concerne, plus précisément, une protéine qui est caractérisée en ce qu'elle peut être l'une des quatre protéines (a) à (d) suivantes : (a) une protéine encodée par un gène comprenant la séquence nucléotidique présentée par les SEQ ID NO : 2, 4, 6 ou 8 ; (b) une protéine comprenant la séquence d'acides aminés présentée par les SEQ ID NO : 1, 3, 5 ou 7 ; (c) une protéine dotée de l'activité D-succinylase et encodée par un polynucléotide capable de s'hybrider avec une séquence nucléotidique complémentaire de la séquence nucléotidique présentée par les SEQ ID NO : 2, 4, 6 ou 8 dans des conditions de stringence ; et (d) une protéine dotée d'une activité D-succinylase et comprenant une séquence d'acides aminés obtenue par substitution, délétion, insertion et/ou addition d'un ou plusieurs acides aminés dans la protéine contenant la séquence d'acides aminés présentée par les SEQ ID NO : 1, 3, 5 ou 7.
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US9795678B2 (en) 2014-05-14 2017-10-24 Adocia Fast-acting insulin composition comprising a substituted anionic compound and a polyanionic compound
WO2018088434A1 (fr) * 2016-11-10 2018-05-17 東洋紡株式会社 Procédé de production d'acide n-succinyl-hydroxy-d-amino et/ou d'acide hydroxy-d-amino
US10525133B2 (en) 2014-05-14 2020-01-07 Adocia Aqueous composition comprising at least one protein and one solubilizing agent, preparation thereof and uses thereof
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US10881716B2 (en) 2012-11-13 2021-01-05 Adocia Rapid-acting insulin formulation comprising a substituted anionic compound
US11324808B2 (en) 2012-11-13 2022-05-10 Adocia Rapid-acting insulin formulation comprising a substituted anionic compound
US9795678B2 (en) 2014-05-14 2017-10-24 Adocia Fast-acting insulin composition comprising a substituted anionic compound and a polyanionic compound
US10525133B2 (en) 2014-05-14 2020-01-07 Adocia Aqueous composition comprising at least one protein and one solubilizing agent, preparation thereof and uses thereof
US10792335B2 (en) 2015-11-16 2020-10-06 Adocia Rapid-acting insulin composition comprising a substituted citrate
WO2018088434A1 (fr) * 2016-11-10 2018-05-17 東洋紡株式会社 Procédé de production d'acide n-succinyl-hydroxy-d-amino et/ou d'acide hydroxy-d-amino
JPWO2018088434A1 (ja) * 2016-11-10 2019-10-03 東洋紡株式会社 Ｎ−サクシニル−ヒドロキシ−ｄ−アミノ酸及び／又はヒドロキシ−ｄ−アミノ酸の製造方法
JP6996714B2 (ja) 2016-11-10 2022-01-17 東洋紡株式会社 Ｎ－サクシニル－ヒドロキシ－ｄ－アミノ酸及び／又はヒドロキシ－ｄ－アミノ酸の製造方法

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