WO2012124513A1 - Modified d-succinylase having improved d-form selectivity for n-succinyl-dl-amino acid - Google Patents

Modified d-succinylase having improved d-form selectivity for n-succinyl-dl-amino acid Download PDF

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WO2012124513A1
WO2012124513A1 PCT/JP2012/055409 JP2012055409W WO2012124513A1 WO 2012124513 A1 WO2012124513 A1 WO 2012124513A1 JP 2012055409 W JP2012055409 W JP 2012055409W WO 2012124513 A1 WO2012124513 A1 WO 2012124513A1
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residue
amino acid
substitution
succinyl
base sequence
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PCT/JP2012/055409
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French (fr)
Japanese (ja)
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洋輔 角田
幸夫 岩井
西矢　芳昭
伸弥 熊谷
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東洋紡績株式会社
積水メディカル株式会社
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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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • 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
    • 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

Definitions

  • the present invention relates to a protein (hereinafter referred to as D-succinylase) which has an activity of producing D-amino acid by desuccinylation of N-succinyl-D-amino acid, which can be used for the production of D-amino acid as a raw material for intermediates such as pharmaceuticals.
  • D-succinylase a protein which has an activity of producing D-amino acid by desuccinylation of N-succinyl-D-amino acid, which can be used for the production of D-amino acid as a raw material for intermediates such as pharmaceuticals.
  • a modified type in which D-amino acid is efficiently generated from N-succinyl-DL-amino acid by improving D-form selectivity by amino acid substitution of wild-type D-succinylase
  • the present invention relates to a D-succinylase and an efficient method for producing a D-amino acid using such a modified D-succin
  • the present invention also relates to a method for more efficiently producing a D-amino acid from N-succinyl-DL-amino acid by using such a modified D-succinylase in combination with an 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-succinyl-DL-amino acid N-succinyl-DL-amino acid
  • D-form N-succinyl-D-amino acid
  • Patent Document 1 A method for obtaining D-amino acid by selective hydrolysis of D-succinyl with D-succinylase was proposed (Patent Document 1). Further, by using N-succinylamino acid racemase in addition to D-succinylase, the L form (N-succinyl-L-amino acid) remaining in the system without reacting with D-succinylase is racemized and generated. In addition, a method for improving the yield of D-amino acid by using the racemate again as a raw material was also proposed (see Patent Document 1).
  • the D-succinylase used in Patent Document 1 is a wild-type D-succinylase produced by a bacterium of the genus Cupriavidus (Cupriavidas). Although these wild-type D-succinylases have a considerably high activity of hydrolyzing N-succinyl-D-amino acids, the present inventors further researched that these wild-type D-succinylases Inability to completely recognize the steric form of N-succinyl-DL-amino acid serving as a substrate, that is, when wild-type D-succinylase is allowed to act on a racemic form of N-succinyl amino acid, it reacts with not only D-form but also L-form Thus, it was found that not only D-amino acids but also L-amino acids were slightly produced. Therefore, in order to efficiently implement the method of Patent Document 1, it was considered desirable to further improve the D-form selectivity by further improving the wild-type D-succinylase used therein
  • the present invention was devised in view of the current state of the prior art, and the object thereof is to further improve the efficiency of D-amino acids by further improving wild-type D-succinylase and improving D-form selectivity. It is to be able to be manufactured.
  • the present inventors have produced a D. produced by the Cupriavidus sp. P4-10-C (Cupriavidus sp. P4-10-C) strain, which is a cell line derived from the genus Cupriavidus. -In the amino acid sequence of succinylase, specific sites are involved in steric recognition of the reaction substrate, and by substituting amino acid residues at these specific sites with other specific amino acid residues, It has been found that D-form selectivity can be remarkably improved.
  • the D. succinylase of the related species Cupriavidus metallidurans Cupriavidas Metalidurans
  • Cupriavidus sp is also known as Cupriavidus sp. It was found that D-form selectivity can be remarkably improved by the same amino acid residue substitution at the same site as the P4-10-C strain.
  • a protein comprising the following amino acid sequence (A) or (B): (A) an amino acid sequence having a substitution of at least one amino acid residue selected from the following (a) to (k) in the amino acid sequence shown in SEQ ID NO: 2 (a) an arginine residue of glutamine residue at position 72 (B) substitution of glycine residue at position 181 with tryptophan residue, lysine residue, arginine residue, aspartic acid or glutamic acid residue (c) tryptophan residue at position 182, tryptophan residue, serine residue Group, cysteine residue, tyrosine residue, lysine residue, arginine residue, aspartic acid residue, glutamic acid residue or proline residue (d) threonine residue at position 183, proline residue, leucine residue Or substitution to asparagine residue (e) substitution of leucine residue at position 184 to proline residue (f) proline
  • a protein comprising the following amino acid sequence (A) or (B): (A) an amino acid sequence having a substitution of at least one amino acid residue selected from the following (a) to (e) in the amino acid sequence shown in SEQ ID NO: 4 (a) an arginine residue of a leucine residue at position 177 (B) Substitution of asparagine residue at position 180 to aspartic acid residue (c) Substitution of leucine residue at position 344 to proline residue (d) To isoleucine residue of phenylalanine residue at position 347 (E) Substitution of asparagine residue at position 457 to isoleucine residue (B) In the amino acid sequence of (A) above, at positions other than positions 177, 180, 344, 347, and 457, An amino acid sequence having substitutions, deletions, insertions, additions and / or inversions of one or several amino acid residues, which is D-form selective for N-succinyl-DL-amino acids Amino acid
  • a gene comprising the following base sequence (A) or (B): (A) a base sequence having a substitution of at least one base sequence selected from the following (a) to (e) in the base sequence shown in SEQ ID NO: 3 (a) cgt of the base sequence ctg at positions 529 to 531 , Cgc, cga, cgg, aga or agg substitution (b) substitution of base sequence aac at positions 538 to 540 to gat or gac (c) cct, ccc, cca of base sequence ctg at positions 1030 to 1032 Or substitution with ccg (d) substitution of base sequence ttc at positions 1039 to 1041 with att, atc or ata (e) substitution of base sequence aat at positions 1369 to 1371 with att, atc or ata (B) A base sequence that hybridizes with the base sequence of (A) above under stringent conditions and selectively acts on D-forms against N-succinyl
  • Nucleotide sequence encoding a protein having the activity to form D- amino Te In the amino acid sequence having 70% or more homology with SEQ ID NO: 2, any one of positions 72, 181-185, 305, 348, 351, 461, and 539 of SEQ ID NO: 2
  • a protein comprising an amino acid sequence in which the amino acid residue at the equivalent position is substituted with the amino acid residue shown in (1) of (1), which is D-form selective for N-succinyl-DL-amino acid
  • an amino acid residue at a position equivalent to any of positions 177, 180, 344, 347, and 457 of SEQ ID NO: 4 in the amino acid sequence having 70% or more homology with SEQ ID NO: 4 Is a protein comprising an amino acid sequence substituted with the amino acid residue shown in (3) (A), wherein the D-amino acid acts selectively on N-succinyl-DL-amino acid to A protein characterized by having an activity to produce.
  • a recombinant vector is prepared by inserting the gene described in (3), (4), (6), (8) or (10) into a vector, and a host cell is transformed with this recombinant vector.
  • N-succinyl-D-amino acid in N-succinyl-DL-amino acid is specifically hydrolyzed using the protein according to (1), (2), (5), (7) or (9)
  • the modified D-succinylase of the present invention has an amino acid residue at a specific site in the amino acid sequence of the wild-type D-succinylase substituted with another specific amino acid residue. D body selectivity is remarkably improved. Therefore, if the modified D-succinylase of the present invention is used, a D-amino acid useful as a raw material for intermediates such as pharmaceuticals can be produced more efficiently.
  • 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 is a diagram showing a screening method for modified D-succinylase.
  • FIG. 3 is a diagram showing an alignment result of amino acid sequences of D-succinylase derived from the genus Cupriavidus.
  • the present invention provides a modified D-succinylase in which D-type selectivity is significantly improved by substituting wild-type D-succinylase with an amino acid at a specific site as compared with wild-type D-succinylase.
  • the wild-type D-succinylase used as the base of modification is mainly D-succinylase of two types of bacteria belonging to the genus Cupriavidus (Cupriavidus sp. P4-10-C and Cupriavidus metallidurans).
  • Cupriavidus sp. P4-10-C and Cupriavidus metallidurans are examples of each modified D-succinylase.
  • the first aspect of the present invention relates to Cupriavidus sp.
  • the present invention relates to a modified D-succinylase based on D-succinylase of the P4-10-C strain. That is, according to the first aspect of the present invention, (A) an amino acid sequence having a substitution of at least one amino acid residue selected from the following (a) to (k) in the amino acid sequence shown in SEQ ID NO: 2
  • a protein characterized by comprising: (A) Replacement of glutamine residue at position 72 with arginine residue (b) Replacement of glycine residue at position 181 with tryptophan residue, lysine residue, arginine residue, aspartic acid or glutamic acid residue (c) Replacement of leucine residue at position 182 with tryptophan residue, serine residue, cysteine residue, tyrosine residue, lysine residue, arginine residue, aspartic acid residue, glutamic acid residue or proline residue (d) 183 Substitution of a
  • the protein of (A) above has a substitution of a leucine residue at position 182 with a glutamic acid residue and a leucine residue at position 348 into an isoleucine residue. It consists of an amino acid sequence having the following substitutions.
  • L348I single mutation does not improve the D-form selectivity, but when combined with the L182E single mutation, a synergistic effect is exhibited and the D-form selectivity is further improved. Can be made.
  • the protein (A) has a feature that it has an activity of producing D-amino acid by selectively acting on D-form with respect to N-succinyl-DL-amino acid.
  • “acting selectively in D form” means that N-succinyl-D-amino acid (D form) is more N-succinyl-L-amino acid (L form) than wild-type D-succinylase.
  • the property which is easy to react is generally called, and this is synonymous with “D-form selectivity is improved”.
  • “acting selectively in D-form” means that the degradation ratio of L-form to D-form (L / D) is lower than that of wild-type D-succinylase. Cupriavidus sp.
  • N-succinyl-L-tryptophan is used according to the method described in Example 2.
  • N-succinyl-D-tryptophan, L-form to D-form when the L-form is 4 and D-form is 1, ie, L: D 4: 1 in the amount of protein for each substrate. It means that the decomposition ratio (L / D) is less than 0.91.
  • the decomposition ratio (L / D) of the L isomer to the D isomer is preferably 0.5 or less, and more preferably 0.3 or less.
  • N-succinylphenylalanine was used as a substrate
  • 4 L-forms were obtained for each of N-succinyl-L-phenylalanine and N-succinyl-D-phenylalanine according to the method described in Example 2.
  • the decomposition ratio (L / D) of the L isomer to the D isomer is preferably 0.4 or less, and more preferably 0.2 or less.
  • N-succinylbiphenylalanine was used as the substrate, the L-form was decomposed into the D-form when reacted with the racemic N-succinyl-DL-biphenylalanine according to the method described in Example 3. It means that the ratio (L / D) is less than 0.09.
  • the decomposition ratio (L / D) of the L isomer to the D isomer is preferably 0.05 or less, and more preferably 0.02 or less.
  • N-succinyl-L-tryptophan is used according to the method described in Example 2.
  • N-succinyl-D-tryptophan, L-form relative to D-form when the L-form is reacted with the L-form, and the L-form is 1, ie, L: D 4: 1. It means that the decomposition ratio (L / D) is less than 0.54.
  • the decomposition ratio (L / D) of the L isomer to the D isomer is preferably 0.3 or less, and more preferably 0.1 or less. From a practical viewpoint, not only the decomposition ratio of the L isomer to the D isomer but also the values of the L isomer decomposition rate and the D isomer decomposition rate should be considered. Specifically, the L-isomer decomposition rate is preferably 12% or less, the L-isomer decomposition rate is preferably 12% or less, and the D-isomer decomposition rate is more preferably 25% or more, and the L-isomer decomposition rate is 0. % And the D-form decomposition rate is particularly preferably 25% or more.
  • stereoselectivity in the present invention is as shown in the examples, but the numerical value may vary depending on the reaction conditions such as the substrate concentration, enzyme amount, reaction temperature, reaction time and reaction pH.
  • D-form selectivity for N-succinyl-DL-tryptophan, N-succinyl-DL-phenylalanine and N-succinyl-DL-biphenylalanine.
  • the type D-succinylase has D-form selectivity not only for these specific N-succinyl-DL-amino acids but also for any N-succinyl-DL-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.
  • the conventionally known wild-type D-succinylase cannot strictly discriminate optical isomerism of N-succinyl-DL-amino acid, which is a precursor of D-amino acid, and acts slightly on L-form, and only D-amino acid In addition, L-amino acids are also produced.
  • the modified D-succinylase of the present invention changes the stereoselectivity by substituting a part of the amino acid residues of the wild-type D-succinylase, and selects D-form for N-succinyl-DL-amino acid.
  • D-amino acids can be selectively generated from N-succinyl-DL-amino acids since they have been modified to act in an effective manner.
  • genes which are a gene corresponding to the protein of said (A) are also provided.
  • the gene (a) 214 to 216 characterized by comprising a base sequence having at least one base sequence substitution selected from the following (a) to (k) in the base sequence shown in SEQ ID NO: 1.
  • the gene corresponding to the protein of the above (A) is the base sequence shown in SEQ ID NO: 1, the substitution of the base sequence ctg at positions 544 to 546 to gaa or gag, and the positions 1042 to 1044
  • the base sequence ctg is a base sequence having substitution to att, atc or ata. This is a gene corresponding to a double mutant of L182E + L348I.
  • the protein of the modified D-succinylase according to the first aspect of the present invention is not limited to the above (A), and in the amino acid sequence of (B) (A), positions 72, 181-185, 305,
  • An amino acid sequence having substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues at positions other than positions 348, 351, 461, and 539, and N-succinyl -Also includes a protein characterized by comprising an amino acid sequence encoding a protein having an activity of producing a D-amino acid by acting D-selectively on a DL-amino acid.
  • the modified D-succinylase gene according to the first aspect of the present invention is not limited to the gene of (A) above, and (B) a nucleotide sequence that hybridizes with the nucleotide sequence of (A) under stringent conditions. And a gene characterized by comprising a base sequence encoding a protein having an activity of producing a D-amino acid by selectively acting on D-form to N-succinyl-DL-amino acid. 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.
  • modified D-succinylase gene of the present invention when the modified D-succinylase gene of the present invention is incorporated into a host organism (such as E. coli) other than the organism from which the modified D-succinylase gene of the present invention is expressed, This is because the base sequence may be changed according to the codon usage.
  • “one or several” is a range that does not significantly impair the three-dimensional structure of the amino acid residue protein, the D-succinylase activity, and the D-form selectivity to N-succinyl-DL-amino acid. Specifically, the number is 1 to 50, preferably 1 to 30, more preferably 1 to 20, and still more preferably 1 to 10. However, in the case of an amino acid sequence containing substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing, 37 ° C., pH 7.
  • stringent conditions refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
  • DNAs having high homology for example, DNAs having a homology of 85% or more, preferably 90% or more, more preferably 95% or more, are present.
  • the conditions are such that the DNAs that hybridize with each other and the DNAs with lower homology do not hybridize with each other (here, the homology is homologous (homology)). Or 37 ° C., 0.1 ⁇ SSC, 0.1% SDS, preferably 60 ° C., 0.1 ⁇ SSC, 0.8%, which are washing conditions for normal Southern hybridization.
  • Conditions include hybridization at a salt concentration corresponding to 1% SDS, more preferably 65 ° C., 0.1 ⁇ SSC, corresponding to 0.1% SDS.
  • a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing the sequence number in the sequence listing under conditions of 37 ° C and pH 7.0 3% or more, preferably 10% or more, more preferably 30% or more, still more preferably 50% or more, particularly preferably 70% or more of the D-succinylase activity of the protein having the amino acid sequence described in 2. It is desirable.
  • the protein of the first aspect of the present invention and its gene are, for example, Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Selection Kit; Can be obtained by modifying the base sequence described in SEQ ID NO: 1 using a commercially available kit 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. Confirmation is made by adding the cell disruption solution of this transformant or purified enzyme protein to N-succinyl-DL-amino acid and measuring the degradation ratio of L-form to D-form by the method described in the Examples. Can do.
  • the second aspect of the present invention relates to a modified D-succinylase based on the D. succinylase of Cupriavidus metallidurans. That is, according to the second aspect of the present invention, (A) an amino acid sequence having a substitution of at least one amino acid residue selected from the following (a) to (e) in the amino acid sequence shown in SEQ ID NO: 4 A protein characterized by comprising: (A) Substitution of leucine residue at position 177 to arginine residue (b) Substitution of asparagine residue at position 180 to aspartic acid residue (c) Substitution of leucine residue at position 344 to proline residue ( d) Substitution of phenylalanine residue at position 347 to isoleucine residue (e) Substitution of asparagine residue at position 457 to isoleucine residue SEQ ID NO: 4 is the amino acid sequence of D-succinylase of Cupriavidus metallidrans.
  • the protein (A) has a feature that it has an activity of producing D-amino acid by selectively acting on D-form with respect to N-succinyl-DL-amino acid.
  • the definition of “acting selectively on D body” and the like are the same as in the first aspect, and thus description thereof is omitted.
  • a gene (a) 529 to 531 comprising a base sequence having substitution of at least one base sequence selected from the following (a) to (e) in the base sequence shown in SEQ ID NO: 3 Substitution of base sequence ctg to cgt, cgc, cga, cgg, aga or agg (b) Substitution of base sequence aac from positions 538 to 540 to gat or gac (c) Base sequence from positions 1030 to 1032 Substitution of ctg to cct, ccc, cca or ccg (d) Substitution of nucleotide sequence ttc at positions 1039 to 1041 to att, atc or ata (e) Att, atc of nucleotide sequence aat at positions 1369 to 1371 Alternatively, substitution to atata SEQ ID NO: 3 is the following (a) to (e) in the base sequence shown in SEQ ID NO: 3 Substitution of base sequence ctg to cgt, cg
  • the modified D-succinylase protein of the second aspect of the present invention is not limited to the above (A), and (B) in the amino acid sequence of (A), positions 177, 180, 344, 347 And an amino acid sequence having substitutions, deletions, insertions, additions and / or inversions of one or several amino acid residues at positions other than position 457, wherein D is an N-succinyl-DL-amino acid
  • a protein comprising an amino acid sequence encoding a protein having an activity of producing a D-amino acid by acting selectively on the body.
  • the modified D-succinylase gene of the second aspect of the present invention is not limited to the gene of (A) above, and (B) a nucleotide sequence that hybridizes with the nucleotide sequence of (A) under stringent conditions. And a gene characterized by comprising a base sequence encoding a protein having an activity of producing a D-amino acid by selectively acting on D-form to N-succinyl-DL-amino acid.
  • the definition of “one or several” and “stringent conditions”, and the method for creating and confirming the activity of the protein and gene of the second aspect are the same as those of the first aspect, and thus the description thereof is omitted. .
  • the modified D-succinylase based on D-succinylase of P4-10-C strain and Cupriavidus metallidurans D-succinylase has been described.
  • the modified D-succinylase of the present invention is based on the D-succinylase of these bacteria. And those based on D-succinylase of related species including other bacteria of the genus Cupriavidus.
  • D-succinylase of the close relative of P4-10-C strain or Cupriavidus metallidurans Cupriavidus sp.
  • D-form selectivity is improved by substituting amino acid residues at positions equivalent to the amino acid residues involved in stereoselectivity in the amino acid sequence of P4-10-C strain or Cupriavidus metallidurans D-succinylase it is conceivable that.
  • Transmuter Mutagenesis Kit manufactured by Clonetech; EXOIII / Mung Bean Deletion Kit; manufactured by Stratagene; QuickChange Site Directed Mutesisis Kit;
  • the base sequence corresponding to the amino acid sequence of the base D-succinylase can be mutated site-specifically.
  • 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 purified enzyme protein is added to N-succinyl-DL amino acid, and the degradation ratio of L-form to D-form is measured by the method described in the Examples. it can.
  • 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.
  • 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.
  • the culture was carried out for 2 days at 35 ° C., 150 r / min, with rotary stirring, using 27 mediums that were autoclaved by adding 200 mL of the above medium to a 500 mL flask.
  • the turbidity (ABS 660 nm) at the end of the culture was 2.7, and the pH was 8.6.
  • the cells were collected by centrifugation at 8000 r / min for 30 minutes using a cooled centrifuge (manufactured by Hitachi Koki Co., Ltd.). The collected cells were washed with 20 mM HEPES-NaOH (pH 7.5) buffer, and then centrifuged again to obtain 36 g of cells.
  • 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. Furthermore, the following operation was performed to obtain a full-length sequence. Using TAKARA LA PCR In Vitro Cloning Kit (manufactured by Takara Bio Inc.), the operation was performed according to the protocol, and a DNA fragment from a known sequence toward the C-terminal was successfully amplified. The base sequence up to the partial gene sequence was determined.
  • a DNA primer (SEQ ID NO: 9 in Table 1) having a sequence in which an NdeI cleavage site is bound 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
  • a DNA primer (SEQ ID NO: 10 in Table 1) having a sequence to which the sites are bound
  • the DNA between this sequence is amplified by PCR using the previously obtained DNA as a template, thereby including the full length of the succinylase gene.
  • a DNA fragment 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.
  • the obtained base sequence and amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively.
  • penicillin acylase family to which this penicillin amidase belongs is an enzyme that hydrolyzes penicillin G, cephalosporin C, etc., and it is presumed that such an enzyme hydrolyzes N-succinyl-D-amino acid. It was difficult, and it was almost impossible to obtain this gene by homology search predicted from the function.
  • CmDSA gene D-succinylase gene derived from Cupriavidus metallidurans strain and Cupriavidus sp. Cloning of D-succinylase from Cupriavidus metallidurans, a related species of P4-10-C, was also performed.
  • 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 pCmDSA.
  • pCmDSA was transformed into Escherichia coli DH5 ⁇ competent cell (manufactured by Toyobo) to obtain the 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 obtained base sequence and amino acid sequence are shown in SEQ ID NOs: 3 and 4, respectively.
  • the ligated DNA was transformed into Escherichia coli DH5 ⁇ strain competent cells (Toyobo Competent High DH5 ⁇ ) according to the protocol attached to the product to obtain the transformants.
  • Escherichia coli DH5 ⁇ strain competent cells Toyobo Competent High DH5 ⁇
  • pBSKP4DSA designed to express a large amount of the D-succinylase gene was obtained.
  • the plasmid was extracted from the mutant which improved stereoselectivity, the P4DSA gene whole base sequence was confirmed, and the mutation location was identified.
  • the 72nd, 176th, 181 to 185th, 286th, 305th, 348th, 351st, 388th, 461st, 518th and 539th amino acid residues contribute to the improvement of stereoselectivity. I was listed as a candidate.
  • modified plasmid (optimization of substituted amino acids) Among the mutant strains obtained in (1), there were mutant strains having a plurality of amino acid mutations. In addition, in order to examine the types of amino acids to be substituted, site-directed mutations were mutated. A modified DSA expression plasmid was prepared. For the production of site-specific mutations, QuikChange Site-Directed Mutagenesis Kit manufactured by STRATAGENE was used. In addition, since the substitution amino acid is also optimized when mutating, the 72nd, 181st to 185th, 305th, 348th, 351 estimated to have contributed to the improvement of stereoselectivity in (2).
  • the name of the modified enzyme is expressed in the order of “amino acid residue ⁇ residue number ⁇ substituted amino acid residue in the amino acid sequence of the wild-type enzyme”.
  • the L182R modified enzyme means a modified enzyme in which the 182nd Leu (L) residue in the amino acid sequence of the wild-type enzyme is substituted with an Arg (R) residue.
  • X in L182X means any amino acid among the 20 types of amino acids.
  • Example 2 Evaluation of stereoselectivity for N-succinyltryptophan and N-succinylphenylalanine using modified P4DSA Using the crude enzyme solution prepared in Example 1, stereoselectivity for N-succinyltryptophan and N-succinylphenylalanine was evaluated. Specifically, the crude enzyme solution was added to each of the N-succinyl-D-amino acid solution and the N-succinyl-L-amino acid solution having the following composition.
  • N-succinyl-D-tryptophan solution 25 mM KPB (pH 7.0), 1% N-succinyl-D-tryptophan N-succinyl-L-tryptophan solution: 25 mM KPB (pH 7.0), 1% N-succinyl-L -Tryptophan N-succinyl-D-phenylalanine solution: 25 mM KPB (pH 7.0), 1% N-succinyl-D-phenylalanine N-succinyl-L-phenylalanine solution: 25 mM KPB (pH 7.0), 1% N-succinyl -L-Phenylalanine After incubating the obtained reaction solution at 40 ° C.
  • the decomposition rate and the decomposition ratio of L-form to D-form were calculated from the remaining substrate concentration.
  • the reaction was performed at a protein ratio of 1: 1.
  • the enzyme protein concentration is adjusted so that the final enzyme protein concentration is 2.0 mg / ml in the case of L-form reaction, and the final enzyme protein concentration is 0.5 mg / ml in the case of D-form reaction. Adjusted as follows. The results are shown in Table 3.
  • Example 3 Evaluation of Stereoselectivity for N-Succinylbiphenylalanine Using Modified P4DSA
  • Example 2 it was clarified that it was involved in stereoselectivity for N-succinyltryptophan and N-succinylphenylalanine. It was confirmed whether the modified P4DSA acted selectively to other aromatic amino acids on the D form.
  • N-succinylbiphenylalanine which is a kind of non-natural amino acid, is selected and the crude enzyme solution prepared in Example 1 is used to perform N-succinyl in the same procedure as in Example 2. The stereoselectivity for biphenylalanine was evaluated.
  • Example 4 N-succinyl-DL-tryptophan to D-tryptophan, N-succinyl-DL-phenylalanine to D-phenylalanine and N-succinyl-DL-, using modified P4DSA and N-succinyl amino acid racemase from Chloroflexus aurantiacus Synthesis of D-biphenylalanine from biphenylalanine (1) Preparation of Modified P4DSA (L182E, L182P, R305N) A colony of E.
  • Example 2 coli transformants expressing the modified P4DSA (L182E, L182P, R305N) obtained in Example 1 was placed in 5 mL of LB medium ( Inoculating ampicillin (containing 50 ⁇ g / mL), and culturing for 16 hours at 180 ° C. and 30 ° C. to obtain a seed culture solution.
  • This seed culture was inoculated into 60 mL of TB medium (containing 50 ⁇ g / mL of ampicillin) contained in a 500 mL Sakaguchi flask, and cultured at a shaking speed of 310 rpm and 30 ° C. for 18 hours.
  • Turbidity (Abs 660 nm) at the end of the culture was 15.0, 16.2, and 15.5.
  • the obtained cells were collected by centrifugation, suspended in a 25 mM phosphate buffer solution (pH 7.0), and crushed using an ultrasonic cell crusher under ice cooling. Thereafter, heat treatment is performed at 65 ° C. for 1 hour, and then desalting is performed by a general method, and an enzyme sample for amino acid synthesis (L182E: 27.9 mg / mL, 5.2 U / mL, L182P: 7. 3 mg / mL, 1.8 U / mL, R305N: 28.5 mg / mL, 4.3 U / mL). Note that N-succinyl-D-tryptophan was used as the enzyme activity substrate.
  • R305N enzyme solution (0.16 U, 0.2 U, 0.08 U) was added to each substrate, and 25 mg (6400 U) of an N-succinyl amino acid racemase solution derived from Chloroflexus aurantiacus strain prepared in Reference Example 5 was added to each substrate. And reacted at 45 ° C. for 3 days.
  • a wild-type DSA enzyme solution prepared by the method described in the examples of PCT / JP2011 / 064943 was used, and reacted in the same manner to calculate the conversion rate and the optical purity of D-amino acid.
  • the conversion rate is determined by measuring the peak area value of the substrate before and after the enzyme reaction by high performance liquid chromatography using “Inertsil ODS-3” (5 ⁇ m, 4.6 ⁇ 100 mm) manufactured by GL Sciences, Inc. Calculated by The optical purity was calculated by measuring the optical purity of the free amino acid produced by the above-described optical resolution column “CROWNPAK CR (+)” (5 ⁇ m, 4.0 ⁇ 150 mm) manufactured by Daicel Chemical Industries, Ltd. The results are shown in Tables 5-7.
  • the conversion rate on the third day of the reaction in D-tryptophan synthesis was 83%, 87%, 86%, and 89% in the order of wild-type DSA, L182E, L182P, and R305N.
  • the conversion rate on the third day of the reaction in the synthesis of phenylalanine is 81%, 84%, 83%, 84% in the order of wild-type DSA, L182E, L182P, and R305N, and the third day of the reaction in the synthesis of D-biphenylalanine.
  • the conversion rates were 96%, 91%, 96%, and 91% in the order of wild type DSA, L182E, L182P, and R305N, and the reaction proceeded without any problem. Further, as shown in Tables 5 to 7, in any modified DSA, when N-succinyl-DL-amino acid constituted by an aromatic amino acid is used as a substrate, compared to the case where wild-type DSA is used. It was confirmed that a highly pure aromatic D-amino acid could be produced. In particular, when L-182E is used to produce D-tryptophan and D-biphenylalanine, 99.6% ee, 99.2% ee, and L182P are used to produce D-biphenylalanine, respectively.
  • Example 5 D-Tryptophan from N-succinyl-DL-tryptophan using N-succinyl-DL-tryptophan using double mutant modified P4DSA (L182E + L348I) and N-succinyl amino acid racemase from Chloroflexus aurantiacus D-phenylalanine from N-succinyl-DL-phenylalanine And D-biphenylalanine synthesis from N-succinyl-DL-biphenylalanine (1) Construction of double-mutant modified plasmids To obtain a plasmid that expresses double-mutant variants, they were obtained in Example 1.
  • PCR was carried out by the method described above.
  • the 182nd leucine was replaced with glutamic acid, and the 348th leucine was replaced with isoleucine.
  • the expression plasmid containing the heavy variant modified P4DSA (L182E + L348I) was constructed.
  • the D-succinylase of the present invention is used for the production of D-amino acids on an industrial scale, the slight improvement in the optical purity of the produced D-amino acids means a significant improvement resulting in a reduction in production costs. You can say that.
  • the single mutation of L348I does not show an effect of improving the selectivity compared to the wild type, and it is confirmed that the combination with L182E contributes to a synergistic improvement of stereoselectivity. It was done.
  • the D-succinylase of the present invention is used for the production of D-amino acids on an industrial scale, the slight improvement in the optical purity of the produced D-amino acids means a significant improvement resulting in a reduction in production costs. You can say that.
  • the single mutation of L348I does not show an effect of improving the selectivity compared to the wild type, and it is confirmed that the combination with L182E contributes to a synergistic improvement of stereoselectivity. It was done.
  • the optical purity of D-biphenylalanine on the third day of the reaction is 88.3% ee for the wild type, whereas it is 99.2% ee for the single mutation type of L182E, and the double mutation of L182E + L348I.
  • the modified type is 99.4% ee, and the optical purity of each modified type is markedly improved compared to the wild type.
  • the double mutant modified type has a further improved optical purity than the single mutant modified type.
  • the D-succinylase of the present invention is used for the production of D-amino acids on an industrial scale, the slight improvement in the optical purity of the produced D-amino acids means a significant improvement resulting in a reduction in production costs. You can say that.
  • the single mutation of L348I does not show an effect of improving the selectivity compared to the wild type, and it is confirmed that the combination with L182E contributes to a synergistic improvement of stereoselectivity. It was done.
  • Example 6 Cupriavidus sp. Comparison of homology of amino acid sequences between D-succinylase derived from P4-10-C strain and D-succinylase of related species Cupriavidus sp. In order to examine whether amino acid residues involved in stereoselectivity revealed in D-succinylase derived from P4-10-C strain are conserved in other D. succinylases of the genus Cupriavidus, Cupriavidus sp . A homology comparison of the amino acid sequences of D4 succinylases of P4-10-C, Cupriavidus metallidrans, Cupriavidus necator and Cupriavidus taiwanensis was performed. Cupriavidus sp.
  • Example 7 Preparation of Modified Cupriavidus metallidurans-derived D-succinylase (hereinafter referred to as CmDSA gene) Based on the alignment results of the amino acid sequence of Example 6, the related species are also involved in the stereoselectivity of P4DSA. Substituents, 181 to 185, 305, 348, 351, 461, 539, and D-form selective action on N-succinyl-DL-amino acid by amino acid substitution at equivalent sites In order to show that this is the case, it was decided to create a modified CmDSA of Cupriavidus metallidurans, a kind of related species, and evaluate the stereoselectivity.
  • CmDSA gene Modified Cupriavidus metallidurans-derived D-succinylase
  • modified CmDSA gene expression plasmid A modified CmDSA gene expression plasmid was constructed in the same manner as described in Example 1 (3). Table 12 shows the sequences of the prepared modified enzymes and the synthetic oligo DNA primers used for introducing the mutation.
  • Example 8 Evaluation of stereoselectivity for N-succinyltryptophan using modified CmDSA Stereoselection for N-succinyltryptophan using the crude enzyme solution prepared in Example 7 under the same conditions and procedures as in Example 1. Sexuality was evaluated. The results are shown in Table 13.
  • the modified D-succinylase of the present invention has a significantly improved D-form selectivity compared to the wild-type D-succinylase, so that it can efficiently produce a D-amino acid useful as a raw material for intermediates such as pharmaceuticals. Useful for.
  • SEQ ID Nos: 5 to 54 are the sequences of the designed polynucleotides described in the examples.

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Abstract

The present invention enables the production of a D-amino acid at an improved efficiency by further modifying wild type D-succinylase to improve the D-form selectivity thereof. A modified D-succinylase obtained by, in the amino acid sequence of wild type D-succinylase derived from Cupriavidus sp. P4-10-C or Cupriavidus metallidurans, substituting an amino acid residue at a specific position by a specific amino acid residue.

Description

Ｎ－サクシニル－ＤＬ－アミノ酸に対する向上されたＤ体選択性を有する改変型Ｄ－サクシニラーゼModified D-succinylase with improved D-form selectivity for N-succinyl-DL-amino acids
　本発明は、医薬品等の中間体の原料となるＤ－アミノ酸の製造に利用できる、Ｎ－サクシニル－Ｄ－アミノ酸を脱サクシニル化してＤ－アミノ酸を生成する活性を有するタンパク質（以下、Ｄ－サクシニラーゼと呼ぶ）に関し、詳しくは、野生型Ｄ－サクシニラーゼをアミノ酸置換することにより、Ｄ体選択性を向上させて、Ｎ－サクシニル－ＤＬ－アミノ酸からＤ－アミノ酸を効率よく生成できるようにした改変型Ｄ－サクシニラーゼ、及びかかる改変型Ｄ－サクシニラーゼを使用した効率的なＤ－アミノ酸の製造方法に関する。また、本発明は、かかる改変型Ｄ－サクシニラーゼと、Ｎ－サクシニルアミノ酸ラセマーゼを組み合わせて使用することにより、Ｎ－サクシニル－ＤＬ－アミノ酸からＤ－アミノ酸をさらに効率的に製造する方法に関する。 The present invention relates to a protein (hereinafter referred to as D-succinylase) which has an activity of producing D-amino acid by desuccinylation of N-succinyl-D-amino acid, which can be used for the production of D-amino acid as a raw material for intermediates such as pharmaceuticals. Specifically, a modified type in which D-amino acid is efficiently generated from N-succinyl-DL-amino acid by improving D-form selectivity by amino acid substitution of wild-type D-succinylase The present invention relates to a D-succinylase and an efficient method for producing a D-amino acid using such a modified D-succinylase. The present invention also relates to a method for more efficiently producing a D-amino acid from N-succinyl-DL-amino acid by using such a modified D-succinylase in combination with an N-succinyl amino acid racemase.
　光学活性アミノ酸は、医薬品、農薬及び食品などの分野で多くの需要があるが、Ｄ－アミノ酸は、光学的に純粋なアミノ酸、特に発酵法などの産物（天然のアミノ酸）として得ることが難しい。従って、Ｄ－アミノ酸を、いかにして効率的に製造するかは、産業上重要な課題となっている。 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.
　この課題に対して、本発明者らは、既に、Ｎ－サクシニルアミノ酸のラセミ体（Ｎ－サクシニル－ＤＬ－アミノ酸）を合成し、このラセミ体中のＤ体（Ｎ－サクシニル－Ｄ－アミノ酸）をＤ－サクシニラーゼでＤ体選択的に加水分解することによりＤ－アミノ酸を得る方法を提案した（特許文献１）。また、Ｄ－サクシニラーゼに加えてＮ－サクシニルアミノ酸ラセマーゼを使用することにより、Ｄ－サクシニラーゼに対して反応せずに系中に残存したＬ体（Ｎ－サクシニル－Ｌ－アミノ酸）をラセミ化し、生じたラセミ体を再度原料として使用することにより、Ｄ－アミノ酸の収率を向上させる方法も提案した（特許文献１参照）。 In response to this problem, the present inventors have already synthesized a racemate of N-succinyl amino acid (N-succinyl-DL-amino acid), and D-form (N-succinyl-D-amino acid) in this racemate. A method for obtaining D-amino acid by selective hydrolysis of D-succinyl with D-succinylase was proposed (Patent Document 1). Further, by using N-succinylamino acid racemase in addition to D-succinylase, the L form (N-succinyl-L-amino acid) remaining in the system without reacting with D-succinylase is racemized and generated. In addition, a method for improving the yield of D-amino acid by using the racemate again as a raw material was also proposed (see Patent Document 1).
　特許文献１で使用したＤ－サクシニラーゼは、Ｃｕｐｒｉａｖｉｄｕｓ属（クプリアビダス属）の細菌が生産する野生型のＤ－サクシニラーゼである。これらの野生型のＤ－サクシニラーゼは、Ｎ－サクシニル－Ｄ－アミノ酸を加水分解する活性がかなり高いが、本発明者らがさらに研究を進めたところ、これらの野生型のＤ－サクシニラーゼは、原料基質となるＮ－サクシニル－ＤＬ－アミノ酸の立体を完全に認識できないこと、すなわち、野生型のＤ－サクシニラーゼをＮ－サクシニルアミノ酸のラセミ体に作用させた場合、Ｄ体だけでなくＬ体とも反応して、Ｄ－アミノ酸だけでなくＬ－アミノ酸もわずかに生成してしまうことが判明した。従って、特許文献１の方法を効率的に実施するためには、そこで使用する野生型のＤ－サクシニラーゼをさらに改良してＤ体選択性を向上させることが望ましいと考えられた。 The D-succinylase used in Patent Document 1 is a wild-type D-succinylase produced by a bacterium of the genus Cupriavidus (Cupriavidas). Although these wild-type D-succinylases have a considerably high activity of hydrolyzing N-succinyl-D-amino acids, the present inventors further researched that these wild-type D-succinylases Inability to completely recognize the steric form of N-succinyl-DL-amino acid serving as a substrate, that is, when wild-type D-succinylase is allowed to act on a racemic form of N-succinyl amino acid, it reacts with not only D-form but also L-form Thus, it was found that not only D-amino acids but also L-amino acids were slightly produced. Therefore, in order to efficiently implement the method of Patent Document 1, it was considered desirable to further improve the D-form selectivity by further improving the wild-type D-succinylase used therein.
特願２０１０－１４８７９５号Japanese Patent Application No. 2010-148895
　本発明は、かかる従来技術の現状に鑑み創案されたものであり、その目的は、野生型のＤ－サクシニラーゼをさらに改良してＤ体選択性を向上させることにより、Ｄ－アミノ酸をさらに効率的に製造することができるようにすることにある。 The present invention was devised in view of the current state of the prior art, and the object thereof is to further improve the efficiency of D-amino acids by further improving wild-type D-succinylase and improving D-form selectivity. It is to be able to be manufactured.
　本発明者らは、上記目標を達成するために鋭意検討した結果、Ｃｕｐｒｉａｖｉｄｕｓ属由来の細胞株であるＣｕｐｒｉａｖｉｄｕｓ　ｓｐ.Ｐ４－１０－Ｃ（クプリアビダス・エスピー．Ｐ４－１０－Ｃ）株の生産するＤ－サクシニラーゼのアミノ酸配列において、特定の部位が反応基質の立体認識に関与すること、そして、これらの特定の部位のアミノ酸残基を別の特定のアミノ酸残基に置換することにより、Ｄ－サクシニラーゼのＤ体選択性を著しく向上させることができることを見出した。また、近縁種のＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓ（クプリアビダス・メタリデュランス）のＤ－サクシニラーゼにおいても、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株と同等の部位における同様のアミノ酸残基置換により、Ｄ体選択性を著しく向上することができることを見出した。 As a result of diligent studies to achieve the above-mentioned goal, the present inventors have produced a D. produced by the Cupriavidus sp. P4-10-C (Cupriavidus sp. P4-10-C) strain, which is a cell line derived from the genus Cupriavidus. -In the amino acid sequence of succinylase, specific sites are involved in steric recognition of the reaction substrate, and by substituting amino acid residues at these specific sites with other specific amino acid residues, It has been found that D-form selectivity can be remarkably improved. In addition, the D. succinylase of the related species Cupriavidus metallidurans (Cupriavidas Metalidurans) is also known as Cupriavidus sp. It was found that D-form selectivity can be remarkably improved by the same amino acid residue substitution at the same site as the P4-10-C strain.
　本発明は、上記の見地に基づいて完成されたものであり、以下の（１）～（１３）を要旨とするものである。
（１）下記（Ａ）又は（Ｂ）のアミノ酸配列からなることを特徴とするタンパク質。
（Ａ）配列番号２に示すアミノ酸配列において、下記（ａ）～（ｋ）から選択される少なくとも１個のアミノ酸残基の置換を有するアミノ酸配列
（ａ）７２位のグルタミン残基のアルギニン残基への置換
（ｂ）１８１位のグリシン残基のトリプトファン残基、リジン残基、アルギニン残基、アスパラギン酸又はグルタミン酸残基への置換
（ｃ）１８２位のロイシン残基のトリプトファン残基、セリン残基、システイン残基、チロシン残基、リジン残基、アルギニン残基、アスパラギン酸残基、グルタミン酸残基又はプロリン残基への置換
（ｄ）１８３位のスレオニン残基のプロリン残基、ロイシン残基又はアスパラギン残基への置換
（ｅ）１８４位のロイシン残基のプロリン残基への置換
（ｆ）１８５位のアスパラギン残基のプロリン残基、フェニルアラニン残基、セリン残基又はアスパラギン酸残基への置換
（ｇ）３０５位のアルギニン残基のスレオニン残基、アラニン残基、グリシン残基、ヒスチジン残基、グルタミン残基、セリン残基、アスパラギン残基又はバリン残基への置換
（ｈ）３４８位のロイシン残基のグルタミン酸残基、プロリン残基、メチオニン残基、トリプトファン残基、セリン残基、スレオニン残基、システイン残基、リジン残基、ヒスチジン残基又はグルタミン残基への置換
（ｉ）３５１位のフェニルアラニン残基のロイシン残基、イソロイシン残基、メチオニン残基、アスパラギン残基又はグルタミン残基への置換
（ｊ）４６１位のアスパラギン残基のイソロイシン残基、フェニルアラニン残基、スレオニン残基、リジン残基又はアルギニン残基への置換
（ｋ）５３９位のグリシン残基のプロリン残基、バリン残基、メチオニン残基、スレオニン残基又はアスパラギン残基への置換
（Ｂ）上記（Ａ）のアミノ酸配列において、７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位、及び５３９位以外の箇所に、１若しくは数個のアミノ酸残基の置換、欠失、挿入、付加および／または逆位を有するアミノ酸配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードするアミノ酸配列。
（２）（１）の（Ａ）のアミノ酸配列が、配列番号２に示すアミノ酸配列において、１８２位のロイシン残基のグルタミン酸残基への置換、及び３４８位のロイシン残基のイソロイシン残基への置換を有するアミノ酸配列であることを特徴とする（１）に記載のタンパク質。
（３）下記（Ａ）又は（Ｂ）の塩基配列からなることを特徴とする遺伝子。
（Ａ）配列番号１に示す塩基配列において、下記（ａ）～（ｋ）から選択される少なくとも１個の塩基配列の置換を有する塩基配列
（ａ）２１４～２１６位の塩基配列ｃａａの、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ又はａｇｇへの置換
（ｂ）５４１～５４３位の塩基配列ｇｇｃの、ｔｇｇ、ａａａ、ａａｇ、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ、ａｇｇ、ｇａｔ、ｇａｃ、ｇａａ又はｇａｇへの置換
（ｃ）５４４～５４６位の塩基配列ｃｔｇの、ｔｇｇ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ｔｇｔ、ｔｇｃ、ｔａｔ、ｔａｃ、ａａａ、ａａｇ、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ、ａｇｇ、ｇａｔ、ｇａｃ、ｇａａ、ｇａｇ、ｃｃｔ、ｃｃｃ、ｃｃａ又はｃｃｇへの置換
（ｄ）５４７～５４９位の塩基配列ａｃｇの、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ｔｔａ、ｔｔｇ、ｃｔｔ、ｃｔｃ、ｃｔａ、ｃｔｇ、ａａｔ又はａａｃへの置換
（ｅ）５５０～５５２位の塩基配列ｃｔｇの、ｃｃｔ、ｃｃｃ、ｃｃａ又はｃｃｇへの置換
（ｆ）５５３～５５５位の塩基配列ａａｔの、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ｔｔｔ、ｔｔｃ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ｇａｔ又はｇａｃへの置換
（ｇ）９１３～９１５位の塩基配列ｃｇｇの、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ｇｃｔ、ｇｃｃ、ｇｃａ、ｇｃｇ、ｇｇｔ、ｇｇｃ、ｇｇａ、ｇｇｇ、ｃａｔ、ｃａｃ、ｃａａ、ｃａｇ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ａａｔ、ａａｃ、ｇｔｔ、ｇｔｃ、ｇｔａ又はｇｔｇへの置換
（ｈ）１０４２～１０４４位の塩基配列ｃｔｇの、ｇａａ、ｇａｇ、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ａｔｇ、ｔｇｇ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ｔｇｔ、ｔｇｃ、ａａａ、ａａｇ、ｃａｔ、ｃａｃ、ｃａａ又はｃａｇへの置換
（ｉ）１０５１～１０５３位の塩基配列ｔｔｃの、ｔｔａ、ｔｔｇ、ｃｔｔ、ｃｔｃ、ｃｔａ、ｃｔｇ、ａｔｔ、ａｔｃ、ａｔａ、ａｔｇ、ａａｔ、ａａｃ、ｃａａ又はｃａｇへの置換
（ｊ）１３８１～１３８３位の塩基配列ａａｃの、ａｔｔ、ａｔｃ、ａｔａ、ｔｔｔ、ｔｔｃ、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ａａａ、ａａｇ、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ又はａｇｇへの置換
（ｋ）１６１５～１６１７位の塩基配列ｇｇｃの、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ｇｔｔ、ｇｔｃ、ｇｔａ、ｇｔｇ、ａｔｇ、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ａａｔ又はａａｃへの置換
（Ｂ）上記（Ａ）の塩基配列とストリンジェントな条件でハイブリダイズする塩基配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードする塩基配列。
（４）（３）の（Ａ）の塩基配列が、配列番号１に示す塩基配列において、５４４～５４６位の塩基配列ｃｔｇの、ｇａａ又はｇａｇへの置換、及び１０４２～１０４４位の塩基配列ｃｔｇの、ａｔｔ，ａｔｃ又はａｔａへの置換を有する塩基配列であることを特徴とする（３）に記載の遺伝子。
（５）下記（Ａ）又は（Ｂ）のアミノ酸配列からなることを特徴とするタンパク質。
（Ａ）配列番号４に示すアミノ酸配列において、下記（ａ）～（ｅ）から選択される少なくとも１個のアミノ酸残基の置換を有するアミノ酸配列
（ａ）１７７位のロイシン残基のアルギニン残基への置換
（ｂ）１８０位のアスパラギン残基のアスパラギン酸残基への置換
（ｃ）３４４位のロイシン残基のプロリン残基への置換
（ｄ）３４７位のフェニルアラニン残基のイソロイシン残基への置換
（ｅ）４５７位のアスパラギン残基のイソロイシン残基への置換
（Ｂ）上記（Ａ）のアミノ酸配列において、１７７位、１８０位、３４４位、３４７位、及び４５７位以外の箇所に、１若しくは数個のアミノ酸残基の置換、欠失、挿入、付加および／または逆位を有するアミノ酸配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードするアミノ酸配列。
（６）下記（Ａ）又は（Ｂ）の塩基配列からなることを特徴とする遺伝子。
（Ａ）配列番号３に示す塩基配列において、下記（ａ）～（ｅ）から選択される少なくとも１個の塩基配列の置換を有する塩基配列
（ａ）５２９～５３１位の塩基配列ｃｔｇの、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ又はａｇｇへの置換
（ｂ）５３８～５４０位の塩基配列ａａｃの、ｇａｔ又はｇａｃへの置換
（ｃ）１０３０～１０３２位の塩基配列ｃｔｇの、ｃｃｔ、ｃｃｃ、ｃｃａ又はｃｃｇへの置換
（ｄ）１０３９～１０４１位の塩基配列ｔｔｃの、ａｔｔ、ａｔｃ又はａｔａへの置換
（ｅ）１３６９～１３７１位の塩基配列ａａｔの、ａｔｔ、ａｔｃ又はａｔａへの置換
（Ｂ）上記（Ａ）の塩基配列とストリンジェントな条件でハイブリダイズする塩基配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードする塩基配列。
（７）配列番号２と７０％以上の相同性を有するアミノ酸配列において、配列番号２の７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位、及び５３９位のうちのいずれかと同等な位置のアミノ酸残基が、（１）の（Ａ）に示すアミノ酸残基に置換されているアミノ酸配列からなるタンパク質であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有することを特徴とするタンパク質。
（８）（７）に記載のタンパク質をコードすることを特徴とする遺伝子。
（９）配列番号４と７０％以上の相同性を有するアミノ酸配列において、配列番号４の１７７位、１８０位、３４４位、３４７位、及び４５７位のうちのいずれかと同等な位置のアミノ酸残基が、（３）の（Ａ）に示すアミノ酸残基に置換されているアミノ酸配列からなるタンパク質であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有することを特徴とするタンパク質。
（１０）（９）に記載のタンパク質をコードすることを特徴とする遺伝子。
（１１）（３），（４），（６），（８）又は（１０）に記載の遺伝子をベクターに挿入して組換えベクターを調製し、この組換えベクターで宿主細胞を形質転換して形質転換体を調製し、この形質転換体を培養する工程を含むことを特徴とする（１），（２），（５），（７）又は（９）に記載のタンパク質の製造方法。
（１２）（１），（２），（５），（７）又は（９）に記載のタンパク質を用いてＮ－サクシニル－ＤＬ－アミノ酸中のＮ－サクシニル－Ｄ－アミノ酸を特異的に加水分解する工程を含むことを特徴とするＤ－アミノ酸の製造方法。
（１３）Ｎ－サクシニルアミノ酸ラセマーゼを用いてＮ－サクシニル－Ｌ－アミノ酸をラセミ化してＮ－サクシニル－Ｄ－アミノ酸を生成させる工程をさらに含むことを特徴とする（１２）に記載の方法。 The present invention has been completed based on the above viewpoint, and has the following (1) to (13).
(1) A protein comprising the following amino acid sequence (A) or (B):
(A) an amino acid sequence having a substitution of at least one amino acid residue selected from the following (a) to (k) in the amino acid sequence shown in SEQ ID NO: 2 (a) an arginine residue of glutamine residue at position 72 (B) substitution of glycine residue at position 181 with tryptophan residue, lysine residue, arginine residue, aspartic acid or glutamic acid residue (c) tryptophan residue at position 182, tryptophan residue, serine residue Group, cysteine residue, tyrosine residue, lysine residue, arginine residue, aspartic acid residue, glutamic acid residue or proline residue (d) threonine residue at position 183, proline residue, leucine residue Or substitution to asparagine residue (e) substitution of leucine residue at position 184 to proline residue (f) proline residue of asparagine residue at position 185, Substituting with a phenylalanine residue, a serine residue or an aspartic acid residue (g) a threonine residue of the arginine residue at position 305, an alanine residue, a glycine residue, a histidine residue, a glutamine residue, a serine residue, Substitution to asparagine residue or valine residue (h) glutamic acid residue, proline residue, methionine residue, tryptophan residue, serine residue, threonine residue, cysteine residue, lysine residue of 348th leucine residue Group, substitution to histidine residue or glutamine residue (i) substitution of phenylalanine residue at position 351 to leucine residue, isoleucine residue, methionine residue, asparagine residue or glutamine residue (j) position 461 Placement of asparagine residues on isoleucine, phenylalanine, threonine, lysine or arginine residues (K) Replacement of the glycine residue at position 539 with a proline residue, valine residue, methionine residue, threonine residue or asparagine residue (B) position 72, 181 to 185 in the amino acid sequence of (A) above An amino acid sequence having substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues at positions other than positions 305, 348, 351, 461 and 539 An amino acid sequence encoding a protein having an activity of producing a D-amino acid by selectively acting on D-form to N-succinyl-DL-amino acid.
(2) In the amino acid sequence of (A) in (1), in the amino acid sequence shown in SEQ ID NO: 2, substitution of a leucine residue at position 182 with a glutamic acid residue, and conversion of a leucine residue at position 348 to an isoleucine residue The protein according to (1), which is an amino acid sequence having the following substitution:
(3) A gene comprising the following base sequence (A) or (B):
(A) a base sequence having the substitution of at least one base sequence selected from the following (a) to (k) in the base sequence shown in SEQ ID NO: 1 (a) cgt of the base sequence caa at positions 214 to 216 (B) Substitution to cgg, cga, cgg, aga or agg (b) tgg, aaa, aag, cgt, cgg, cga, cgg, aga, agg, gat, gac, gaa or the base sequence ggg at positions 541 to 543 Substitution to gag (c) tgg, tct, tcc, tca, tcg, agt, agc, tgt, tgc, tat, tac, aaa, aag, cgt, cgc, cga, cgg of nucleotide sequence ctg at positions 544 to 546 , Aga, agg, gat, gac, gaa, gag, cct, ccc, cca or ccg substitution (d) nucleotide sequence from position 547 to 549 Substitution of acg to cct, ccc, cca, ccg, tta, ttg, ctt, ctc, cta, ctg, aat or aac (e) cct, ccc, cca or ccg of nucleotide sequence ctg at positions 550 to 552 (F) Substitution of base sequence aat at positions 553 to 555 to cct, ccc, cca, ccg, ttt, ttt, tct, tcc, tca, tcg, agt, agc, gat or gac (g) 913 to The base sequence cgg at position 915, act, acc, aca, acg, gct, gcc, gca, gcg, ggt, ggg, gga, ggg, cat, cac, caa, cag, tct, tcc, tca, tcg, agt, Substitution to agc, aat, aac, gtt, gtt, gta or gtg (h) bases at positions 1042 to 1044 Ctg, gaa, gag, cct, ccc, cca, ccg, atg, tgg, tct, tcc, tca, tcg, agt, agc, act, acc, aca, acg, tgt, tgc, aaa, aag, cat, Substitution to cac, caa or cag (i) substitution of base sequence ttc at positions 1051 to 1053 to tta, ttg, ctt, ctc, cta, ctg, att, atc, ata, atg, aat, aac, caa or cag Substitution (j) Substitution of nucleotide sequence aac at positions 1381 to 1383 to att, atc, ata, ttt, ttc, act, acc, aca, acg, aaa, aag, cgt, cgg, cga, cgg, aga or agg (K) cct, ccc, cca, ccg, gt of the base sequence ggc at positions 1615 to 1617 Substitution to t, gtc, gta, gtg, atg, act, acc, aca, acg, aat or aac (B) A nucleotide sequence that hybridizes with the nucleotide sequence of (A) above under stringent conditions, and N A base sequence encoding a protein having an activity of producing a D-amino acid by selectively acting on a D-form to succinyl-DL-amino acid.
(4) In the base sequence of (A) in (3), the base sequence ctg at positions 544 to 546 in the base sequence shown in SEQ ID NO: 1 is replaced with gaa or gag, and the base sequence ctg at positions 1042 to 1044 The gene according to (3), which is a base sequence having substitution to att, atc or ata.
(5) A protein comprising the following amino acid sequence (A) or (B):
(A) an amino acid sequence having a substitution of at least one amino acid residue selected from the following (a) to (e) in the amino acid sequence shown in SEQ ID NO: 4 (a) an arginine residue of a leucine residue at position 177 (B) Substitution of asparagine residue at position 180 to aspartic acid residue (c) Substitution of leucine residue at position 344 to proline residue (d) To isoleucine residue of phenylalanine residue at position 347 (E) Substitution of asparagine residue at position 457 to isoleucine residue (B) In the amino acid sequence of (A) above, at positions other than positions 177, 180, 344, 347, and 457, An amino acid sequence having substitutions, deletions, insertions, additions and / or inversions of one or several amino acid residues, which is D-form selective for N-succinyl-DL-amino acids Amino acid sequence encoding a protein having an activity to produce the effect to D- amino acids.
(6) A gene comprising the following base sequence (A) or (B):
(A) a base sequence having a substitution of at least one base sequence selected from the following (a) to (e) in the base sequence shown in SEQ ID NO: 3 (a) cgt of the base sequence ctg at positions 529 to 531 , Cgc, cga, cgg, aga or agg substitution (b) substitution of base sequence aac at positions 538 to 540 to gat or gac (c) cct, ccc, cca of base sequence ctg at positions 1030 to 1032 Or substitution with ccg (d) substitution of base sequence ttc at positions 1039 to 1041 with att, atc or ata (e) substitution of base sequence aat at positions 1369 to 1371 with att, atc or ata (B) A base sequence that hybridizes with the base sequence of (A) above under stringent conditions and selectively acts on D-forms against N-succinyl-DL-amino acids. Nucleotide sequence encoding a protein having the activity to form D- amino Te.
(7) In the amino acid sequence having 70% or more homology with SEQ ID NO: 2, any one of positions 72, 181-185, 305, 348, 351, 461, and 539 of SEQ ID NO: 2 A protein comprising an amino acid sequence in which the amino acid residue at the equivalent position is substituted with the amino acid residue shown in (1) of (1), which is D-form selective for N-succinyl-DL-amino acid A protein characterized in that it has an activity of acting on D to produce a D-amino acid.
(8) A gene encoding the protein according to (7).
(9) an amino acid residue at a position equivalent to any of positions 177, 180, 344, 347, and 457 of SEQ ID NO: 4 in the amino acid sequence having 70% or more homology with SEQ ID NO: 4 Is a protein comprising an amino acid sequence substituted with the amino acid residue shown in (3) (A), wherein the D-amino acid acts selectively on N-succinyl-DL-amino acid to A protein characterized by having an activity to produce.
(10) A gene encoding the protein according to (9).
(11) A recombinant vector is prepared by inserting the gene described in (3), (4), (6), (8) or (10) into a vector, and a host cell is transformed with this recombinant vector. The method for producing a protein according to (1), (2), (5), (7) or (9), comprising the steps of preparing a transformant and culturing the transformant.
(12) N-succinyl-D-amino acid in N-succinyl-DL-amino acid is specifically hydrolyzed using the protein according to (1), (2), (5), (7) or (9) A method for producing D-amino acid, comprising a step of decomposing.
(13) The method according to (12), further comprising the step of racemizing N-succinyl-L-amino acid with N-succinylamino acid racemase to produce N-succinyl-D-amino acid.
　本発明の改変型Ｄ－サクシニラーゼは、野生型Ｄ－サクシニラーゼのアミノ酸配列の特定の部位のアミノ酸残基が別の特定のアミノ酸残基に置換されているので、野生型Ｄ－サクシニラーゼと比較してＤ体選択性が著しく向上している。従って、本発明の改変型Ｄ－サクシニラーゼを使用すれば、医薬品等の中間体の原料として有用なＤ－アミノ酸をさらに効率良く製造することができる。 The modified D-succinylase of the present invention has an amino acid residue at a specific site in the amino acid sequence of the wild-type D-succinylase substituted with another specific amino acid residue. D body selectivity is remarkably improved. Therefore, if the modified D-succinylase of the present invention is used, a D-amino acid useful as a raw material for intermediates such as pharmaceuticals can be produced more efficiently.
図１は、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株の生産するＤ－サクシニラーゼの電気泳動法による電気泳動像を示す図である。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. 図２は、改変型Ｄ－サクシニラーゼのスクリーニング方法を示す図である。FIG. 2 is a diagram showing a screening method for modified D-succinylase. 図３は、Ｃｕｐｒｉａｖｉｄｕｓ属由来Ｄ－サクシニラーゼのアミノ酸配列のアライメント結果を示す図である。FIG. 3 is a diagram showing an alignment result of amino acid sequences of D-succinylase derived from the genus Cupriavidus.
　本発明は、野生型Ｄ－サクシニラーゼを特定の部位でアミノ酸置換することにより、野生型Ｄ－サクシニラーゼと比較してＤ体選択性が著しく向上した改変型Ｄ－サクシニラーゼを提供するものである。本発明において、改変のベースとする野生型Ｄ－サクシニラーゼは、主にＣｕｐｒｉａｖｉｄｕｓ属の２種類の細菌（Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株及びＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓ）のＤ－サクシニラーゼである。以下、各改変型Ｄ－サクシニラーゼについて説明する。 The present invention provides a modified D-succinylase in which D-type selectivity is significantly improved by substituting wild-type D-succinylase with an amino acid at a specific site as compared with wild-type D-succinylase. In the present invention, the wild-type D-succinylase used as the base of modification is mainly D-succinylase of two types of bacteria belonging to the genus Cupriavidus (Cupriavidus sp. P4-10-C and Cupriavidus metallidurans). Hereinafter, each modified D-succinylase will be described.
　本発明の第１の側面は、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株のＤ－サクシニラーゼをベースとした改変型Ｄ－サクシニラーゼに関する。即ち、本発明の第１の側面によれば、（Ａ）配列番号２に示すアミノ酸配列において、下記（ａ）～（ｋ）から選択される少なくとも１個のアミノ酸残基の置換を有するアミノ酸配列からなることを特徴とするタンパク質が提供される。
（ａ）７２位のグルタミン残基のアルギニン残基への置換
（ｂ）１８１位のグリシン残基のトリプトファン残基、リジン残基、アルギニン残基、アスパラギン酸又はグルタミン酸残基への置換
（ｃ）１８２位のロイシン残基のトリプトファン残基、セリン残基、システイン残基、チロシン残基、リジン残基、アルギニン残基、アスパラギン酸残基、グルタミン酸残基又はプロリン残基への置換
（ｄ）１８３位のスレオニン残基のプロリン残基、ロイシン残基又はアスパラギン残基への置換
（ｅ）１８４位のロイシン残基のプロリン残基への置換
（ｆ）１８５位のアスパラギン残基のプロリン残基、フェニルアラニン残基、セリン残基又はアスパラギン酸残基への置換
（ｇ）３０５位のアルギニン残基のスレオニン残基、アラニン残基、グリシン残基、ヒスチジン残基、グルタミン残基、セリン残基、アスパラギン酸残基又はバリン残基への置換
（ｈ）３４８位のロイシン残基のグルタミン酸残基、プロリン残基、メチオニン残基、トリプトファン残基、セリン残基、スレオニン残基、システイン残基、リジン残基、ヒスチジン残基又はグルタミン残基への置換
（ｉ）３５１位のフェニルアラニン残基のロイシン残基、イソロイシン残基、メチオニン残基、アスパラギン残基又はグルタミン残基への置換
（ｊ）４６１位のアスパラギン残基のイソロイシン残基、フェニルアラニン残基、スレオニン残基、リジン残基又はアルギニン残基への置換
（ｋ）５３９位のグリシン残基のプロリン残基、バリン残基、メチオニン残基、スレオニン残基又はアスパラギン残基への置換
　配列番号２は、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株のＤ－サクシニラーゼのアミノ酸配列である。 The first aspect of the present invention relates to Cupriavidus sp. The present invention relates to a modified D-succinylase based on D-succinylase of the P4-10-C strain. That is, according to the first aspect of the present invention, (A) an amino acid sequence having a substitution of at least one amino acid residue selected from the following (a) to (k) in the amino acid sequence shown in SEQ ID NO: 2 A protein characterized by comprising:
(A) Replacement of glutamine residue at position 72 with arginine residue (b) Replacement of glycine residue at position 181 with tryptophan residue, lysine residue, arginine residue, aspartic acid or glutamic acid residue (c) Replacement of leucine residue at position 182 with tryptophan residue, serine residue, cysteine residue, tyrosine residue, lysine residue, arginine residue, aspartic acid residue, glutamic acid residue or proline residue (d) 183 Substitution of a threonine residue at the position with a proline residue, a leucine residue or an asparagine residue (e) a substitution of a leucine residue at position 184 with a proline residue (f) a proline residue at the position 185 of asparagine; Substitution to phenylalanine residue, serine residue or aspartic acid residue (g) Threonine residue, alanine residue, glycine of arginine residue at position 305 Substitution to residue, histidine residue, glutamine residue, serine residue, aspartate residue or valine residue (h) glutamic acid residue of leucine residue at position 348, proline residue, methionine residue, tryptophan residue Group, serine residue, threonine residue, cysteine residue, lysine residue, histidine residue or glutamine residue substitution (i) leucine residue, isoleucine residue, methionine residue of phenylalanine residue at position 351, Substitution to asparagine residue or glutamine residue (j) Substitution of asparagine residue at position 461 to isoleucine residue, phenylalanine residue, threonine residue, lysine residue or arginine residue (k) Residue of glycine at position 539 Substitution of a group with a proline residue, valine residue, methionine residue, threonine residue or asparagine residue Cupriavidus sp. Amino acid sequence of D-succinylase of the P4-10-C strain.
　好ましい実施態様によれば、上記（Ａ）のタンパク質は、配列番号２に示すアミノ酸配列において、１８２位のロイシン残基のグルタミン酸残基への置換、及び３４８位のロイシン残基のイソロイシン残基への置換を有するアミノ酸配列からなる。これは、Ｌ１８２Ｅ＋Ｌ３４８Ｉの二重変異体に相当する。後述の実施例に示す通り、Ｌ３４８Ｉの単変異のみでは、Ｄ体選択性の向上効果は見られないが、Ｌ１８２Ｅの単変異と組み合わせることにより、相乗効果が発揮され、Ｄ体選択性をさらに向上させることができる。 According to a preferred embodiment, in the amino acid sequence shown in SEQ ID NO: 2, the protein of (A) above has a substitution of a leucine residue at position 182 with a glutamic acid residue and a leucine residue at position 348 into an isoleucine residue. It consists of an amino acid sequence having the following substitutions. This corresponds to a double mutant of L182E + L348I. As shown in the examples described later, only the L348I single mutation does not improve the D-form selectivity, but when combined with the L182E single mutation, a synergistic effect is exhibited and the D-form selectivity is further improved. Can be made.
　上記（Ａ）のタンパク質は、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するという特徴を有する。 The protein (A) has a feature that it has an activity of producing D-amino acid by selectively acting on D-form with respect to N-succinyl-DL-amino acid.
　ここで、「Ｄ体選択的に作用する」とは、野生型のＤ－サクシニラーゼと比較して、Ｎ－サクシニル－Ｌ－アミノ酸（Ｌ体）よりもＮ－サクシニル－Ｄ－アミノ酸（Ｄ体）に反応しやすい性質のことを総じて称し、これは「Ｄ体選択性が向上している」と同義である。具体的には、「Ｄ体選択的に作用する」とは、Ｄ体に対するＬ体の分解比率（Ｌ／Ｄ）が野生型のＤ－サクシニラーゼより低いことを意味する。Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－ＣのＤ－サクシニラーゼをベースとした改変型Ｄ－サクシニラーゼの場合、基質として、例えばＮ－サクシニルトリプトファンを用いたときは、実施例２に記載の方法に従ってＮ－サクシニル－Ｌ－トリプトファン、Ｎ－サクシニル－Ｄ－トリプトファン、それぞれの基質に対して、Ｌ体を４、Ｄ体を１、すなわちＬ：Ｄ＝４：１の比率のタンパク量で反応させたときのＤ体に対するＬ体の分解比率（Ｌ／Ｄ）が０．９１未満であることを意味する。Ｄ体に対するＬ体の分解比率（Ｌ／Ｄ）は０．５以下であることが好ましく、０．３以下であることがさらに好ましい。また、基質として、Ｎ－サクシニルフェニルアラニンを用いたときは、実施例２に記載の方法に従ってＮ－サクシニル－Ｌ－フェニルアラニン、Ｎ－サクシニル－Ｄ－フェニルアラニン、それぞれの基質に対して、Ｌ体を４、Ｄ体を１、すなわちＬ：Ｄ＝４：１の比率のタンパク量で反応させたときのＤ体に対するＬ体の分解比率（Ｌ／Ｄ）が０．６９未満であることを意味する。Ｄ体に対するＬ体の分解比率（Ｌ／Ｄ）は０．４以下であることが好ましく、０．２以下であることがさらに好ましい。また、基質として、Ｎ－サクシニルビフェニルアラニンを用いたときは、実施例３に記載の方法に従ってＮ－サクシニル－ＤＬ－ビフェニルアラニンのラセミ体に対し、反応させたときのＤ体に対するＬ体の分解比率（Ｌ／Ｄ）が０．０９未満であることを意味する。Ｄ体に対するＬ体の分解比率（Ｌ／Ｄ）は０．０５以下であることが好ましく、０．０２以下であることがさらに好ましい。また、後述するＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼをベースとした改変型Ｄ－サクシニラーゼの場合、基質として、Ｎ－サクシニルトリプトファンを用いたときは、実施例２に記載の方法に従ってＮ－サクシニル－Ｌ－トリプトファン、Ｎ－サクシニル－Ｄ－トリプトファン、それぞれの基質に対して、Ｌ体を４、Ｌ体を１、すなわちＬ：Ｄ＝４：１の比率のタンパク量で反応させたときのＤ体に対するＬ体の分解比率（Ｌ／Ｄ）が０．５４未満であることを意味する。Ｄ体に対するＬ体の分解比率（Ｌ／Ｄ）は０．３以下であることが好ましく、０．１以下であることがさらに好ましい。
　なお、実用上の観点からは、Ｄ体に対するＬ体の分解比率だけではなく、Ｌ体分解率及びＤ体分解率それぞれの値も考慮すべきである。具体的には、Ｌ体分解率が１２％以下であることが好ましく、Ｌ体分解率が１２％以下でかつＤ体分解率が２５％以上であることがさらに好ましく、Ｌ体分解率が０％でかつＤ体分解率が２５％以上であることが特に好ましい。 Here, “acting selectively in D form” means that N-succinyl-D-amino acid (D form) is more N-succinyl-L-amino acid (L form) than wild-type D-succinylase. The property which is easy to react is generally called, and this is synonymous with “D-form selectivity is improved”. Specifically, “acting selectively in D-form” means that the degradation ratio of L-form to D-form (L / D) is lower than that of wild-type D-succinylase. Cupriavidus sp. In the case of a modified D-succinylase based on P4-10-C D-succinylase, when N-succinyltryptophan is used as a substrate, for example, N-succinyl-L-tryptophan is used according to the method described in Example 2. , N-succinyl-D-tryptophan, L-form to D-form when the L-form is 4 and D-form is 1, ie, L: D = 4: 1 in the amount of protein for each substrate. It means that the decomposition ratio (L / D) is less than 0.91. The decomposition ratio (L / D) of the L isomer to the D isomer is preferably 0.5 or less, and more preferably 0.3 or less. In addition, when N-succinylphenylalanine was used as a substrate, 4 L-forms were obtained for each of N-succinyl-L-phenylalanine and N-succinyl-D-phenylalanine according to the method described in Example 2. , It means that the decomposition ratio (L / D) of L-form to D-form is less than 0.69 when D-form is reacted at a protein amount of 1, ie L: D = 4: 1. The decomposition ratio (L / D) of the L isomer to the D isomer is preferably 0.4 or less, and more preferably 0.2 or less. When N-succinylbiphenylalanine was used as the substrate, the L-form was decomposed into the D-form when reacted with the racemic N-succinyl-DL-biphenylalanine according to the method described in Example 3. It means that the ratio (L / D) is less than 0.09. The decomposition ratio (L / D) of the L isomer to the D isomer is preferably 0.05 or less, and more preferably 0.02 or less. Further, in the case of modified D-succinylase based on Cupriavidus metallidurans D-succinylase described later, when N-succinyltryptophan is used as a substrate, N-succinyl-L-tryptophan is used according to the method described in Example 2. , N-succinyl-D-tryptophan, L-form relative to D-form when the L-form is reacted with the L-form, and the L-form is 1, ie, L: D = 4: 1. It means that the decomposition ratio (L / D) is less than 0.54. The decomposition ratio (L / D) of the L isomer to the D isomer is preferably 0.3 or less, and more preferably 0.1 or less.
From a practical viewpoint, not only the decomposition ratio of the L isomer to the D isomer but also the values of the L isomer decomposition rate and the D isomer decomposition rate should be considered. Specifically, the L-isomer decomposition rate is preferably 12% or less, the L-isomer decomposition rate is preferably 12% or less, and the D-isomer decomposition rate is more preferably 25% or more, and the L-isomer decomposition rate is 0. % And the D-form decomposition rate is particularly preferably 25% or more.
　本発明における立体選択性の評価は、実施例に示した通りであるが、基質濃度や酵素量、反応温度、反応時間、反応ｐＨなどの反応条件によっては、数値が変動する場合もある。 The evaluation of stereoselectivity in the present invention is as shown in the examples, but the numerical value may vary depending on the reaction conditions such as the substrate concentration, enzyme amount, reaction temperature, reaction time and reaction pH.
　なお、上記のＤ体選択性の説明において、Ｎ－サクシニル－ＤＬ－トリプトファン、Ｎ－サクシニル－ＤＬ－フェニルアラニン及びＮ－サクシニル－ＤＬ－ビフェニルアラニンに対するＤ体選択性に言及したが、本発明の改変型Ｄ－サクシニラーゼは、これらの特定のＮ－サクシニル－ＤＬ－アミノ酸だけでなく、下記一般式（Ｉ）に示す任意のＮ－サクシニル－ＤＬ－アミノ酸に対しても同様にＤ体選択性を有する。
Figure JPOXMLDOC01-appb-I000001
（一般式（Ｉ）中、Ｒは、置換基を有してもよい炭素数４～２０のアリール基、または置換基を有してもよい炭素数５～２０のアラルキル基を示す。）
　上記Ｒにおける置換基を有していてもよい炭素数６～２０のアリール基としては、フェニル基、４－ヒドロキシフェニル基などが挙げられる。置換基としては、アミノ基、ヒドロキシル基、ニトロ基、シアノ基、カルボキシル基、アルキル基、アラルキル基、アリール基、アルカノイル基、アルケニル基、アルキニル基、アルコキシル基、又はハロゲン原子等が挙げられる。また、置換基を有していてもよい炭素数７～２０のアラルキル基としては、特に限定されず、例えば、ベンジル基、インドリルメチル基、４－フェニルベンジル基、４－ヒドロキシベンジル基等が挙げられる。 In the above description of D-form selectivity, reference was made to D-form selectivity for N-succinyl-DL-tryptophan, N-succinyl-DL-phenylalanine and N-succinyl-DL-biphenylalanine. The type D-succinylase has D-form selectivity not only for these specific N-succinyl-DL-amino acids but also for any N-succinyl-DL-amino acid represented by the following general formula (I). .
Figure JPOXMLDOC01-appb-I000001
(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 for 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. Further, 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.
　従来公知の野生型Ｄ－サクシニラーゼは、Ｄ－アミノ酸の前駆体であるＮ－サクシニル－ＤＬ－アミノ酸の光学異性を厳密には区別できず、Ｌ体にもわずかに作用して、Ｄ－アミノ酸のみならず、Ｌ－アミノ酸も生じてしまう。しかし、本発明の改変型Ｄ－サクシニラーゼは、野生型Ｄ－サクシニラーゼの一部のアミノ酸残基を置換することにより立体選択性を変化させて、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用するよう改変を加えているので、Ｎ－サクシニル－ＤＬ－アミノ酸からＤ－アミノ酸を選択的に生成することができる。 The conventionally known wild-type D-succinylase cannot strictly discriminate optical isomerism of N-succinyl-DL-amino acid, which is a precursor of D-amino acid, and acts slightly on L-form, and only D-amino acid In addition, L-amino acids are also produced. However, the modified D-succinylase of the present invention changes the stereoselectivity by substituting a part of the amino acid residues of the wild-type D-succinylase, and selects D-form for N-succinyl-DL-amino acid. D-amino acids can be selectively generated from N-succinyl-DL-amino acids since they have been modified to act in an effective manner.
　また、本発明の第１の側面によれば、上記（Ａ）のタンパク質に対応する遺伝子である、以下の遺伝子も提供される。
（Ａ）配列番号１に示す塩基配列において、下記（ａ）～（ｋ）から選択される少なくとも１個の塩基配列の置換を有する塩基配列からなることを特徴とする遺伝子
（ａ）２１４～２１６位の塩基配列ｃａａの、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ又はａｇｇへの置換
（ｂ）５４１～５４３位の塩基配列ｇｇｃの、ｔｇｇ、ａａａ、ａａｇ、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ、ａｇｇ、ｇａｔ、ｇａｃ、ｇａａ又はｇａｇへの置換
（ｃ）５４４～５４６位の塩基配列ｃｔｇの、ｔｇｇ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ｔｇｔ、ｔｇｃ、ｔａｔ、ｔａｃ、ａａａ、ａａｇ、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ、ａｇｇ、ｇａｔ、ｇａｃ、ｇａａ、ｇａｇ、ｃｃｔ、ｃｃｃ、ｃｃａ又はｃｃｇへの置換
（ｄ）５４７～５４９位の塩基配列ａｃｇの、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ｔｔａ、ｔｔｇ、ｃｔｔ、ｃｔｃ、ｃｔａ、ｃｔｇ、ａａｔ又はａａｃへの置換
（ｅ）５５０～５５２位の塩基配列ｃｔｇの、ｃｃｔ、ｃｃｃ、ｃｃａ又はｃｃｇへの置換
（ｆ）５５３～５５５位の塩基配列ａａｔの、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ｔｔｔ、ｔｔｃ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ｇａｔ又はｇａｃへの置換
（ｇ）９１３～９１５位の塩基配列ｃｇｇの、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ｇｃｔ、ｇｃｃ、ｇｃａ、ｇｃｇ、ｇｇｔ、ｇｇｃ、ｇｇａ、ｇｇｇ、ｃａｔ、ｃａｃ、ｃａａ、ｃａｇ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ａａｔ、ａａｃ、ｇｔｔ、ｇｔｃ、ｇｔａ又はｇｔｇへの置換
（ｈ）１０４２～１０４４位の塩基配列ｃｔｇの、ｇａａ、ｇａｇ、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ａｔｇ、ｔｇｇ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ｔｇｔ、ｔｇｃ、ａａａ、ａａｇ、ｃａｔ、ｃａｃ、ｃａａ又はｃａｇへの置換
（ｉ）１０５１～１０５３位の塩基配列ｔｔｃの、ｔｔａ、ｔｔｇ、ｃｔｔ、ｃｔｃ、ｃｔａ、ｃｔｇ、ａｔｔ、ａｔｃ、ａｔａ、ａｔｇ、ａａｔ、ａａｃ、ｃａａ又はｃａｇへの置換
（ｊ）１３８１～１３８３位の塩基配列ａａｃの、ａｔｔ、ａｔｃ、ａｔａ、ｔｔｔ、ｔｔｃ、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ａａａ、ａａｇ、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ又はａｇｇへの置換
（ｋ）１６１５～１６１７位の塩基配列ｇｇｃの、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ｇｔｔ、ｇｔｃ、ｇｔａ、ｇｔｇ、ａｔｇ、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ａａｔ又はａａｃへの置換
　配列番号１は、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株のＤ－サクシニラーゼの塩基配列である。 Moreover, according to the 1st side surface of this invention, the following genes which are a gene corresponding to the protein of said (A) are also provided.
(A) The gene (a) 214 to 216 characterized by comprising a base sequence having at least one base sequence substitution selected from the following (a) to (k) in the base sequence shown in SEQ ID NO: 1. (C) substitution of the base sequence caa with cgt, cgg, cga, cgg, aga or agg (b) tgg, aaa, aag, cgt, cgg, cga, cgg, aga, Substitution to agg, gat, gac, gaa or gag (c) tgg, tct, tcc, tca, tcg, agt, agc, tgt, tgc, tat, tac, aaa, aag of the nucleotide sequence ctg at positions 544 to 546 , Cgt, cgc, cga, cgg, aga, agg, gat, gac, gaa, gag, cct, ccc, cca or ccg d) Substitution of base sequence acg at positions 547 to 549 to cct, ccc, cca, ccg, tta, ttg, ctt, ctc, cta, ctg, aat or aac (e) base sequence ctg at positions 550 to 552 (C), ccc, ccc, cca or ccg substitution (f) cct, ccc, cca, ccg, ttt, ttc, tct, tcc, tca, tcg, agt, agc, gat or Substitution to gac (g) Act, acc, aca, acg, gct, gcc, gca, gcg, ggt, ggg, gga, ggg, cat, cac, caa, cag, tct of base sequence cgg at positions 913 to 915 , Tcc, tca, tcg, agt, agc, aat, aac, gtt, gtt, gta or gtg ( ) The base sequence ctg at positions 1042 to 1044, gaa, gag, cct, ccc, cca, ccg, atg, tgg, tct, tcc, tca, tcg, agt, agc, act, acc, aca, acg, tgt, tgc , Aaa, aag, cat, cac, caa or cag (i) tta, ttg, ctt, ctc, cta, ctg, att, atc, ata, atg, aat, of the nucleotide sequence ttc at positions 1051 to 1053 Substitution with aac, caa or cag (j) att, atc, ata, ttt, ttc, act, acc, aca, acg, aaa, aag, cgt, cgg, cga, cgg of the base sequence aac at positions 1381 to 1383 , Substitution to aga or agg (k) cct of the nucleotide sequence ggc at positions 1615 to 1617 , Ccc, cca, ccg, gtt, gtc, gta, gtg, atg, act, acc, aca, acg, aat, or aac substitution SEQ ID NO: 1 is Cupriavidus sp. This is the base sequence of D-succinylase of the P4-10-C strain.
　好ましい実施態様によれば、上記（Ａ）のタンパク質に対応する遺伝子は、配列番号１に示す塩基配列において、５４４～５４６位の塩基配列ｃｔｇの、ｇａａ又はｇａｇへの置換、及び１０４２～１０４４位の塩基配列ｃｔｇの、ａｔｔ，ａｔｃ又はａｔａへの置換を有する塩基配列からなる。これは、Ｌ１８２Ｅ＋Ｌ３４８Ｉの二重変異体に相当する遺伝子である。 According to a preferred embodiment, the gene corresponding to the protein of the above (A) is the base sequence shown in SEQ ID NO: 1, the substitution of the base sequence ctg at positions 544 to 546 to gaa or gag, and the positions 1042 to 1044 The base sequence ctg is a base sequence having substitution to att, atc or ata. This is a gene corresponding to a double mutant of L182E + L348I.
　本発明の第１の側面の改変型Ｄ－サクシニラーゼのタンパク質は、上記（Ａ）のものに限定されず、（Ｂ）（Ａ）のアミノ酸配列において、７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位、及び５３９位以外の箇所に、１若しくは数個のアミノ酸残基の置換、欠失、挿入、付加および／または逆位を有するアミノ酸配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードするアミノ酸配列からなることを特徴とするタンパク質も含む。また、本発明の第１の側面の改変型Ｄ－サクシニラーゼの遺伝子は、上記（Ａ）のものに限定されず、（Ｂ）（Ａ）の塩基配列とストリンジェントな条件でハイブリダイズする塩基配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードする塩基配列からなることを特徴とする遺伝子も含む。これは、タンパク質をコードする遺伝子の塩基配列の一部に変異が生じたり、またその結果としてタンパク質のアミノ酸配列の一部に変異が生じても、機能的には同等のタンパク質であることが多いからである。また、本発明の改変型Ｄ－サクシニラーゼの遺伝子を、由来生物以外の宿主生物（大腸菌など）に組込んで本発明の改変型Ｄ－サクシニラーゼを発現させる場合、発現効率向上のため、宿主生物のコドンユーセージに合わせて塩基配列を変更することもあるからである。 The protein of the modified D-succinylase according to the first aspect of the present invention is not limited to the above (A), and in the amino acid sequence of (B) (A), positions 72, 181-185, 305, An amino acid sequence having substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues at positions other than positions 348, 351, 461, and 539, and N-succinyl -Also includes a protein characterized by comprising an amino acid sequence encoding a protein having an activity of producing a D-amino acid by acting D-selectively on a DL-amino acid. The modified D-succinylase gene according to the first aspect of the present invention is not limited to the gene of (A) above, and (B) a nucleotide sequence that hybridizes with the nucleotide sequence of (A) under stringent conditions. And a gene characterized by comprising a base sequence encoding a protein having an activity of producing a D-amino acid by selectively acting on D-form to N-succinyl-DL-amino acid. 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 modified D-succinylase gene of the present invention is incorporated into a host organism (such as E. coli) other than the organism from which the modified D-succinylase gene of the present invention is expressed, This is because the base sequence may be changed according to the codon usage.
　ここで、「１若しくは数個」とは、アミノ酸残基のタンパク質の立体構造や、Ｄ－サクシニラーゼ活性及びＮ－サクシニル－ＤＬ－アミノ酸に対するＤ体選択性を大きく損なわない範囲のものであり、具体的には、１～５０個、好ましくは１～３０個、より好ましくは１～２０個、さらに好ましくは１～１０個である。ただし、配列表の配列番号２に記載のアミノ酸配列において１又は数個のアミノ酸残基の置換、欠失、挿入、付加及び／又は逆位を含むアミノ酸配列の場合には、３７℃、ｐＨ７．０の条件下で配列表の配列番号２に記載のアミノ酸配列を有するタンパク質の３％以上、好ましくは１０％以上、より好ましくは３０％以上、さらに好ましくは５０％以上、特に好ましくは７０％以上のＤ－サクシニラーゼ活性を保持していることが望ましい。 Here, “one or several” is a range that does not significantly impair the three-dimensional structure of the amino acid residue protein, the D-succinylase activity, and the D-form selectivity to N-succinyl-DL-amino acid. Specifically, the number is 1 to 50, preferably 1 to 30, more preferably 1 to 20, and still more preferably 1 to 10. However, in the case of an amino acid sequence containing substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing, 37 ° C., pH 7. 3% or more, preferably 10% or more, more preferably 30% or more, still more preferably 50% or more, particularly preferably 70% or more of the protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing under the condition of 0 It is desirable to retain the D-succinylase activity.
　また、「ストリンジェントな条件」とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。この条件を明確に数値化することは困難であるが、一例を示せば、相同性が高いＤＮＡ同士、例えば８５％以上、好ましくは９０％以上、より好ましくは９５％以上の相同性を有するＤＮＡ同士がハイブリダイズし、それより相同性が低いＤＮＡ同士がハイブリダイズしない条件（ここでいう相同性（ｈｏｍｏｌｏｇｙ）は、比較する配列間において一致する塩基の数が最大となるような並べ方にして演算された値であることが望ましい）、あるいは通常のサザンハイブリダイゼーションの洗いの条件である３７℃、０．１×ＳＳＣ、０．１％　ＳＤＳ、好ましくは６０℃、０．１×ＳＳＣ、０．１％　ＳＤＳ、さらに好ましくは６５℃、０．１×ＳＳＣ、０．１％　ＳＤＳに相当するに相当する塩濃度でハイブリダイズする条件が挙げられる。ただし、配列表の配列番号１に記載の塩基配列と相補的な塩基配列とストリンジェントな条件でハイブリダイズする塩基配列の場合には、３７℃、ｐＨ７．０の条件下で配列表の配列番号２に記載のアミノ酸配列を有するタンパク質の３％以上、好ましくは１０％以上、より好ましくは３０％以上、さらに好ましくは５０％以上、特に好ましくは７０％以上のＤ－サクシニラーゼ活性を保持していることが望ましい。 Also, “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 these conditions clearly, for example, DNAs having high homology, for example, DNAs having a homology of 85% or more, preferably 90% or more, more preferably 95% or more, are present. The conditions are such that the DNAs that hybridize with each other and the DNAs with lower homology do not hybridize with each other (here, the homology is homologous (homology)). Or 37 ° C., 0.1 × SSC, 0.1% SDS, preferably 60 ° C., 0.1 × SSC, 0.8%, which are washing conditions for normal Southern hybridization. Conditions include hybridization at a salt concentration corresponding to 1% SDS, more preferably 65 ° C., 0.1 × SSC, corresponding to 0.1% SDS.However, in the case of a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing, the sequence number in the sequence listing under conditions of 37 ° C and pH 7.0 3% or more, preferably 10% or more, more preferably 30% or more, still more preferably 50% or more, particularly preferably 70% or more of the D-succinylase activity of the protein having the amino acid sequence described in 2. It is desirable.
　本発明の第１の側面のタンパク質及びその遺伝子は、例えばＴｒａｎｓｆｏｒｍｅｒＭｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ；Ｃｌｏｎｅｔｅｃｈ製、ＥＸＯＩＩＩ／Ｍｕｎｇ　Ｂｅａｎ　Ｄｅｌｅｔｉｏｎ　Ｋｉｔ；Ｓｔｒａｔａｇｅｎｅ製、ＱｕｉｃｋＣｈａｎｇｅ　Ｓｉｔｅ　Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ；Ｓｔｒａｔａｇｅｎｅ製、ＫＯＤ－Ｐｌｕｓ－Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ；東洋紡製などの市販のキットやＰＣＲ法を利用して配列番号１に記載の塩基配列を改変することによって得ることができる。得られた遺伝子によってコードされるタンパク質の活性は、例えば、得られた遺伝子を大腸菌に導入して形質転換体を作成し、この形質転換体を培養して酵素タンパク質を生成させ、この形質転換体、この形質転換体の菌体破砕液もしくは精製した酵素タンパク質をＮ－サクシニル－ＤＬ－アミノ酸に添加してＤ体に対するＬ体の分解比率を実施例に記載の方法で測定することによって確認することができる。 The protein of the first aspect of the present invention and its gene are, for example, Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Selection Kit; Can be obtained by modifying the base sequence described in SEQ ID NO: 1 using a commercially available kit 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. Confirmation is made by adding the cell disruption solution of this transformant or purified enzyme protein to N-succinyl-DL-amino acid and measuring the degradation ratio of L-form to D-form by the method described in the Examples. Can do.
　本発明の第２の側面は、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼをベースとした改変型Ｄ－サクシニラーゼに関する。即ち、本発明の第２の側面によれば、（Ａ）配列番号４に示すアミノ酸配列において、下記（ａ）～（ｅ）から選択される少なくとも１個のアミノ酸残基の置換を有するアミノ酸配列からなることを特徴とするタンパク質が提供される。
（ａ）１７７位のロイシン残基のアルギニン残基への置換
（ｂ）１８０位のアスパラギン残基のアスパラギン酸残基への置換
（ｃ）３４４位のロイシン残基のプロリン残基への置換
（ｄ）３４７位のフェニルアラニン残基のイソロイシン残基への置換
（ｅ）４５７位のアスパラギン残基のイソロイシン残基への置換
　配列番号４は、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼのアミノ酸配列である。 The second aspect of the present invention relates to a modified D-succinylase based on the D. succinylase of Cupriavidus metallidurans. That is, according to the second aspect of the present invention, (A) an amino acid sequence having a substitution of at least one amino acid residue selected from the following (a) to (e) in the amino acid sequence shown in SEQ ID NO: 4 A protein characterized by comprising:
(A) Substitution of leucine residue at position 177 to arginine residue (b) Substitution of asparagine residue at position 180 to aspartic acid residue (c) Substitution of leucine residue at position 344 to proline residue ( d) Substitution of phenylalanine residue at position 347 to isoleucine residue (e) Substitution of asparagine residue at position 457 to isoleucine residue SEQ ID NO: 4 is the amino acid sequence of D-succinylase of Cupriavidus metallidrans.
　上記（Ａ）のタンパク質は、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するという特徴を有する。「Ｄ体選択的に作用する」の定義などは、第１の側面と同様であるので、説明を省略する。 The protein (A) has a feature that it has an activity of producing D-amino acid by selectively acting on D-form with respect to N-succinyl-DL-amino acid. The definition of “acting selectively on D body” and the like are the same as in the first aspect, and thus description thereof is omitted.
　また、本発明の第２の側面によれば、上記（Ａ）のタンパク質に対応する遺伝子である、以下の遺伝子も提供される。
（Ａ）配列番号３に示す塩基配列において、下記（ａ）～（ｅ）から選択される少なくとも１個の塩基配列の置換を有する塩基配列からなることを特徴とする遺伝子
（ａ）５２９～５３１位の塩基配列ｃｔｇの、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ又はａｇｇへの置換
（ｂ）５３８～５４０位の塩基配列ａａｃの、ｇａｔ又はｇａｃへの置換
（ｃ）１０３０～１０３２位の塩基配列ｃｔｇの、ｃｃｔ、ｃｃｃ、ｃｃａ又はｃｃｇへの置換
（ｄ）１０３９～１０４１位の塩基配列ｔｔｃの、ａｔｔ、ａｔｃ又はａｔａへの置換
（ｅ）１３６９～１３７１位の塩基配列ａａｔの、ａｔｔ、ａｔｃ又はａｔａへの置換
　配列番号３は、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼの塩基配列である。 Moreover, according to the 2nd side surface of this invention, the following genes which are a gene corresponding to the protein of said (A) are also provided.
(A) A gene (a) 529 to 531 comprising a base sequence having substitution of at least one base sequence selected from the following (a) to (e) in the base sequence shown in SEQ ID NO: 3 Substitution of base sequence ctg to cgt, cgc, cga, cgg, aga or agg (b) Substitution of base sequence aac from positions 538 to 540 to gat or gac (c) Base sequence from positions 1030 to 1032 Substitution of ctg to cct, ccc, cca or ccg (d) Substitution of nucleotide sequence ttc at positions 1039 to 1041 to att, atc or ata (e) Att, atc of nucleotide sequence aat at positions 1369 to 1371 Alternatively, substitution to atata SEQ ID NO: 3 is the base sequence of D-succinylase of Cupriavidus metallidurans.
　本発明の第２の側面の改変型Ｄ－サクシニラーゼのタンパク質は、上記（Ａ）のものに限定されず、（Ｂ）（Ａ）のアミノ酸配列において、１７７位、１８０位、３４４位、３４７位、及び４５７位以外の箇所に、１若しくは数個のアミノ酸残基の置換、欠失、挿入、付加および／または逆位を有するアミノ酸配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードするアミノ酸配列からなることを特徴とするタンパク質も含む。また、本発明の第２の側面の改変型Ｄ－サクシニラーゼの遺伝子は、上記（Ａ）のものに限定されず、（Ｂ）（Ａ）の塩基配列とストリンジェントな条件でハイブリダイズする塩基配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードする塩基配列からなることを特徴とする遺伝子も含む。「１若しくは数個」及び「ストリンジェントな条件」の定義など、並びに第２の側面のタンパク質及びその遺伝子の作成方法や活性確認方法は、第１の側面と同様であるので、説明を省略する。 The modified D-succinylase protein of the second aspect of the present invention is not limited to the above (A), and (B) in the amino acid sequence of (A), positions 177, 180, 344, 347 And an amino acid sequence having substitutions, deletions, insertions, additions and / or inversions of one or several amino acid residues at positions other than position 457, wherein D is an N-succinyl-DL-amino acid Also included is a protein comprising an amino acid sequence encoding a protein having an activity of producing a D-amino acid by acting selectively on the body. The modified D-succinylase gene of the second aspect of the present invention is not limited to the gene of (A) above, and (B) a nucleotide sequence that hybridizes with the nucleotide sequence of (A) under stringent conditions. And a gene characterized by comprising a base sequence encoding a protein having an activity of producing a D-amino acid by selectively acting on D-form to N-succinyl-DL-amino acid. The definition of “one or several” and “stringent conditions”, and the method for creating and confirming the activity of the protein and gene of the second aspect are the same as those of the first aspect, and thus the description thereof is omitted. .
　以上、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株のＤ－サクシニラーゼ及びＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼをベースとした改変型Ｄ－サクシニラーゼについて説明したが、本発明の改変型Ｄ－サクシニラーゼはこれらの細菌のＤ－サクシニラーゼをベースとしたものに限定されず、Ｃｕｐｒｉａｖｉｄｕｓ属の他の細菌を含む近縁種のＤ－サクシニラーゼをベースとしたものも含む。 As described above, Cupriavidus sp. The modified D-succinylase based on D-succinylase of P4-10-C strain and Cupriavidus metallidurans D-succinylase has been described. The modified D-succinylase of the present invention is based on the D-succinylase of these bacteria. And those based on D-succinylase of related species including other bacteria of the genus Cupriavidus.
　Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株のＤ－サクシニラーゼとＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼは、７５％の相同性しか有さないが、実施例で示すように、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株のＤ－サクシニラーゼのアミノ酸配列において立体選択性に関与することが今回見出されたアミノ酸残基は全て、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼのアミノ酸配列でも保存されている。また、実際に、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼにおいても、これらの保存アミノ酸残基を、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株のＤ－サクシニラーゼベースの改変型Ｄ－サクシニラーゼと同様にアミノ酸置換することによって、Ｄ体選択性が向上することが確認されている。さらに、立体選択性に関与するアミノ酸残基は全て、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株とＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓの間だけではなく、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株とＣｕｐｒｉａｖｉｄｕｓ属の他の細菌との間でも保存されている。これらのことから、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株のＤ－サクシニラーゼ及びＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼに限らず、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株又はＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓの近縁種のＤ－サクシニラーゼにおいて、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株又はＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼのアミノ酸配列中の立体選択性に関与するアミノ酸残基と同等な位置のアミノ酸残基をアミノ酸置換することによって、Ｄ体選択性が向上するものと考えられる。 Cupriavidus sp. The P4-10-C strain D-succinylase and Cupriavidus metallidrans D-succinylase have only 75% homology, but as shown in the Examples, Cupriavidus sp. All amino acid residues found this time to be involved in stereoselectivity in the amino acid sequence of D-succinylase of the P4-10-C strain are also conserved in the amino acid sequence of Cupriavidus metallidrans D-succinylase. In fact, the conserved amino acid residues in Cupriavidus metallidrans D-succinylase were also converted to Cupriavidus sp. It has been confirmed that D-form selectivity is improved by amino acid substitution in the same manner as the D-succinylase-based modified D-succinylase of the P4-10-C strain. In addition, all amino acid residues involved in stereoselectivity are Cupriavidus sp. Not only between the P4-10-C strain and Cupriavidus metallidurans, but also Cupriavidus sp. It is also conserved between the P4-10-C strain and other bacteria of the genus Cupriavidus. From these facts, Cupriavidus sp. Not only D-succinylase of P4-10-C strain and D-succinylase of Cupriavidus metallidurans, but also Cupriavidus sp. In D-succinylase of the close relative of P4-10-C strain or Cupriavidus metallidurans, Cupriavidus sp. D-form selectivity is improved by substituting amino acid residues at positions equivalent to the amino acid residues involved in stereoselectivity in the amino acid sequence of P4-10-C strain or Cupriavidus metallidurans D-succinylase it is conceivable that.
　従って、本発明の第３の側面は、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株の近縁種のＤ－サクシニラーゼをベースとした改変型Ｄ－サクシニラーゼ及びその遺伝子に関する。即ち、本発明の第３の側面によれば、配列番号２と７０％以上の相同性を有するアミノ酸配列において、配列番号２の７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位、及び５３９位のうちのいずれかと同等な位置のアミノ酸残基が、本発明の第１の側面のタンパク質（Ａ）に示すアミノ酸残基に置換されているアミノ酸配列からなるタンパク質であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有することを特徴とするタンパク質、及びこのタンパク質をコードすることを特徴とする遺伝子が提供される。 Therefore, the third aspect of the present invention relates to Cupriavidus sp. The present invention relates to a modified D-succinylase based on D-succinylase of a close relative of the P4-10-C strain and a gene thereof. That is, according to the third aspect of the present invention, in the amino acid sequence having 70% or more homology with SEQ ID NO: 2, positions 72, 181-185, 305, 348, 351 of SEQ ID NO: 2, A protein comprising an amino acid sequence in which an amino acid residue at a position equivalent to any of positions 461 and 539 is substituted with the amino acid residue shown in the protein (A) of the first aspect of the present invention, , A protein characterized by having an activity of producing a D-amino acid by selectively acting on D-form against N-succinyl-DL-amino acid, and a gene encoding the protein The
　本発明の第３の側面の改変型Ｄ－サクシニラーゼは、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株のＤ－サクシニラーゼのアミノ酸配列である配列番号２と７０％以上、好ましくは７４％以上、より好ましくは８０％以上、さらに好ましくは９０％以上、特に好ましくは９５％以上の相同性を有するＤ－サクシニラーゼをベースとする。このような相同性レベルを有するアミノ酸配列は、公知のアミノ酸配列のデータベースに基づく相同性検索や、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株のＤ－サクシニラーゼの遺伝子のＤＮＡ断片をプローブとしたハイブリダイゼーションにより得ることができる。例えば、相同性検索により得られるアミノ酸配列としては、Ｃｕｐｒｉａｖｉｄｕｓ　ｎｅｃａｔｏｒ及びＣｕｐｒｉａｖｉｄｕｓ　ｔａｉｗａｎｅｎｓｉｓのＤ－サクシニラーゼのアミノ酸配列が挙げられる。これらのアミノ酸配列は、ＮＣＢＩ（ｈｔｔｐ：／／ｂｌａｓｔ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／）に公開されている。なお、ＮＣＢＩでは、これらのアミノ酸配列は、Ｄ－サクシニラーゼの配列ではなくペニシリンＧアシラーゼ又はペニシリンＧアミダーゼの配列としてそれぞれ登録されている。また、プローブハイブリダイゼーションの場合は、以下に示すようなＣｕｐｒｉａｖｉｄｕｓ属の細菌から、該当する相同性レベルのＤ－サクシニラーゼのアミノ酸配列を得ることができる可能性が高い。
Ｃｕｐｒｉａｖｉｄｕｓ　ｏｘａｌａｔｉｃｕｓ；
Ｃｕｐｒｉａｖｉｄｕｓ　ｇｉｌａｒｄｉｉ；
Ｃｕｐｒｉａｖｉｄｕｓ　ｂａｓｉｌｅｎｓｉｓ；
Ｃｕｐｒｉａｖｉｄｕｓ　ｃａｍｐｉｎｅｎｓｉｓ；
Ｃｕｐｒｉａｖｉｄｕｓ　ｐａｕｃｕｌｕｓ；
Ｃｕｐｒｉａｖｉｄｕｓ　ｐｉｎａｔｕｂｏｎｅｎｓｉｓ；
Ｃｕｐｒｉａｖｉｄｕｓ　ｒｅｓｐｉｒａｃｕｌｉ；
Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ。
　なお、上述のＣｕｐｒｉａｖｉｄｕｓ属の細菌の中には、Ｒａｌｓｔｏｎｉａ（ラルストニア）属の細菌として文献に表記されているものもあるが、Ｃｕｐｒｉａｖｉｄｕｓ属が正式な属名として一般的に使用されてきている。これらの細菌の学術名は、将来の再分類により、統一又は変更される可能性がある。 The modified D-succinylase of the third aspect of the present invention is Cupriavidus sp. SEQ ID NO: 2 which is the amino acid sequence of D-succinylase of the P4-10-C strain and 70% or more, preferably 74% or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more Based on homologous D-succinylase. An amino acid sequence having such a homology level is obtained by homology search based on a database of known amino acid sequences, or by Cupriavidus sp. It can be obtained by hybridization using a DNA fragment of the D-succinylase gene of the P4-10-C strain as a probe. For example, the amino acid sequence obtained by homology search includes the amino acid sequence of D-succinylase of Cupriavidus necator and Cupriavidus taiwanensis. These amino acid sequences are published in NCBI (http://blast.ncbi.nlm.nih.gov/). In NCBI, these amino acid sequences are registered as penicillin G acylase or penicillin G amidase sequences, not D-succinylase sequences. In the case of probe hybridization, it is highly possible that an amino acid sequence of D-succinylase having a corresponding homology level can be obtained from a bacterium belonging to the genus Cupriavidus as shown below.
Cupriavidus oxalaticus;
Cupriavidus gillardii;
Cupriavidus basilensis;
Cupriavidus campinensis;
Cupriavidus pauculus;
Cupriavidus pinatobonensis;
Cupriavidus respiraculi;
Cupriavidus sp.
Some bacteria of the genus Cupriavidus are described in the literature as bacteria of the genus Ralstonia, but the genus Cupriavidus has been generally used as an official genus name. The scientific names of these bacteria may be unified or changed due to future reclassification.
　本発明の第３の側面において、改変のベースとするＤ－サクシニラーゼは、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株の近縁種からの野生型Ｄ－サクシニラーゼに限定されず、これらの野生型Ｄ－サクシニラーゼに何らかの変異を生じさせたものも、配列番号２と７０％以上の相同性を有する限り、改変のベースとして使用することができる。 In the third aspect of the present invention, the D-succinylase used as a modification base is Cupriavidus sp. Not limited to wild-type D-succinylases from closely related species of the P4-10-C strain, those having some mutations in these wild-type D-succinylases also have a homology of at least 70% with SEQ ID NO: 2. As long as it has, it can be used as a base for modification.
　本発明の第３の側面において、改変は、これらのベースとするＤ－サクシニラーゼのアミノ酸配列中の、配列番号２の７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位、及び５３９位のうちのいずれかと同等な位置のアミノ酸残基を、本発明の第１の側面の改変型Ｄ－サクシニラーゼと同じアミノ酸残基に置換することによって行われる。ここで、「配列番号２の７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位、及び５３９位のうちのいずれかと同等な位置」は、ベースとするＤ－サクシニラーゼのアミノ酸配列を配列番号２のアミノ酸配列とアラインメントさせることによって特定することができる。アラインメントは、Ｂｌａｓｔなどの公知のアルゴリズムに基づく市販のアミノ酸配列解析ソフトを使用して容易に行うことができる。例えば、先に例示したＣｕｐｒｉａｖｉｄｕｓ属の細菌のうち、Ｃｕｐｒｉａｖｉｄｕｓ　ｎｅｃａｔｏｒ及びＣｕｐｒｉａｖｉｄｕｓ　ｔａｉｗａｎｅｎｓｉｓのＤ－サクシニラーゼのアミノ酸配列の場合、配列番号２のアミノ酸配列とのアラインメント結果は、図３に示す通りであり、図３中の網掛けされた位置のアミノ酸残基が「配列番号２の７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位、及び５３９位のうちのいずれかと同等な位置」のアミノ酸残基に相当する。アミノ酸置換は、例えばＴｒａｎｓｆｏｒｍｅｒＭｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ；Ｃｌｏｎｅｔｅｃｈ製、ＥＸＯＩＩＩ／Ｍｕｎｇ　Ｂｅａｎ　Ｄｅｌｅｔｉｏｎ　Ｋｉｔ；Ｓｔｒａｔａｇｅｎｅ製、ＱｕｉｃｋＣｈａｎｇｅ　Ｓｉｔｅ　Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ；Ｓｔｒａｔａｇｅｎｅ製、ＫＯＤ－Ｐｌｕｓ－Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ；東洋紡製などの市販のキットやＰＣＲ法を利用して、ベースとするＤ－サクシニラーゼのアミノ酸配列に相当する塩基配列を部位特異的に変異させることによって行うことができる。得られた遺伝子によってコードされるタンパク質の活性は、例えば、得られた遺伝子を大腸菌に導入して形質転換体を作成し、この形質転換体を培養して酵素タンパク質を生成させ、この形質転換体、この形質転換体の菌体破砕液もしくは精製した酵素タンパク質をＮ－サクシニル－ＤＬアミノ酸に添加してＤ体に対するＬ体の分解比率を実施例に記載の方法で測定することによって確認することができる。 In the third aspect of the present invention, the modification is performed in the amino acid sequence of these base D-succinylases at positions 72, 181-185, 305, 348, 351, 461 of SEQ ID NO: 2, And the amino acid residue at a position equivalent to any of positions 539 is replaced with the same amino acid residue as the modified D-succinylase of the first aspect of the present invention. Here, “positions equivalent to any of positions 72, 181-185, 305, 348, 351, 461, and 539 of SEQ ID NO: 2” are amino acids of the base D-succinylase The sequence can be identified by aligning it with the amino acid sequence of SEQ ID NO: 2. The alignment can be easily performed using commercially available amino acid sequence analysis software based on a known algorithm such as Blast. For example, among the bacteria of the genus Cupriavidus exemplified above, in the case of the amino acid sequence of Cupriavidus necator and Cupriavidus taiwanensis D-succinylase, the alignment result with the amino acid sequence of SEQ ID NO: 2 is as shown in FIG. The amino acid residue at the shaded position in “the position equivalent to any of positions 72, 181-185, 305, 348, 351, 461, and 539 of SEQ ID NO: 2” Corresponds to an amino acid residue. For example, Transmuter Mutagenesis Kit; manufactured by Clonetech; EXOIII / Mung Bean Deletion Kit; manufactured by Stratagene; QuickChange Site Directed Mutesisis Kit; The base sequence corresponding to the amino acid sequence of the base D-succinylase can be mutated site-specifically. 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 purified enzyme protein is added to N-succinyl-DL amino acid, and the degradation ratio of L-form to D-form is measured by the method described in the Examples. it can.
　また、本発明の第４の側面は、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓの近縁種のＤ－サクシニラーゼをベースとした改変型Ｄ－サクシニラーゼ及びその遺伝子に関する。即ち、本発明の第４の側面によれば、配列番号４と７０％以上の相同性を有するアミノ酸配列において、配列番号４の１７７位、１８０位、３４４位、３４７位、及び４５７位のうちのいずれかと同等な位置のアミノ酸残基が、本発明の第２の側面のタンパク質（Ａ）に示すアミノ酸残基に置換されているアミノ酸配列からなるタンパク質であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有することを特徴とするタンパク質、及びこのタンパク質をコードすることを特徴とする遺伝子が提供される。 In addition, the fourth aspect of the present invention relates to a modified D-succinylase based on D-succinylase of a close relative of Cupriavidus metallidurans and a gene thereof. That is, according to the fourth aspect of the present invention, among amino acid sequences having 70% or more homology with SEQ ID NO: 4, among positions 177, 180, 344, 347, and 457 of SEQ ID NO: 4 A protein comprising an amino acid sequence in which an amino acid residue at a position equivalent to any one of the above is substituted with the amino acid residue shown in the protein (A) of the second aspect of the present invention, the N-succinyl-DL-amino acid There is provided a protein characterized by having an activity of selectively producing D-amino acids by acting selectively on D-form, and a gene characterized by encoding this protein.
　本発明の第４の側面の改変型Ｄ－サクシニラーゼは、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼのアミノ酸配列である配列番号４と７０％以上、好ましくは７４％以上、より好ましくは８０％以上、さらに好ましくは９０％以上、特に好ましくは９５％以上の相同性を有するＤ－サクシニラーゼをベースとする。このような相同性レベルを有するアミノ酸配列は、公知のアミノ酸配列のデータベースに基づく相同性検索や、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓのＤ－サクシニラーゼの遺伝子のＤＮＡ断片をプローブとしたハイブリダイゼーションにより得ることができ、具体的なアミノ酸配列としては、本発明の第３の側面で例示したものと同様のものが例示される。
　本発明の第４の側面において、改変のベースとするＤ－サクシニラーゼは、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓの近縁種からの野生型Ｄ－サクシニラーゼに限定されず、これらの野生型Ｄ－サクシニラーゼに何らかの変異を生じさせたものも、配列番号４と７０％以上の相同性を有する限り、改変のベースとして使用することができる。
　本発明の第４の側面において、改変は、これらのベースとするＤ－サクシニラーゼのアミノ酸配列中の、配列番号４の１７７位、１８０位、３４４位、３４７位、及び４５７位のうちのいずれかと同等な位置のアミノ酸残基を、本発明の第２の側面の改変型Ｄ－サクシニラーゼと同じアミノ酸残基に置換することによって行われる。ここで、「配列番号４の１７７位、１８０位、３４４位、３４７位、及び４５７位のうちのいずれかと同等な位置」は、ベースとするＤ－サクシニラーゼのアミノ酸配列を配列番号４のアミノ酸配列とアラインメントさせることによって特定することができる。アラインメントは、Ｂｌａｓｔなどの公知のアルゴリズムに基づく市販のアミノ酸配列解析ソフトを使用して容易に行うことができる。例えば、先に例示したＣｕｐｒｉａｖｉｄｕｓ属の細菌のうち、Ｃｕｐｒｉａｖｉｄｕｓ　ｎｅｃａｔｏｒ及びＣｕｐｒｉａｖｉｄｕｓ　ｔａｉｗａｎｅｎｓｉｓのＤ－サクシニラーゼのアミノ酸配列の場合、配列番号４のアミノ酸配列とのアラインメント結果は、図３に示す通りであり、図３中の網掛けされた位置のアミノ酸残基が「配列番号４の１７７位、１８０位、３４４位、３４７位、及び４５７位のうちのいずれかと同等な位置」のアミノ酸残基に相当する。アミノ酸置換の方法、及び得られたタンパク質の活性の確認方法は、第３の側面と同様であるので、説明を省略する。 The modified D-succinylase according to the fourth aspect of the present invention is 70% or more, preferably 74% or more, more preferably 80% or more, more preferably 80% or more, with SEQ ID NO: 4, which is the amino acid sequence of D-succinylase of Cupriavidus metallidurans It is based on D-succinylase having a homology of 90% or more, particularly preferably 95% or more. An amino acid sequence having such a homology level can be obtained by homology search based on a database of known amino acid sequences or by hybridization using a DNA fragment of the gene of D. succinylase of Cupriavidus metallidurans as a probe. Examples of such amino acid sequences are the same as those exemplified in the third aspect of the present invention.
In the fourth aspect of the present invention, the D-succinylase on which the modification is based is not limited to wild-type D-succinylases from relatives of Cupriavidus metallidurans, but causes any mutation in these wild-type D-succinylases. As long as it has 70% or more homology with SEQ ID NO: 4, it can be used as a base for modification.
In the fourth aspect of the present invention, the modification is any one of positions 177, 180, 344, 347, and 457 of SEQ ID NO: 4 in the amino acid sequence of these base D-succinylases. This is done by substituting an amino acid residue at an equivalent position with the same amino acid residue as the modified D-succinylase of the second aspect of the present invention. Here, “position equivalent to any of positions 177, 180, 344, 347, and 457 of SEQ ID NO: 4” is the amino acid sequence of SEQ ID NO: 4 based on the amino acid sequence of the base D-succinylase And can be specified by aligning. The alignment can be easily performed using commercially available amino acid sequence analysis software based on a known algorithm such as Blast. For example, among the bacteria of the genus Cupriavidus exemplified above, in the case of the amino acid sequence of Cupriavidus necator and Cupriavidus taiwanensis D-succinylase, the alignment result with the amino acid sequence of SEQ ID NO: 4 is as shown in FIG. The amino acid residue at the shaded position corresponds to the amino acid residue at “position equivalent to any of positions 177, 180, 344, 347, and 457 of SEQ ID NO: 4”. Since the method for amino acid substitution and the method for confirming the activity of the obtained protein are the same as those in the third aspect, description thereof is omitted.
　次に、本発明の改変型Ｄ－サクシニラーゼの製造方法について説明する。本発明の改変型Ｄ－サクシニラーゼは、その遺伝子を適当なベクターに挿入して組換えベクターを調製し、この組換えベクターで適当な宿主細胞を形質転換して形質転換体を調製し、この形質転換体を培養することによって容易に製造することができる。 Next, a method for producing the modified D-succinylase of the present invention will be described. The modified D-succinylase of the present invention is prepared by inserting a gene into an appropriate vector to prepare a recombinant vector, and transforming an appropriate host cell with the recombinant vector to prepare a transformant. It can be easily produced by culturing the transformant.
　ベクターとしては、原核および／または真核細胞の各種宿主細胞内で複製保持または自律増殖できるものであれば特に限定されず、プラスミドベクターおよびファージベクター、ウィルスベクター等が包含される。組換えベクターの調製は、常法に従って行えばよく、例えば、これらのベクターに、本発明の改変型Ｄ－サクシニラーゼの遺伝子を適当な制限酵素およびリガーゼ、あるいは必要に応じてさらにリンカーもしくはアダプターＤＮＡを用いて連結することにより容易に行うことができる。また、Ｔａｑポリメラーゼのように増幅末端に一塩基を付加するようなＤＮＡポリメラーゼを用いて増幅作製した遺伝子断片であれば、ＴＡクローニングによるベクターへの接続も可能である。 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 can be prepared according to conventional methods. For example, the modified D-succinylase gene of the present invention is added to these vectors with an appropriate restriction enzyme and ligase, or, if necessary, a linker or adapter DNA. It can carry out easily by using and 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.
　また、宿主細胞としては、従来公知のものが使用可能であり、組換え発現系が確立しているものであれば特に制限されないが、好ましくは大腸菌、枯草菌、放線菌、麹菌、酵母といった微生物ならびに昆虫細胞、動物細胞、高等植物などが挙げられ、より好ましくは微生物が挙げられ、特に好ましくは大腸菌（例えば、Ｋ１２株、Ｂ株など）が挙げられる。形質転換体の調製は、常法に従って行えばよい。 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.
　得られた形質転換体を、その宿主細胞に応じた適当な培養条件で一定期間培養すれば、組込まれた遺伝子から本発明の改変型Ｄ－サクシニラーゼが発現されて、形質転換体中に蓄積する。 When the obtained transformant is cultured for a certain period of time under appropriate culture conditions according to the host cell, the modified D-succinylase of the present invention is expressed from the incorporated gene and accumulated in the transformant. .
　形質転換体中に蓄積した本発明の改変型Ｄ－サクシニラーゼは、未精製のまま用いることができるが、精製したものを使用しても良い。この精製方法としては、従来公知のものが使用可能であり、例えば、培養後の形質転換体あるいはその培養物を適当な緩衝液中でホモジナイズし、超音波処理や界面活性剤処理等により細胞抽出液を得、そこからタンパク質の分離精製に常套的に利用される分離技術を適宜組み合わせることにより行うことができる。このような分離技術としては、塩析、溶媒沈澱法等の溶解度の差を利用する方法、透析、限外濾過、ゲル濾過、非変性ポリアクリルアミドゲル電気泳動（ＰＡＧＥ）、ドデシル硫酸ナトリウム－ポリアクリルアミドゲル電気泳動（ＳＤＳ－ＰＡＧＥ）などの分子量の差を利用する方法、イオン交換クロマトグラフィー、ヒドロキシアパタイトクロマトグラフィーなどの荷電を利用する方法、アフィニティークロマトグラフィーなどの特異的親和性を利用する方法、逆相高速液体クロマトグラフィーなどの疎水性の差を利用する方法、等電点電気泳動などの等電点の差を利用する方法などが挙げられるが、これらに限定されない。 The modified D-succinylase of the present invention accumulated in the transformant can be used as it is, but it 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 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.
　次に、本発明の改変型Ｄ－サクシニラーゼを用いてＤ－アミノ酸を製造する方法について説明する。本発明によるＤ－アミノ酸は、本発明の改変型Ｄ－サクシニラーゼを用いてＮ－サクシニル－ＤＬ－アミノ酸（ラセミ体）中のＮ－サクシニル－Ｄ－アミノ酸（Ｄ体）を特異的に加水分解する工程によって製造される。 Next, a method for producing a D-amino acid using the modified D-succinylase of the present invention will be described. The D-amino acid according to the present invention specifically hydrolyzes N-succinyl-D-amino acid (D form) in N-succinyl-DL-amino acid (racemic form) using the modified D-succinylase of the present invention. Manufactured by process.
　この工程は、具体的には、適当な溶液中に、本発明の改変型Ｄ－サクシニラーゼと原料のＮ－サクシニル－ＤＬ－アミノ酸を溶解させて反応液を調製し、この反応液を適当な条件で反応させることによって行うことができる。 Specifically, in this step, a modified reaction solution is prepared by dissolving the modified D-succinylase of the present invention and the raw material N-succinyl-DL-amino acid in a suitable solution. It can be performed by reacting with.
　使用する溶液は、蒸留水でもよいが、必要により、リン酸塩やトリスなどの緩衝剤を使用してもよい。緩衝剤を使用する場合、その濃度は２０～２００ｍＭであることが好ましく、ｐＨは６～８であることが好ましい。 The solution to be used may be distilled water, but if necessary, a buffer such as phosphate or Tris may be used. When a buffering agent is used, the concentration is preferably 20 to 200 mM, and the pH is preferably 6 to 8.
　本発明の改変型Ｄ－サクシニラーゼは、反応液中１０～３０００ｍｇ／Ｌ（１００～３００００Ｕ／Ｌ）の濃度で使用されることが好ましい。 The modified D-succinylase of the present invention is preferably used at a concentration of 10 to 3000 mg / L (100 to 30000 U / L) in the reaction solution.
　本発明の改変型Ｄ－サクシニラーゼと反応させるＮ－サクシニル－ＤＬ－アミノ酸は、種々の公知の方法によって合成することができ、例えばＳａｋａｉ　Ａ．ｅｔ　ａｌ．，Ｂｉｏｃｈｅｍｉｓｔｒｙ，２００６，４５（１４），４４５５－６２に記載の方法によって合成することができる。原料のＤＬ－アミノ酸の種類は、製造したいＤ－アミノ酸の種類に応じて適宜選択すればよく、天然に存在する２０種のアミノ酸およびビフェニルアラニン、シクロヘキシルグリシン、４－ブロモフェニルアラニンなどの非天然アミノ酸およびその誘導体であることができる。反応液中のＮ－サクシニル－ＤＬ－アミノ酸の濃度は、特に限定されないが、一般的に１重量％～３０重量％である。 N-succinyl-DL-amino acid to be reacted with the modified 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 type of DL-amino acid used as a raw material may be appropriately selected according to the type of D-amino acid to be produced. Twenty naturally occurring amino acids and unnatural amino acids such as biphenylalanine, cyclohexylglycine, 4-bromophenylalanine and the like It can be a derivative 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.
　本発明のＤ－アミノ酸の製造方法では、反応液を反応させる温度は、本発明の改変型Ｄ－サクシニラーゼが十分作用する温度であれば特に限定されないが、一般的には５～６０℃が好ましく、１０～５５℃がより好ましく、３０～５０℃がさらに好ましい。また、反応時のｐＨは、本発明の改変型Ｄ－サクシニラーゼが十分作用するｐＨであれば特に限定されないが、一般的にはｐＨ５～１０が好ましく、ｐＨ６～９がより好ましい。反応時間は、特に限定されないが、一般的に１～１２０時間程度、好ましくは１～７２時間程度、さらに好ましくは１～２４時間程度である。反応時間は、製造したいＤ－アミノ酸の種類と所望の収量、収率、使用する酵素や基質の量、量比、反応温度や反応ｐＨなどを考慮し、実験的に適宜選択することができる。 In the method for producing a D-amino acid of the present invention, the temperature at which the reaction solution is reacted is not particularly limited as long as the modified D-succinylase of the present invention acts sufficiently, but generally 5 to 60 ° C. is preferable. 10 to 55 ° C is more preferable, and 30 to 50 ° C is more preferable. Further, the pH during the reaction is not particularly limited as long as the modified 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.
　なお、本発明のＤ－アミノ酸の製造方法で使用する改変型Ｄ－サクシニラーゼは、精製されたものや未精製のもの（粗酵素液など）に限定されず、形質転換体中に含まれた状態のものであってもよい。この場合は、形質転換体を反応系に添加して、形質転換体を培養させながら同時に反応を進行させるか、又は予め培養された形質転換体を反応系に添加すればよい。 The modified D-succinylase used in the method for producing D-amino acid of the present invention is not limited to a purified or unpurified one (crude enzyme solution, etc.), and is included in the 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.
　好ましくは、本発明のＤ－アミノ酸の製造方法は、Ｎ－サクシニルアミノ酸ラセマーゼを用いてＮ－サクシニル－Ｌ－アミノ酸をラセミ化してＮ－サクシニル－Ｄ－アミノ酸を生成させる工程をさらに含む。本発明の改変型Ｄ－サクシニラーゼは、Ｎ－サクシニル－ＤＬ－アミノ酸（ラセミ体）中のＮ－サクシニル－Ｄ－アミノ酸を主に加水分解するため、このままではラセミ体のうち、Ｎ－サクシニル－Ｌ－アミノ酸の多くが無駄になってしまう。そこで、Ｎ－サクシニルアミノ酸ラセマーゼを用いてＮ－サクシニル－Ｌ－アミノ酸をラセミ化してＮ－サクシニル－Ｄ－アミノ酸を生成させてやれば、残ったＮ－サクシニル－Ｌ－アミノ酸もＤ－アミノ酸に変換することができる。 Preferably, the method for producing D-amino acid of 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 modified D-succinylase of the present invention mainly hydrolyzes the N-succinyl-D-amino acid in the N-succinyl-DL-amino acid (racemate), the N-succinyl-L of the racemate remains as it is. -Many amino acids 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－サクシニルアミノ酸ラセマーゼは、Ｎ－サクシニルアミノ酸のＬ体をＤ体に変換する反応とＤ体をＬ体に変換する反応の両方を触媒してその比率をほぼ等しく（ラセミ化）する酵素である。本発明の製造方法で使用するＮ－サクシニルアミノ酸ラセマーゼは、Ｎ－サクシニルアミノ酸をラセミ化することができれば特に限定されず、特開２００７－８２５３４号公報に記載されたＮ－アシルアミノ酸ラセマーゼや特開２００８－６１６４２号公報に記載されたＮ－アシルアミノ酸ラセマーゼなどの従来公知のものを使用することができる。 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.
　Ｎ－サクシニルアミノ酸ラセマーゼを用いてＮ－サクシニル－Ｄ－アミノ酸をラセミ化する反応は、例えば以下の条件でＮ－サクシニルアミノ酸、Ｎ－サクシニルアミノ酸ラセマーゼおよび緩衝剤を含む反応溶液中で混合することにより行なう。反応温度は、使用するＮ－サクシニルアミノ酸ラセマーゼが十分作用する温度であれば特に限定されないが、一般的には２５～７０℃が好ましく、３７～６０℃がより好ましい。反応時のｐＨは、使用するＮ－サクシニルアミノ酸ラセマーゼが十分作用するｐＨであれば特に限定されないが、一般的にはｐＨ５～９が好ましく、ｐＨ６～８がより好ましい。Ｎ－サクシニルアミノ酸ラセマーゼは、反応液中５０～１５０００ｍｇ／Ｌ（５０００～１５０００００Ｕ／Ｌ）の濃度で使用されることが好ましい。Ｎ－サクシニルアミノ酸ラセマーゼは、２価の金属イオンを０．１ｍＭ～１Ｍ（好ましくは０．１～１ｍＭ）の終濃度で添加することにより、活性が著しく向上する。添加する２価の金属イオンとしては、Ｍｎ２＋，Ｃｏ２＋，Ｍｇ２＋，Ｆｅ２＋およびＮｉ２＋が挙げられる。Ｎ－サクシニルアミノ酸ラセマーゼの反応に用いる緩衝剤は、Ｄ－サクシニラーゼの反応に用いる緩衝剤と同様のものが使用できる。なお、特開２００７－８２５３４号に記載のＮ－アシルアミノ酸ラセマーゼは、その後の研究により、Ｎ－サクシニルアミノ酸をより好適な基質とするＮ－サクシニルアミノ酸ラセマーゼであることが判明している。従って、特開２００７－８２５３４号に記載のＮ－アシルアミノ酸ラセマーゼは、本発明のＤ－サクシニラーゼと組合せて使用することができる。 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). 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-succinyl amino acid racemase, the same buffer as that used for the reaction of D-succinylase can be used. The 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.
　前述のＮ－サクシニルアミノ酸ラセマーゼによるラセミ化反応とＤ－サクシニラーゼによる加水分解反応は、別々に行なうことも可能であるが、同時に行なわれることが好ましい。同時に行われる場合、微視的に見ると、まずＮ－サクシニル－ＤＬ－アミノ酸のうちＤ体が本発明のＤ－サクシニラーゼにより加水分解（脱サクシニル化）され、目的のＤ－アミノ酸が生成する。基質のＤ体が消費されるとラセミ状態が解消されるため、Ｎ－サクシニルアミノ酸ラセマーゼはＬ体をＤ体に変換する反応をより促進する。Ｎ－サクシニルアミノ酸ラセマーゼにより生成したＮ－サクシニル－Ｄ－アミノ酸は、本発明のＤ－サクシニラーゼにより順次Ｄ－アミノ酸へと分解される。この繰返しにより、理論的にはほぼすべてのＮ－サクシニル－ＤＬ－アミノ酸をＤ－アミノ酸に変換することができる。ラセミ化反応および加水分解反応を同時に行う場合の反応条件は、Ｎ－サクシニルアミノ酸ラセマーゼおよび本発明のＤ－サクシニラーゼが活性を発揮する条件の範囲内であれば特に限定しないが、基質濃度１重量％～３０重量％、ｐＨ６～８、温度４０～５０℃で行うことが好ましい。ラセミ化反応および加水分解反応に要する時間は、原料として用いるＮ－サクシニル－ＤＬ－アミノ酸が、所望する量のＤ－アミノ酸へと変換し得るまでの時間であれば特に制限されず、仕込み量によっても異なるが、一般的に１日～７日程度である。 The racemization reaction using N-succinylamino acid racemase and the hydrolysis reaction using 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, 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. By repeating this, theoretically almost all N-succinyl-DL-amino acids can be converted to D-amino acids. The reaction conditions when the racemization reaction and the hydrolysis reaction are carried out simultaneously are not particularly limited as long as the N-succinylamino acid racemase and the D-succinylase of the present invention are within the range of activity, but the substrate concentration is 1% by weight. It is preferable to carry out at 30 to 30% by weight, pH 6 to 8 and temperature 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.
　以下、本発明を実施例により、具体的に説明するが、本発明の範囲は下記の実施例に限定されることはない。 Hereinafter, the present invention will be specifically described by way of examples. However, the scope of the present invention is not limited to the following examples.
　本実施例において、Ｎ－サクシニルトリプトファン、Ｎ－サクシニルフェニルアラニン、Ｎ－サクシニルビフェニルアラニンの定量は、特に記載がない場合は、目的に応じて、以下の分析条件で定量した。 In this example, N-succinyltryptophan, N-succinylphenylalanine, and N-succinylbiphenylalanine were quantified under the following analysis conditions depending on the purpose unless otherwise specified.
　Ｎ－サクシニル－Ｄ－アミノ酸又はＮ－サクシニル－Ｌ－アミノ酸などの光学活性体を定量する場合は、ジーエルサイエンス社製「Ｉｎｅｒｔｓｉｌ　ＯＤＳ－３」（５μｍ、４．６×１００ｍｍ）を利用した高速液体クロマトグラフィーにより行った。 When quantifying optically active substances such as N-succinyl-D-amino acid or N-succinyl-L-amino acid, a high-speed liquid using “Inertsil ODS-3” (5 μm, 4.6 × 100 mm) manufactured by GL Sciences Inc. Performed by chromatography.
Ｎ－サクシニルトリプトファンの分析
移動相：ｐＨ２．３リン酸水溶液／アセトニトリル＝７５／２５
流速：０．７ｍｌ／ｍｉｎ
カラム温度：４０℃
検出：ＵＶ２８０ｎｍ Analytical mobile phase of N-succinyltryptophan: pH 2.3 aqueous phosphoric acid / acetonitrile = 75/25
Flow rate: 0.7 ml / min
Column temperature: 40 ° C
Detection: UV280nm
Ｎ－サクシニルフェニルアラニンの分析
移動相：ｐＨ２．３リン酸水溶液／アセトニトリル＝７５／２５
流速：０．７ｍｌ／ｍｉｎ
カラム温度：４０℃
検出：ＵＶ２１０ｎｍ Analytical mobile phase of N-succinylphenylalanine: pH 2.3 aqueous phosphoric acid solution / acetonitrile = 75/25
Flow rate: 0.7 ml / min
Column temperature: 40 ° C
Detection: UV210nm
Ｎ－サクシニルビフェニルアラニンの分析
移動相：ｐＨ２．３リン酸水溶液／アセトニトリル＝７５／２５
流速：１．５ｍｌ／ｍｉｎ
カラム温度：４０℃
検出：ＵＶ２５４ｎｍ Analytical mobile phase of N-succinylbiphenylalanine: pH 2.3 aqueous phosphoric acid / acetonitrile = 75/25
Flow rate: 1.5 ml / min
Column temperature: 40 ° C
Detection: UV254nm
　また、光学純度を測定する際には、ダイセル化学工業株式会社製光学分割カラム「ＣＨＩＲＡＬＰＡＫ　ＱＮ－ＡＸ」（５μｍ、４．６×１５０ｍｍ）により行った。分析条件は以下に示した通りである。 Further, when measuring the optical purity, it was performed with an optical resolution column “CHIRALPAK QN-AX” (5 μm, 4.6 × 150 mm) manufactured by Daicel Chemical Industries, Ltd. The analysis conditions are as shown below.
Ｎ－サクシニルトリプトファン、Ｎ－サクシニルフェニルアラニン及びＮ－サクシニルビフェニルアラニンの分析
移動相：メタノール／０．２Ｍ酢酸＝９０／１０（Ｖ／Ｖ）アンモニアでｐＨ７．０
に調製
流速：１．０ｍｌ／ｍｉｎ
カラム温度：３５℃
検出：ＵＶ２１０ｎｍ Analytical mobile phase of N-succinyltryptophan, N-succinylphenylalanine and N-succinylbiphenylalanine: methanol / 0.2M acetic acid = 90/10 (V / V) pH 7.0 with ammonia
Preparation flow rate: 1.0 ml / min
Column temperature: 35 ° C
Detection: UV210nm
　また、Ｎ－サクシニルアミノ酸が加水分解（脱サクシニル化）されて生成する遊離体のＤ－アミノ酸及びＬ－アミノ酸を定量する際は、ダイセル化学工業株式会社製光学分割カラム「ＣＲＯＷＮＰＡＫ　ＣＲ（＋）」（５μｍ、４．０×１５０ｍｍ）により行った。分析条件は以下に示した通りである。 In addition, when quantifying free D-amino acids and L-amino acids produced by hydrolysis (desuccinylation) of N-succinylamino acids, an optical resolution column “CROWNPAK CR (+)” manufactured by Daicel Chemical Industries, Ltd. (5 μm, 4.0 × 150 mm). The analysis conditions are as shown below.
ＤＬ－トリプトファンの分析
移動相：過塩素酸水溶液（ｐＨ２．０）／メタノール＝９０／１０
流速：０．７ｍｌ／ｍｉｎ
カラム温度：１５℃
検出：ＵＶ２８０ｎｍ Analytical mobile phase of DL-tryptophan: aqueous perchloric acid solution (pH 2.0) / methanol = 90/10
Flow rate: 0.7 ml / min
Column temperature: 15 ° C
Detection: UV280nm
ＤＬ－フェニルアラニンの分析
移動相：過塩素酸水溶液（ｐＨ２．０）
流速：１．２ｍｌ／ｍｉｎ
カラム温度：１５℃
検出：ＵＶ２１０ｎｍ Analysis mobile phase of DL-phenylalanine: aqueous perchloric acid solution (pH 2.0)
Flow rate: 1.2 ml / min
Column temperature: 15 ° C
Detection: UV210nm
ＤＬ－ビフェニルアラニンの分析
移動相：過塩素酸水溶液（ｐＨ２．０）／メタノール＝８５／１５
流速：１．５ｍｌ／ｍｉｎ
カラム温度：５０℃
検出：ＵＶ２５４ｎｍ Analysis mobile phase of DL-biphenylalanine: aqueous perchloric acid solution (pH 2.0) / methanol = 85/15
Flow rate: 1.5 ml / min
Column temperature: 50 ° C
Detection: UV254nm
　本発明におけるＤ－サクシニラーゼの活性は、以下のＤ－アミノ酸オキシダーゼ法により測定することができる。
＜試薬＞
１４ｍＭ　Ｎ－サクシニル－Ｄ－バリン
２．５（Ｕ／ｍＬ）　ペルオキシダーゼ（東洋紡製ＰＥＯ－３０２）
１．５ｍＭ　４－アミノアンチピリン（第一化学薬品製）
２．４ｍＭ　ＴＯＯＳ（同人化学研究所製）
６．０（Ｕ／ｍＬ）　Ｄ－アミノ酸オキシダーゼ（バイオザイム製ＤＯＸ２）
を含む２５ｍＭリン酸緩衝溶液を反応試薬とする。
　なお、Ｎ－サクシニル－Ｄ－トリプトファンに対する酵素活性を測定する際には、１４ｍＭ　Ｎ－サクシニル－Ｄ－バリンの代わりに、３３ｍＭ　Ｎ－サクシニル－Ｄ－トリプトファンを使用した。
　Ｎ－サクシニル－Ｄ－バリンの合成は以下のようにして行った。Ｄ－バリン（ナカライテスク製、４．７ｇ）と無水コハク酸（ナカライテスク製、４．０ｇ）を４０ｍＬの酢酸（ナカライテスク製）に溶解した。溶液を５０～６０℃に加熱し、溶媒を揮発させて結晶化した。次に、結晶化した白い沈殿を集めて、酢酸エチル２０ｍＬとメタノール２０ｍＬの混合液にて再結晶化した。沈殿を乳鉢で破砕し、乾燥させ、Ｎ－サクシニル－Ｄ－バリンを得た。詳細はＳａｋａｉ　Ａ．ｅｔ　ａｌ．，Ｂｉｏｃｈｅｍｉｓｔｒｙ，２００６，４５（１４），４４５５－６２に記載されている。
＜測定条件＞
　反応試薬３．０ｍＬを３７℃で５分間予備加温する。酵素溶液０．１ｍＬを添加しゆるやかに混和後、水を対照に３７℃に制御された分光光度計で、５５５ｎｍの吸光度変化を５分記録し、直線部分から１分間あたりの吸光度変化（ΔＯＤＴＥＳＴ）を測定する。盲検は、酵素溶液の代わりに酵素を溶解する溶液を試薬混液に加えて同様に１分間あたりの吸光度変化（ΔＯＤＢＬＡＮＫ）を測定する。これらの値から次の式に従ってＤ－サクシニラーゼ活性を求める。ここでＤ－サクシニラーゼ活性における１単位（Ｕ）とは、上記条件下で１分間に１マイクロモルのＤ－アミノ酸を生成する酵素量として定義する。
 
活性（Ｕ／ｍＬ）＝
　　　　｛（ΔＯＤＴＥＳＴ－ΔＯＤＢＬＡＮＫ）×３．１×希釈倍率｝／｛３１．０×１／２×０．１×１．０｝
 
　なお、式中の３．１は反応試薬＋酵素溶液の液量（ｍＬ）、３１．０は本活性測定条件におけるミリモル分子吸光係数（ｃｍ２／マイクロモル）、１／２は酵素反応で生成したＨ２Ｏ２の１分子から形成するＱｕｉｎｏｎｅｉｍｉｎｅ色素が１／２分子であることによる係数、０．１は酵素溶液の液量（ｍＬ）、１．０はセルの光路長（ｃｍ）を示す。 The activity of D-succinylase in the present invention can be measured by the following D-amino acid oxidase method.
<Reagent>
14 mM N-succinyl-D-valine 2.5 (U / mL) peroxidase (Toyobo PEO-302)
1.5 mM 4-aminoantipyrine (Daiichi Chemical)
2.4 mM TOOS (manufactured by Doujin Chemical Laboratory)
6.0 (U / mL) D-amino acid oxidase (Biozyme DOX2)
A 25 mM phosphate buffer solution containing
In measuring the enzyme activity for N-succinyl-D-tryptophan, 33 mM N-succinyl-D-tryptophan was used instead of 14 mM N-succinyl-D-valine.
N-succinyl-D-valine was synthesized as follows. D-valine (Nacalai Tesque, 4.7 g) and succinic anhydride (Nacalai Tesque, 4.0 g) were dissolved in 40 mL of acetic acid (Nacalai Tesque). The solution was heated to 50-60 ° C. and the solvent was evaporated and crystallized. Next, the crystallized white precipitate was collected and recrystallized with a mixed solution of 20 mL of ethyl acetate and 20 mL of methanol. The precipitate was crushed in a mortar and dried to obtain N-succinyl-D-valine. For details, see Sakai A. et al. Biochemistry, 2006, 45 (14), 4455-62.
<Measurement conditions>
Prewarm 3.0 mL of reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 mL of the enzyme solution and mixing gently, the absorbance change at 555 nm was recorded for 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control, and the absorbance change per minute (ΔOD TEST) ). In the blind test, a solution that dissolves the enzyme instead of the enzyme solution is added to the reagent mixture, and the change in absorbance per minute (ΔOD BLANK ) is similarly measured. From these values, D-succinylase activity is determined according to the following formula. Here, 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.

Activity (U / mL) =
{(ΔOD TEST −ΔOD BLANK ) × 3.1 × dilution ratio} / {31.0 × 1/2 × 0.1 × 1.0}

In the formula, 3.1 is the amount of the reaction reagent + enzyme solution (mL), 31.0 is the millimolar 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, 0.1 is the volume of the enzyme solution (mL), 1.0 is the optical path length (cm) of the cell .
　また、Ｎ－サクシニルアミノ酸ラセマーゼの活性測定は、以下に述べる方法で行った。
ＨＥＰＥＳ（１．０Ｍ／ｐＨ７．９）を１０μｌ（終濃度１００ｍＭ）、酢酸コバルトを１μｌ（終濃度０．１ｍＭ）滅菌蒸留水を４９μｌを混和し、この溶液に酵素液１０μｌを加え、この反応液に、基質となるＮ－サクシニル－Ｄ－フェニルアラニン溶液を３０μｌ（終濃度６０ｍＭ）を加えて３０℃で反応を行い、適当な時間で移動相（メタノール／０．２Ｍ酢酸＝９０／１０（Ｖ／Ｖ）アンモニアでｐＨ７．０に調製したもの）により反応停止させた。生成したＮ－サクシニル－Ｌ－フェニルアラニンを高速液体クロマトグラフィーにより定量し、酵素活性を算出する。酵素活性はＮ－サクシニル－Ｄ－フェニルアラニンからＮ－サクシニル－Ｌ－フェニルアラニンが１分間に１μｍｏｌｅ生成された場合を１ｕｎｉｔ（Ｕ）と定義した。 The activity of N-succinyl amino acid racemase was measured by the method described below.
Mix 10 μl of HEPES (1.0 M / pH 7.9) (final concentration 100 mM), 1 μl of cobalt acetate (final concentration 0.1 mM) and 49 μl of sterile distilled water, add 10 μl of enzyme solution to this solution, and add this reaction solution. 30 μl (final concentration 60 mM) of N-succinyl-D-phenylalanine solution as a substrate was added and reacted at 30 ° C., and mobile phase (methanol / 0.2 M acetic acid = 90/10 (V / V) The reaction was stopped with ammonia adjusted to pH 7.0). The produced N-succinyl-L-phenylalanine is quantified by high performance liquid chromatography, and the enzyme activity is calculated. The enzyme activity was defined as 1 unit (U) when 1 μmole of N-succinyl-L-phenylalanine was produced from N-succinyl-D-phenylalanine per minute.
参考例１　Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株由来Ｄ－サクシニラーゼ遺伝子（以下、Ｐ４ＤＳＡ遺伝子と称する）のクローニング
（１）野生型Ｄ－サクシニラーゼの精製
　Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株（日本国茨城県つくば市東１丁目１番地１　中央第６の独立行政法人産業技術総合研究所　特許生物寄託センターに受託番号ＦＥＲＭ　ＢＰ－１１３８７として２０１１年６月２８日に国際寄託済）を、普通ブイヨン“栄研”１．８％に、粉末酵母エキスＤ－３　０．２％（和光純薬工業（株）製、Ｃｏｄｅ：３９０－００５３１）、カザミノ酸「ダイゴ」０．１％（和光純薬工業（株）製、Ｃｏｄｅ：３９２－００６５５）、リン酸水素二カリウム０．３％、及びグルコース０．２％を添加したｐＨ７．５の培地で培養した。具体的には、５００ｍＬフラスコに上記培地２００ｍＬを加えオートクレーブ滅菌した培地２７本を用い、３５℃、１５０ｒ／ｍｉｎ、回転攪拌で２日間培養した。培養終了時の濁度（ＡＢＳ６６０ｎｍ）は２．７であり、ｐＨは８．６であった。培養後、冷却遠心分離機（日立工機（株）製）を用い、８０００ｒ／ｍｉｎで３０分間遠心分離を行い、集菌した。集菌した菌体を、２０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ（ｐＨ７．５）緩衝液で洗浄した後、再度遠心分離し、菌体を３６ｇ得た。取得菌体量の約３倍の２０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ（ｐＨ７．５）緩衝液１００ｍＬで懸濁後、氷冷下の超音波細胞破砕装置　ＢＤ－１（東湘電気（株）製）を用い、５分サイクルで１０回超音波破砕を行った。破砕後、高速冷却遠心機（日立工機（株）製）で８０００ｒ／ｍｉｎ、４℃、６０分間遠心分離し、上清液１７８ｍＬを得た。これを粗酵素液とした。なお、本菌株は培養時にＮ－サクシニル－Ｄ－フェニルアラニンを添加しなくともＤ－サクシニラーゼを産生した。粗酵素液を透析チューブに詰め、２０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ（ｐＨ７．５）１．２Ｍ硫酸アンモニウム含有緩衝液中に投入し、低温室内（４℃）で攪拌を行いながら、数回緩衝液を交換し、一昼夜透析を行った。透析終了後、高速冷却遠心機（日立工機（株）製）で４０００ｒ／ｍｉｎ、４℃、３０分間遠心分離し、上清液１００ｍＬを得た。この上清液１００ｍＬを、予め２０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ（ｐＨ７．５）１．２Ｍ硫酸アンモニウム含有緩衝液で平衡化したＢｕｔｙｌ－ＴＯＹＯＰＥＡＲＬ　６５０Ｍカラム（東ソー（株）製）（３．２ｃｍφ×２０ｃｍ）に供して目的酵素を吸着させ、２０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ（ｐＨ７．５）１．２Ｍ硫酸アンモニウム含有緩衝液と２０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ（ｐＨ７．５）緩衝液を総量２０００ｍＬ用い、直線濃度勾配法で酵素を溶出し、Ｄ－サクシニラーゼ活性のある画分を回収した。Ｂｕｔｙｌ－ＴＯＹＯＰＥＡＲＬで得られた活性画分を７０％硫酸アンモニウム飽和にて濃縮し、遠心分離１０，０００ｒ／ｍｉｎ、４℃、６０分間にて沈殿を回収した。回収した硫安沈殿を、５ｍＭリン酸緩衝液（ｐＨ７．２）で透析した。この透析した酵素液３３ｍＬを、予め５ｍＭリン酸緩衝液（ｐＨ７．２）で平衡化したＭａｃｒｏ－Ｐｒｅｐ　ＣＨＴ　ＴｙｐｅＩ（ＢＩＯ－ＲＡＤ社製）ハイドロキシアパタイトカラム（１．６ｃｍφ×２０ｃｍ）に吸着させた。次に、５ｍＭリン酸緩衝液（ｐＨ７．２）と３００ｍＭ　リン酸緩衝液（ｐＨ７．２）を総量１５０ｍＬ用いて直線濃度勾配法で酵素を溶出し、Ｄ－サクシニラーゼ活性のある画分を回収した。Ｍａｃｒｏ－Ｐｒｅｐ　ＣＨＴ　ＴｙｐｅＩにより得られた活性画分１３０ｍＬを、ビバスピン　２０（ザルトリウス（株）製）分画分子量１００００の限外ろ過膜を用いて、２０ｍＬに濃縮した。回収した濃縮液を、２０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ（ｐＨ７．５）酸緩衝液で透析した。この透析液を、予め２０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ（ｐＨ７．５）緩衝液で平衡化したＨｉ　Ｔｒａｐ　Ｑ　Ｆ．Ｆ．カラム５ｍＬ（ＧＥヘルスケア　バイオサイエンス社製）に供し、続いて２０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ（ｐＨ７．５）と２０ｍＭ　ＨＥＰＥＳ－ＮａＯＨ（ｐＨ７．５）０．３Ｍ塩化ナトリウム酸緩衝液を総量２００ｍＬ用いて直線濃度勾配法で酵素を溶出し、Ｄ－サクシニラーゼ活性のある画分を回収した。Ｄ－サクシニラーゼ活性が確認された画分の少量を定法のＳＤＳ－ポリアクリルアミドゲル電気泳動（ＳＤＳ－ＰＡＧＥ）分析に供し、不純蛋白質が混在しない画分を回収し、新規Ｄ－サクシニラーゼを得た。精製後の電気泳動結果を図１に示す。 Reference Example 1 Cupriavidus sp. Cloning of D-succinylase gene derived from P4-10-C strain (hereinafter referred to as P4DSA gene) (1) Purification of wild-type D-succinylase Cupriavidus sp. P4-10-C (1st East, 1-chome, Tsukuba, Ibaraki, Japan 1st International Administrative Institute, National Institute of Advanced Industrial Science and Technology, Patent Biology Depositary Center on June 28, 2011 as FERM BP-11387 Deposited) is 1.8% for ordinary bouillon “Eiken”, 0.2% for powdered yeast extract D-3 (Code: 390-00531 manufactured by Wako Pure Chemical Industries, Ltd.), “Daigo” casamino acid 0 The cells were cultured in a medium at pH 7.5 supplemented with 1% (Wako Pure Chemical Industries, Ltd., Code: 392-00655), dipotassium hydrogen phosphate 0.3%, and glucose 0.2%. Specifically, the culture was carried out for 2 days at 35 ° C., 150 r / min, with rotary stirring, using 27 mediums that were autoclaved by adding 200 mL of the above medium to a 500 mL flask. The turbidity (ABS 660 nm) at the end of the culture was 2.7, and the pH was 8.6. After culturing, the cells were collected by centrifugation at 8000 r / min for 30 minutes using a cooled centrifuge (manufactured by Hitachi Koki Co., Ltd.). The collected cells were washed with 20 mM 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 Electric 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. This strain produced D-succinylase even when N-succinyl-D-phenylalanine was not added during culture. The crude enzyme solution was 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 was changed several times while stirring in a low temperature chamber (4 ° C.). Dialysis was performed overnight. 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. 100 mL of this supernatant was applied to a Butyl-TOYOPEARL 650M column (manufactured by Tosoh Corporation) (3.2 cmφ × 20 cm) previously equilibrated with a buffer containing 1.2 mM ammonium sulfate of 20 mM HEPES-NaOH (pH 7.5). The target enzyme is adsorbed, and the enzyme is eluted by a linear concentration gradient method using a total volume of 2000 mL of a 20 mM HEPES-NaOH (pH 7.5) 1.2 M ammonium sulfate-containing buffer and 20 mM HEPES-NaOH (pH 7.5) buffer. -The fraction with 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 recovered ammonium sulfate precipitate was dialyzed against 5 mM phosphate buffer (pH 7.2). 33 mL of the dialyzed enzyme solution was adsorbed onto a Macro-Prep CHT Type I (manufactured by BIO-RAD) hydroxyapatite column (1.6 cmφ × 20 cm) equilibrated in advance with a 5 mM phosphate buffer (pH 7.2). Next, the enzyme was eluted by a linear concentration gradient method using a total volume of 150 mL of 5 mM phosphate buffer (pH 7.2) and 300 mM phosphate buffer (pH 7.2), and a fraction having D-succinylase activity was recovered. . An active fraction (130 mL) obtained by Macro-Prep CHT Type I was concentrated to 20 mL using an ultrafiltration membrane having a molecular weight cut off of 10,000 by 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 Biosciences), 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 in a total volume of 200 mL The enzyme was eluted by a gradient method, and a fraction having D-succinylase activity was collected. 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.
（２）部分アミノ酸配列の取得
　ＳＤＳ－ＰＡＧＥ後に該酵素に相当するバンドをアクリルアミドゲル上からＰＶＤＦ膜（Ｂｉｏ－Ｒａｄ：シーケ－ブロットＰＶＤＦメンブレン）に転写し、Ｎ末端アミノ酸配列分析、及び内部アミノ酸配列分析を行い、約６０ｋＤａのバンドから内部アミノ酸配列；ＳＮＮＷＶＩＡＧＳＲＴＳＴＧＲ、約２３ｋＤａのバンドからＮ末端アミノ酸配列；ＡＰＰＴＤＲＹＡＡＰＧＬＥＫＰと内部アミノ酸配列；ＭＡＲＤＦＧＰＡＹＶＤＧＤＲＲを得た。 (2) Acquisition of partial amino acid sequence After SDS-PAGE, a band corresponding to the enzyme was transferred from an acrylamide gel to a PVDF membrane (Bio-Rad: Sequel-Blot PVDF membrane), N-terminal amino acid sequence analysis, and internal amino acid sequence Analysis was performed to obtain an internal amino acid sequence from the band of about 60 kDa; SNNWVIAGSRSTSTGR; an N-terminal amino acid sequence from the band of about 23 kDa; APPTDRYAAPGLEKP and the internal amino acid sequence; MARDFGPAYVDGDRR.
（３）染色体ＤＮＡの調製
　培養液５０ｍＬの菌体を遠心分離操作に供し、ゲノム抽出キット（東洋紡：Ｇｅｎｏｍｉｃ　ＤＮＡ　ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ）を用いて、そのプロトコールに従ってゲノム精製を行った。しかし、本キットで精製したゲノムＤＮＡでは全長配列のクローニングのためのＰＣＲ反応において、長鎖のＤＮＡ断片を増幅させることができなかった。ＰＣＲ条件を検討しても改善は見られなかったため、これはゲノムＤＮＡの純度に問題があると考え、「ｃｕｒｒｅｎｔ　ｐｒｏｔｏｃｏｌｓ　ｉｎ　ｍｏｌｕｃｕｌａｒ　ｂｉｏｌｏｇｙ」に記載されている公知の方法に基づいて、再びゲノムＤＮＡの抽出を行った。まず、５０ｍＬの培地で培養し、集菌した菌体をＴＥ（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０）、２０ｍＭ　ＥＤＴＡ）に懸濁して洗浄し、遠心分離操作により菌体を回収した後、再びこの菌体を１１．３ｍＬのＴＥに懸濁した。さらに、この懸濁液に０．０６ｍｌの２０ｍｇ／ｍＬプロテイナーゼＫ溶液、０．６ｍＬの１０％ＳＤＳ溶液を加えた後、３７℃で１時間インキュベートした。インキュベート後、２ｍＬの５Ｍ　ＮａＣｌ溶液を加えて十分に攪拌し、１．６ｍＬのＣＴＡＢ／ＮａＣｌ溶液（１０％　ＣＴＡＢ／０．７Ｍ　ＮａＣｌ）を混合し、６５℃で１０分間インキュベートした。インキュベート後、等量の２５／２４／１＝フェノール／クロロホルム／イソアミルアルコール溶液で除タンパクを行った。通常の細菌であれば、この工程で水層に濁りは見られないが、本菌においては濁りが残っていた。そこで、もう一度、等量の２５／２４／１＝フェノール／クロロホルム／イソアミルアルコール溶液で除タンパクを行った。これにより、濁りが完全に除去されたので、続いて、分離した水層に対して等量の２４／１＝クロロホルム／イソアミルアルコールを加えて混合し、水層を回収した。これと等量のイソプロパノールを加えてＤＮＡを沈殿させ、回収した。沈殿したＤＮＡを０．５ｍＬのＴＥに溶解した後、５μｌの１０ｍｇ／ｍＬ　ＲＮａｓｅを加えて、３７℃で一晩反応させた。反応後、等量の２５／２４／１＝フェノール／クロロホルム／イソアミルアルコール溶液で除タンパクを行い、２４／１＝クロロホルム／イソアミルアルコールを加えて攪拌し、水層を回収した。この操作をさらに２回行った後に得られた水層に終濃度０．４Ｍとなるように３Ｍ酢酸ナトリウム溶液（ｐＨ５．２）を加え、さらに２倍容のエタノールを加えた。沈殿となって生じたＤＮＡを回収し、７０％エタノールで洗浄、乾燥させ、１ｍＬのＴＥに溶解させた。 (3) Preparation of chromosomal DNA 50 mL of the culture broth was subjected to centrifugation, and genome purification was performed using 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 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, the cells were cultured in 50 mL of the medium, and the collected cells were suspended in TE (50 mM Tris-HCl (pH 8.0), 20 mM EDTA) and washed. After the cells were collected by centrifugation, the cells were again collected. The cells were suspended in 11.3 mL of TE. Further, 0.06 ml of 20 mg / mL proteinase K solution 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 5M NaCl solution was added and stirred well, 1.6 mL of CTAB / NaCl solution (10% CTAB / 0.7M 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 ordinary bacteria, turbidity was not observed in the aqueous layer in this step, but turbidity remained in this bacterium. 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 thereto 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.
（４）Ｐ４ＤＳＡ遺伝子の全長配列取得
　上記（２）で同定したアミノ酸配列をもとに縮重プライマーを合成し（表１の配列番号５、６）、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃから、上記（３）で抽出したゲノムＤＮＡを鋳型として、ＤＮＡポリメラーゼＫＯＤ－Ｐｌｕｓ（東洋紡製）を用いて推奨する条件のもとＰＣＲを行った。該ＰＣＲで増幅されたＰＣＲ産物をクローニングキットＴａｒｇｅｔ　Ｃｌｏｎｅ－Ｐｌｕｓ（東洋紡製）を用いて、そのプロトコールに従って操作を行い、ベクターｐＢｌｕｅｓｃｒｉｐｔにクローニングし、エシェリヒア・コリー（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＤＨ５α株コンピテントセル（東洋紡製）に形質転換し、該形質転換体を取得した。該形質転換体をＬＢ培地で培養し、プラスミドを抽出し、ＢｉｇＤｙｅ（商標登録）Ｔｅｒｍｉｎａｔｏｒ　ｖ３．１　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）により遺伝子配列を確認し、部分配列５７５ｂｐを取得した。さらに、全長配列を取得するために以下のような操作を行った。ＴＡＫＡＲＡ　ＬＡ　ＰＣＲ　Ｉｎ　Ｖｉｔｒｏ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（タカラバイオ製）を用いて、そのプロトコールに従って操作を行い、既知配列からＣ末端方向へのＤＮＡ断片を増幅させることに成功し、開始コドンを含むＮ末端から既知の部分遺伝子配列までの塩基配列を決定した。しかしながら、上記キットではＣ末端側の塩基配列を決定することができなかったため、既知の塩基配列に基づき、遺伝子の外側方向に向けた２種類のプライマー（表１の配列番号７、８）を新たに設計した。このプライマーを用い、先に得たＤＮＡを鋳型にＩｎｖｅｒｓｅ　ＰＣＲ法を行った。これにより、既に取得した部分遺伝子より外側の遺伝子部分を含むＤＮＡ断片を取得し、Ｃ末端配列を決定した。続いて、酵素のＮ末端より上流と推定される部分にＮｄｅＩの切断部位を結合させた配列を有するＤＮＡプライマー（表１の配列番号９）とＣ末端より下流と推定される部分にＥｃｏＲＩの切断部位を結合させた配列を有するＤＮＡプライマー（表１の配列番号１０）を用いて、この配列の間のＤＮＡを先に得たＤＮＡを鋳型にしたＰＣＲにより増幅することでサクシニラーゼ遺伝子の全長を含むＤＮＡ断片を取得した。得られたＤＮＡ断片の塩基配列を解析し、Ｄ－サクシニラーゼ遺伝子の全長が含まれていることを確認し、そのアミノ酸配列を推定した。得られた塩基配列及びアミノ酸配列をそれぞれ、配列表の配列番号１及び２に示す。 (4) Acquisition of full-length sequence of P4DSA gene Degenerate primers were synthesized based on the amino acid sequence identified in (2) above (SEQ ID NOs: 5 and 6 in Table 1), and Cupriavidus sp. PCR was performed under the recommended conditions using DNA polymerase KOD-Plus (manufactured by Toyobo) from P4-10-C using the genomic DNA extracted in (3) above as a template. 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 DH5α strain competent cell (Toyobo). And the transformant was obtained. 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. Furthermore, the following operation was performed to obtain a full-length sequence. Using TAKARA LA PCR In Vitro Cloning Kit (manufactured by Takara Bio Inc.), the operation was performed according to the protocol, and a DNA fragment from a known sequence toward the C-terminal was successfully amplified. The base sequence up to the 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: 7 and 8 in Table 1) 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: 9 in Table 1) having a sequence in which an NdeI cleavage site is bound 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: 10 in Table 1) having a sequence to which the sites are bound, the DNA between this sequence is amplified by PCR using the previously obtained DNA as a template, thereby including the full length of the succinylase gene. A DNA fragment 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. The obtained base sequence and amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
（５）取得できたＰ４ＤＳＡの塩基配列
　上記（４）で得られたＣｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株由来Ｄ－サクシニラーゼ遺伝子をＮＣＢＩ－ＢＬＡＳＴ（ｈｔｔｐ：／／ｂｌａｓｔ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｂｌａｓｔ．ｃｇｉ）により検索すると、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｌａｌｌｉｄｕｒａｎｓ　ＣＨ３４のｐｅｐｔｉｄａｓｅ　Ｓ４５，ｐｅｎｉｃｉｌｌｉｎ　ａｍｉｄａｓｅ（ＡＣＣＥＳＳＩＯＮ　Ｎｏ．ＹＰ＿５８７７６３）として登録されている遺伝子と７６％の相同性を示した。このｐｅｎｉｃｉｌｌｉｎ　ａｍｉｄａｓｅが属しているｐｅｎｉｃｉｌｌｉｎ　ａｃｙｌａｓｅファミリーは、ペニシリンＧやセファロスポリンＣ等を加水分解する酵素であり、このような酵素がＮ－サクシニル－Ｄ－アミノ酸を加水分解することを推定することは困難であり、機能から予測してホモロジー検索により本遺伝子を取得することは不可能に近いものであった。 (5) The base sequence of the obtained P4DSA Cupriavidus sp. 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 sillAinSlAinSinA45A peptidase SIlAinSinApSidseinSinApS .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 presumed that such an enzyme hydrolyzes N-succinyl-D-amino acid. It was difficult, and it was almost impossible to obtain this gene by homology search predicted from the function.
参考例２　Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓ株由来Ｄ－サクシニラーゼ遺伝子（以下、ＣｍＤＳＡ遺伝子と称する）のクローニング
　また、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃの近縁種であるＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓ由来のＤ－サクシニラーゼのクローニングも行った。 Reference Example 2 Cloning of D-succinylase gene (hereinafter referred to as CmDSA gene) derived from Cupriavidus metallidurans strain and Cupriavidus sp. Cloning of D-succinylase from Cupriavidus metallidurans, a related species of P4-10-C, was also performed.
（１）ＣｍＤＳＡのクローニング
　独立行政法人製品評価技術基盤機構よりＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓ（ＮＢＲＣ番号１０１２７２）の菌株を取得し、液体培地で培養した。培養して得た菌体から、ゲノム抽出キット（東洋紡：Ｇｅｎｏｍｉｃ　ＤＮＡ　ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ）によりゲノムを抽出した。遺伝子特異的プライマーを合成し（表７の配列番号４３、４４）、得られたゲノムを鋳型にＤＮＡポリメラーゼＫＯＤ－Ｐｌｕｓ（東洋紡製）を用いたＰＣＲにより遺伝子を取得した。クローニングキットＴａｒｇｅｔ　Ｃｌｏｎｅ－Ｐｌｕｓ（東洋紡製）を用いて、そのプロトコールに従って操作を行い、ベクターｐＢｌｕｅｓｃｒｉｐｔにクローニングし、組換え発現プラスミドｐＣｍＤＳＡを取得した。ｐＣｍＤＳＡをエシェリヒア・コリー（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＤＨ５α株コンピテントセル（東洋紡製）に形質転換し、該形質転換体を取得した。該形質転換体をＬＢ培地で培養し、プラスミドを抽出し、ＢｉｇＤｙｅ（商標登録）Ｔｅｒｍｉｎａｔｏｒ　ｖ３．１　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）により遺伝子配列を確認し、アミノ酸配列を推定した。得られた塩基配列及びアミノ酸配列をそれぞれ、配列表の配列番号３及び４に示す。 (1) Cloning of CmDSA A strain of Cupriavidus metallidrans (NBRC No. 101272) was obtained from the National Institute of Technology and Evaluation, an independent administrative agency, 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: 43 and 44 in Table 7), 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 the product was cloned into the vector pBluescript to obtain a recombinant expression plasmid pCmDSA. pCmDSA was transformed into Escherichia coli DH5α competent cell (manufactured by Toyobo) to obtain the 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 obtained base sequence and amino acid sequence are shown in SEQ ID NOs: 3 and 4, respectively.
参考例３　Ｐ４ＤＳＡ遺伝子発現プラスミドの構築
　参考例１で得られたＤ－サクシニラーゼ遺伝子のＮ末端、Ｃ末端部分にそれぞれ制限酵素ＮｄｅＩ及びＥｃｏＲＩの切断部位を結合させた配列を有するプライマー（表１の配列番号９、１０）を用いて、この間のＤＮＡを参考例１で得たゲノムＤＮＡを鋳型にしたＰＣＲにより増幅することで、オープンリーディングフレームを含むＤＮＡ断片を取得した。このＤＮＡ断片を制限酵素ＮｄｅＩとＥｃｏＲＩで切断し、同酵素で切断したベクタープラスミドｐＢＳＫと混合し、混合液と等量のライゲーション試薬（東洋紡製ラーゲーションハイ）を加えてインキュベーションすることにより、ライゲーションを実施した。このようにライゲーションしたＤＮＡをエシェリヒア・コリーＤＨ５α株コンピテントセル（東洋紡績製コンピテントハイＤＨ５α）に当製品に添付のプロトコールに従ってそれぞれ形質転換し、該形質転換体を取得した。このようにしてＤ－サクシニラーゼ遺伝子を大量に発現できるように設計されたｐＢＳＫＰ４ＤＳＡを取得した。 Reference Example 3 Construction of P4DSA Gene Expression Plasmid Primer having a sequence in which the cleavage sites of restriction enzymes NdeI and EcoRI are bound to the N-terminal and C-terminal parts of the D-succinylase gene obtained in Reference Example 1, respectively (sequence in Table 1 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 Reference Example 1 as a template. The DNA fragment was cleaved with restriction enzymes NdeI and EcoRI, mixed with vector plasmid pBSK cleaved with the same enzyme, ligation reagent (Toyobo Lagation High) was added to the mixture and incubated, and ligation was performed. Carried out. The ligated DNA was transformed into Escherichia coli DH5α strain competent cells (Toyobo Competent High DH5α) according to the protocol attached to the product to obtain the transformants. Thus, pBSKP4DSA designed to express a large amount of the D-succinylase gene was obtained.
参考例４　Ｐ４ＤＳＡ発現Ｅ．ｃｏｌｉの作製
　参考例３で構築したプラスミド、ｐＢＳＫＰ４ＤＳＡをエシェリヒア・コリーＤＨ５α株コンピテントセル（東洋紡績製コンピテントハイＤＨ５α）に当製品に添付のプロトコールに従ってそれぞれ形質転換し、該形質転換体を取得した。 Reference Example 4 P4DSA expression The plasmid constructed in Reference Example 3 of E. coli, pBSKP4DSA, was transformed into Escherichia coli DH5α strain competent cells (Toyobo Competent High DH5α) according to the protocol attached to this product, and the transformants were obtained. .
参考例５　Ｎ－サクシニルアミノ酸ラセマーゼの調製
　特開２００７－８２５３４号公報の実施例に記載の方法で、Ｃｈｌｏｒｏｆｌｅｘｕｓ　ａｕｒａｎｔｉａｃｕｓ（クロロフレクス・アウランチアクス）由来のＮ－サクシニルアミノ酸ラセマーゼを調製し、２５．６（ＫＵ／ｍＬ）、１００（ｍｇ／ｍＬ）の精製酵素標品を得た。 Reference Example 5 Preparation of N-succinyl amino acid racemase N-succinyl amino acid racemase derived from Chloroflexus aurantiacus (Chloroflex aurantiacus) was prepared by the method described in Examples of Japanese Patent Application Laid-Open No. 2007-82534, and 25.6 ( KU / mL) and 100 (mg / mL) purified enzyme preparations were obtained.
実施例１　改変型Ｐ４ＤＳＡの調製
（１）改変型Ｐ４ＤＳＡ遺伝子を含む発現プラスミドの作成
　参考例１で得られたｐＢＳＫＰ４ＤＳＡのＰ４ＤＳＡ遺伝子全長を増幅するよう設計したプライマーを用いて、Ｐ４ＤＳＡ遺伝子に変異が生じるよう、クロンテック社製、Ｄｉｖｅｒｓｉｆｙ　ＰＣＲ　Ｒａｎｄｏｍ　Ｍｕｔａｇｅｎｅｓｉｓ　ＫｉｔでＰＣＲ反応を実施した。続いて、反応液を制限酵素ＮｄｅＩとＥｃｏＲＩで処理後、アガロースゲル電気泳動に供し、ランダムな変異を生じたＰ４ＤＳＡ遺伝子のＮｄｅＩ＋ＥｃｏＲＩ断片をゲルから回収した。また、ｐＢＳＫＰ４ＤＳＡを制限酵素ＮｄｅＩとＥｃｏＲＩで処理し、同様にしてｐＢＳＫＰ４ＤＳＡのベクターＮｄｅＩとＥｃｏＲＩ断片を回収した。最後に、ランダムな変異を生じたＰ４ＤＳＡ遺伝子のＮｄｅＩ＋ＥｃｏＲＩ断片とｐＢＳＫＰ４ＤＳＡのＮｄｅＩ＋ＥｃｏＲＩベクター断片とをライゲーションし、ランダムな変異を有するＰ４ＤＳＡ遺伝子が挿入されたｐＢＳＫＰ４ＤＳＡのプラスミド集団を作成した。 Example 1 Preparation of Modified P4DSA (1) Preparation of Expression Plasmid Containing Modified P4DSA Gene Using the primer designed to amplify the full length of the P4DSA gene of pBSKP4DSA obtained in Reference Example 1, a mutation occurs in the P4DSA gene As described above, PCR reaction was performed using Diversity PCR Random Mutagenesis Kit manufactured by Clontech. Subsequently, the reaction solution was treated with restriction enzymes NdeI and EcoRI, and then subjected to agarose gel electrophoresis, and the NdeI + EcoRI fragment of the P4DSA gene having a random mutation was recovered from the gel. Further, pBSKP4DSA was treated with restriction enzymes NdeI and EcoRI, and the vector NdeI and EcoRI fragments of pBSKP4DSA were recovered in the same manner. Finally, the NdeI + EcoRI fragment of the P4DSA gene having a random mutation and the NdeI + EcoRI vector fragment of pBSKP4DSA were ligated to create a plasmid population of pBSKP4DSA in which the P4DSA gene having a random mutation was inserted.
（２）Ｄ体選択的に作用する改変型Ｄ－サクシニラーゼのスクリーニング
　（１）で調製したｐＢＳＫＰ４ＤＳＡのプラスミド集団を用いて、エシェリヒア・コリーＤＨ５α株コンピテントセル（東洋紡績製コンピテントハイＤＨ５α）に、当製品に添付のプロトコールに従ってそれぞれ形質転換を行った。そうして得られたコロニー、約５，０００株をＬＢ培地（アンピシリン５０μｇ／ｍＬを含む）にそれぞれ接種し、３７℃で１６時間振とう培養した。その培養液から遠心分離により調製した菌体を、Ｎ－サクシニル－Ｄ－トリプトファン溶液（２５ｍＭ　ＫＰＢ（ｐＨ７．０）、１００ｍＭ　Ｎ－サクシニル－Ｄ－トリプトファン）及びＮ－サクシニル－Ｌ－トリプトファン溶液（２５ｍＭ　ＫＰＢ（ｐＨ７．０）、１００ｍＭ　Ｎ－サクシニル－Ｌ－トリプトファン）とそれぞれ混合し、３７℃で１６時間反応させた。その反応液の一部をＴＬＣプレート（Ｍｅｒｃｋ社製）に供し、ｎ－ブタノール：酢酸：水（３：１：１、ｖ／ｖ）からなる展開溶媒で展開し、ニンヒドリンスプレーにより、Ｄ－トリプトファン、Ｌ－トリプトファンを検出し、Ｄ－トリプトファンのスポットに対するＬ－トリプトファンのスポットの割合が野生型よりも小さい、即ち、野生型Ｐ４ＤＳＡと比較して、Ｄ体に反応しやすい変異株を選抜した。結果の一例を図２に示す。図２中、変異株２や変異株３が望ましい変異体であり、このような変異体を選抜した。そして、立体選択性が向上した変異株よりプラスミドを抽出し、Ｐ４ＤＳＡ遺伝子全塩基配列を確認し、変異箇所を同定した。その結果、７２番目、１７６番目、１８１～１８５番目、２８６番目、３０５番目、３４８番目、３５１番目、３８８番目、４６１番目、５１８番目及び５３９番目のアミノ酸残基が立体選択性向上に寄与している部位として候補に挙がった。 (2) Screening of modified D-succinylase that selectively acts on D body Using the plasmid population of pBSKP4DSA prepared in (1), Escherichia coli DH5α strain competent cell (competitive high DH5α manufactured by Toyobo Co., Ltd.) Transformation was performed according to the protocol attached to this product. The colonies thus obtained, about 5,000 strains, were each inoculated into LB medium (containing 50 μg / mL ampicillin), and cultured with shaking at 37 ° C. for 16 hours. The cells prepared by centrifugation from the culture solution were mixed with N-succinyl-D-tryptophan solution (25 mM KPB (pH 7.0), 100 mM N-succinyl-D-tryptophan) and N-succinyl-L-tryptophan solution (25 mM). Each was mixed with KPB (pH 7.0) and 100 mM N-succinyl-L-tryptophan) and reacted at 37 ° C. for 16 hours. A part of the reaction solution was applied to a TLC plate (manufactured by Merck), developed with a developing solvent consisting of n-butanol: acetic acid: water (3: 1: 1, v / v), and D-tryptophan by ninhydrin spray. Thus, L-tryptophan was detected, and the ratio of the L-tryptophan spot to the D-tryptophan spot was smaller than that of the wild type, that is, a mutant strain that was more susceptible to D-form than the wild type P4DSA was selected. An example of the results is shown in FIG. In FIG. 2, mutant 2 and mutant 3 are desirable mutants, and such mutants were selected. And the plasmid was extracted from the mutant which improved stereoselectivity, the P4DSA gene whole base sequence was confirmed, and the mutation location was identified. As a result, the 72nd, 176th, 181 to 185th, 286th, 305th, 348th, 351st, 388th, 461st, 518th and 539th amino acid residues contribute to the improvement of stereoselectivity. I was listed as a candidate.
（３）改変型プラスミドの構築（置換アミノ酸の至適化）
　（１）で得た変異株の中には、複数のアミノ酸変異をもつ変異株もあったため、また、置換するアミノ酸の種類を検討するために、部位特異的（Ｓｉｔｅ－Ｄｉｒｅｃｔｅｄ　ｍｕｔａｇｅｎｅｓｉｓ）に変異させた改変型ＤＳＡ発現プラスミドを作成した。部位特異的変異の作製には、ＳＴＲＡＴＡＧＥＮＥ社製ＱｕｉｋＣｈａｎｇｅ　Ｓｉｔｅ－Ｄｉｒｅｃｔｅｄ　ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔを使用した。また、変異させる際には、置換アミノ酸の至適化も行うため、（２）で立体選択性向上に寄与していると推測された７２番目、１８１～１８５番目、３０５番目、３４８番目、３５１番目、４６１番目及び５３９番目のアミノ酸残基それぞれを２０種のアミノ酸に置換するように、かつ、２本鎖ＤＮＡのそれぞれの鎖に相補的になるように設計した合成オリゴＤＮＡプライマーをそれぞれ合成した（配列番号１１～４２）。なお、一部の合成オリゴＤＮＡプライマーは、置換されるアミノ酸が１種類になるように、合成した。変異導入に使用した合成オリゴＤＮＡプライマーの配列を表２に示す。 (3) Construction of modified plasmid (optimization of substituted amino acids)
Among the mutant strains obtained in (1), there were mutant strains having a plurality of amino acid mutations. In addition, in order to examine the types of amino acids to be substituted, site-directed mutations were mutated. A modified DSA expression plasmid was prepared. For the production of site-specific mutations, QuikChange Site-Directed Mutagenesis Kit manufactured by STRATAGENE was used. In addition, since the substitution amino acid is also optimized when mutating, the 72nd, 181st to 185th, 305th, 348th, 351 estimated to have contributed to the improvement of stereoselectivity in (2). Synthetic oligo DNA primers designed to substitute the 20th amino acid at each of the first, 461st and 539th amino acid residues and to be complementary to each strand of double-stranded DNA were synthesized. (SEQ ID NO: 11-42). Some synthetic oligo DNA primers were synthesized so that one amino acid was substituted. Table 2 shows the sequences of synthetic oligo DNA primers used for mutagenesis.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　改変型酵素の名称は、「野生型酵素のアミノ酸配列中のアミノ酸残基→残基番号→置換したアミノ酸残基」の順に表記する。例えば、Ｌ１８２Ｒ改変型酵素は野生型酵素のアミノ酸配列中の１８２番目のＬｅｕ（Ｌ）残基をＡｒｇ（Ｒ）残基に置換した改変型酵素であることを意味する。なお、表２中のＬ１８２ＸのＸとは、２０種類のアミノ酸のうちの任意のアミノ酸を意味するものである。 The name of the modified enzyme is expressed in the order of “amino acid residue → residue number → substituted amino acid residue in the amino acid sequence of the wild-type enzyme”. For example, the L182R modified enzyme means a modified enzyme in which the 182nd Leu (L) residue in the amino acid sequence of the wild-type enzyme is substituted with an Arg (R) residue. In Table 2, X in L182X means any amino acid among the 20 types of amino acids.
　これらのプライマーを使用し、上記キットの方法に従い、参考例１にて採取した野生型Ｐ４ＤＳＡ発現プラスミドｐＢＳＫＰ４ＤＳＡを鋳型として、改変型プラスミドを作製した。例えば、Ｌ１８２Ｒの作製にあたっては、ｐＢＳＫＰ４ＤＳＡを鋳型として、配列番号１７及び１８のプライマーを用いて、以下のＰＣＲ条件で改変型Ｐ４ＤＳＡ発現プラスミドを増幅した。
９８℃　１０秒
６０℃　３０秒
６８℃　６分　　　×　２５サイクル Using these primers, a modified plasmid was prepared using the wild-type P4DSA expression plasmid pBSKP4DSA collected in Reference Example 1 as a template according to the method of the above kit. For example, in preparation of L182R, a modified P4DSA expression plasmid was amplified under the following PCR conditions using pBSKP4DSA as a template and primers of SEQ ID NOs: 17 and 18.
98 ° C 10 seconds 60 ° C 30 seconds 68 ° C 6 minutes x 25 cycles
　次に、メチル化ＤＮＡを認識して切断する制限酵素ＤｐｎＩ処理によって鋳型ｐＢＳＫＰ４ＤＳＡを切断し、得られた反応液でＥ．ｃｏｌｉ　ＤＨ５αを形質転換した。形質転換体からプラスミドを回収して塩基配列を決定し、正しく変異が導入されていることを確認すると共に、コードしているアミノ酸残基を確認した。 Next, the template pBSKP4DSA was cleaved by a restriction enzyme DpnI treatment that recognizes and cleaves methylated DNA. E. coli DH5α was transformed. The plasmid was recovered from the transformant, the nucleotide sequence was determined, it was confirmed that the mutation was correctly introduced, and the encoded amino acid residue was confirmed.
（４）改変型Ｐ４ＤＳＡ発現Ｅ．ｃｏｌｉの作製及び粗酵素液の調製
　改変型Ｐ４ＤＳＡ遺伝子搭載プラスミドを持つ大腸菌形質転換体をＬＢ培地３ｍｌ（アンピシリン５０μｇ／ｍＬを含む）に接種し、３７℃で１６時間振とう培養した。その培養液から遠心分離により調製した菌体を超音波破砕し、その遠心後の上清を改変型Ｐ４ＤＳＡの粗酵素液とした。 (4) Modified P4DSA expression Preparation of E. coli and preparation of crude enzyme solution An E. coli transformant having a modified P4DSA gene carrying plasmid was inoculated into 3 ml of LB medium (containing 50 μg / ml of ampicillin), and cultured with shaking at 37 ° C. for 16 hours. The bacterial cells prepared by centrifugation from the culture solution were sonicated, and the supernatant after the centrifugation was used as a modified P4DSA crude enzyme solution.
実施例２　改変型Ｐ４ＤＳＡを用いたＮ－サクシニルトリプトファン及びＮ－サクシニルフェニルアラニンに対する立体選択性の評価
　実施例１で調製した粗酵素液を用いて、Ｎ－サクシニルトリプトファン及びＮ－サクシニルフェニルアラニンに対する立体選択性の評価を行った。具体的には、以下の組成のＮ－サクシニル－Ｄ－アミノ酸溶液及びＮ－サクシニル－Ｌ－アミノ酸溶液のそれぞれに対して粗酵素液を添加した。
Ｎ－サクシニル－Ｄ－トリプトファン溶液：２５ｍＭ　ＫＰＢ（ｐＨ７．０）、１％　Ｎ－サクシニル－Ｄ－トリプトファン
Ｎ－サクシニル－Ｌ－トリプトファン溶液：２５ｍＭ　ＫＰＢ（ｐＨ７．０）、１％　Ｎ－サクシニル－Ｌ－トリプトファン
Ｎ－サクシニル－Ｄ－フェニルアラニン溶液：２５ｍＭ　ＫＰＢ（ｐＨ７．０）、１％　Ｎ－サクシニル－Ｄ－フェニルアラニン
Ｎ－サクシニル－Ｌ－フェニルアラニン溶液：２５ｍＭ　ＫＰＢ（ｐＨ７．０）、１％　Ｎ－サクシニル－Ｌ－フェニルアラニン
　得られた反応液を４０℃で１６時間インキュベートした後に、残存する基質濃度から分解率及びＤ体に対するＬ体の分解比率（Ｌ／Ｄ）を算出した。なお、改変型Ｐ４ＤＳＡは、Ｌ体に対する反応性がかなり低下しており、基質の減少量を定量する方法では、識別しづらかったため、Ｌ体を４、Ｄ体を１、すなわちＬ：Ｄ＝４：１のタンパク質比率で反応させた。また、酵素タンパク質濃度は、Ｌ体の反応の場合は最終酵素タンパク質濃度が２．０ｍｇ／ｍｌになるように調節し、Ｄ体の反応の場合は最終酵素タンパク質濃度が０．５ｍｇ／ｍｌになるように調節した。その結果を表３に示す。 Example 2 Evaluation of stereoselectivity for N-succinyltryptophan and N-succinylphenylalanine using modified P4DSA Using the crude enzyme solution prepared in Example 1, stereoselectivity for N-succinyltryptophan and N-succinylphenylalanine Was evaluated. Specifically, the crude enzyme solution was added to each of the N-succinyl-D-amino acid solution and the N-succinyl-L-amino acid solution having the following composition.
N-succinyl-D-tryptophan solution: 25 mM KPB (pH 7.0), 1% N-succinyl-D-tryptophan N-succinyl-L-tryptophan solution: 25 mM KPB (pH 7.0), 1% N-succinyl-L -Tryptophan N-succinyl-D-phenylalanine solution: 25 mM KPB (pH 7.0), 1% N-succinyl-D-phenylalanine N-succinyl-L-phenylalanine solution: 25 mM KPB (pH 7.0), 1% N-succinyl -L-Phenylalanine After incubating the obtained reaction solution at 40 ° C. for 16 hours, the decomposition rate and the decomposition ratio of L-form to D-form (L / D) were calculated from the remaining substrate concentration. It should be noted that the modified P4DSA has a considerably reduced reactivity to the L-form, and it was difficult to discriminate by the method of quantifying the amount of decrease in the substrate. Therefore, the L-form was 4, and the D-form was 1, that is, L: D = 4 The reaction was performed at a protein ratio of 1: 1. The enzyme protein concentration is adjusted so that the final enzyme protein concentration is 2.0 mg / ml in the case of L-form reaction, and the final enzyme protein concentration is 0.5 mg / ml in the case of D-form reaction. Adjusted as follows. The results are shown in Table 3.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　その結果、実施例１（１）では、複数の箇所にアミノ酸置換が起こることもあり、どの変異が立体選択性に寄与しているのか分からない変異体もあったために、Ｒ１７６ＨやＶ２８６Ａなどの立体選択性の向上の寄与していない改変体も見られたが、表３に示したように、多くの改変型Ｐ４ＤＳＡにおいて、Ｎ－サクシニルトリプトファン及びＮ－サクシニルフェニルアラニンに対して共に、Ｄ体選択的に作用していることが確認できた。特に、Ｎ－サクシニルトリプトファンに対しては、Ｇ１８１Ｅ、Ｌ１８２Ｗ、Ｌ１８２Ｙ、Ｌ１８２Ｋ、Ｌ１８２Ｒ、Ｌ１８２Ｅ、Ｎ１８５Ｆ、Ｒ３０５Ａ、Ｒ３０５Ｇ、Ｒ３０５Ｈ、Ｒ３０５Ｑ、Ｒ３０５Ｓ、Ｒ３０５Ｎ、Ｌ３４８Ｅ、Ｌ３４８Ｗ、Ｌ３４８Ｔ、Ｆ３５１Ｉ、Ｇ５３９Ｖ及びＧ５３９Ｍ、Ｎ－サクシニルフェニルアラニンに対しては、Ｇ１８１Ｗ、Ｇ１８１Ｄ、Ｇ１８１Ｅ、Ｌ１８２Ｒ、Ｒ３０５Ａ、Ｒ３０５Ｇ、Ｒ３０５Ｓ、Ｌ３４８Ｅ及びＬ３４８Ｗにおいて、基質の減少量では検出限界以下（表中では、０．０）となるまでに、Ｌ体に対する反応性が低下し、野生型と比べて顕著にＤ体選択的に作用していることが確認できた。 As a result, in Example 1 (1), amino acid substitutions may occur at a plurality of positions, and some mutations do not know which mutation contributes to stereoselectivity. Therefore, a three-dimensional structure such as R176H or V286A is used. Although some variants did not contribute to the improvement of selectivity, as shown in Table 3, in many modified P4DSAs, both D-form selective to N-succinyltryptophan and N-succinylphenylalanine. It was confirmed that it was acting on. In particular, for N-succinyltryptophan, G181E, L182W, L182Y, L182K, L182R, L182E, N185F, R305A, R305G, R305H, R305Q, R305S, R305N, L348E, L348W, L348T, F351I, G539V, -For succinylphenylalanine, in G181W, G181D, G181E, L182R, R305A, R305G, R305S, L348E, and L348W, the amount of substrate decrease is less than the detection limit (0.0 in the table). It was confirmed that the reactivity to the body decreased, and the D-form selection was remarkably acting compared to the wild type.
実施例３　改変型Ｐ４ＤＳＡを用いたＮ－サクシニルビフェニルアラニンに対する立体選択性の評価
　次に、実施例２でＮ－サクシニルトリプトファン及びＮ－サクシニルフェニルアラニンに対する立体選択性に関与していることが明らかとなった改変型Ｐ４ＤＳＡについて、他の芳香族アミノ酸に対してもＤ体選択的に作用するかどうかを確認した。他の芳香族アミノ酸としては、非天然型アミノ酸の一種であるＮ－サクシニルビフェニルアラニンを選択し、実施例１で調製した粗酵素液を用いて、実施例２と同様の手順で、Ｎ－サクシニルビフェニルアラニンに対する立体選択性の評価を行った。ただし、本実施例３では、以下に示すようなラセミ体溶液を反応基質として使用した。
Ｎ－サクシニル－ＤＬ－ビフェニルアラニン反応溶液：２５ｍＭ　ＫＰＢ（ｐＨ７．０）、２％　Ｎ－サクシニル－ＤＬ－ビフェニルアラニン Example 3 Evaluation of Stereoselectivity for N-Succinylbiphenylalanine Using Modified P4DSA Next, in Example 2, it was clarified that it was involved in stereoselectivity for N-succinyltryptophan and N-succinylphenylalanine. It was confirmed whether the modified P4DSA acted selectively to other aromatic amino acids on the D form. As another aromatic amino acid, N-succinylbiphenylalanine, which is a kind of non-natural amino acid, is selected and the crude enzyme solution prepared in Example 1 is used to perform N-succinyl in the same procedure as in Example 2. The stereoselectivity for biphenylalanine was evaluated. However, in Example 3, a racemic solution as shown below was used as a reaction substrate.
N-succinyl-DL-biphenylalanine reaction solution: 25 mM KPB (pH 7.0), 2% N-succinyl-DL-biphenylalanine
　反応液を４０℃で１６時間インキュベートした後に、残存する基質濃度から分解率及びＤ体に対するＬ体の分解比率（Ｌ／Ｄ）を算出した。なお、Ｎ－サクシニル－ＤＬ－ビフェニルアラニンについては、基質にラセミ体を利用したため、Ｄ体とＬ体を同タンパク量で反応させたことになる。また、酵素タンパク質濃度は、最終酵素タンパク質濃度が１．０ｍｇ／ｍｌになるように調節した。その結果を表４に示す。 After the reaction solution was incubated at 40 ° C. for 16 hours, the decomposition rate and the decomposition ratio (L / D) of L-form to D-form were calculated from the remaining substrate concentration. For N-succinyl-DL-biphenylalanine, a racemate was used as a substrate, so that D form and L form were reacted with the same amount of protein. The enzyme protein concentration was adjusted so that the final enzyme protein concentration was 1.0 mg / ml. The results are shown in Table 4.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　その結果、評価した全ての改変型Ｐ４ＤＳＡにおいて、Ｎ－サクシニル－ＤＬ－ビフェニルアラニンに対してもＤ体選択的に作用していることが確認できた。実施例２及び３に示す結果は、本発明の改変型Ｐ４ＤＳＡはＮ－サクシニルトリプトファン、Ｎ－サクシニルフェニルアラニン及びＮ－サクシニルビフェニルアラニンのみに対してＤ体選択的に作用するのではなく、その他の芳香族アミノ酸や、置換基を有する非天然型の芳香族アミノ酸を含む任意のアミノ酸を構成アミノ酸とするＮ－サクシニル－ＤＬ－アミノ酸に対しても、Ｄ体選択的に作用することを示唆するものである。 As a result, it was confirmed that all the modified P4DSAs evaluated acted selectively to D-form with respect to N-succinyl-DL-biphenylalanine. The results shown in Examples 2 and 3 show that the modified P4DSA of the present invention does not act selectively on D-form only for N-succinyltryptophan, N-succinylphenylalanine and N-succinylbiphenylalanine. N-succinyl-DL-amino acid containing any amino acid, including non-natural aromatic amino acids having a non-natural type having a substituent, as a constituent amino acid. is there.
実施例４　改変型Ｐ４ＤＳＡ並びにＣｈｌｏｒｏｆｌｅｘｕｓ　ａｕｒａｎｔｉａｃｕｓ由来のＮ－サクシニルアミノ酸ラセマーゼを用いたＮ－サクシニル－ＤＬ－トリプトファンからＤ－トリプトファン、Ｎ－サクシニル－ＤＬ－フェニルアラニンからのＤ－フェニルアラニン及びＮ－サクシニル－ＤＬ－ビフェニルアラニンからＤ－ビフェニルアラニンの合成
（１）
改変型Ｐ４ＤＳＡ（Ｌ１８２Ｅ、Ｌ１８２Ｐ、Ｒ３０５Ｎ）の調製
　実施例１で得られた改変型Ｐ４ＤＳＡ（Ｌ１８２Ｅ、Ｌ１８２Ｐ、Ｒ３０５Ｎ）を発現する大腸菌形質転換体のコロニーを、試験管に入った５ｍＬのＬＢ培地（アンピシリン５０μｇ／ｍＬを含む）にそれぞれ植菌し、振とう数１８０ｒｐｍ、３０℃で１６時間培養し、種培養液とした。この種培養液を、５００ｍＬの坂口フラスコに入った６０ｍＬのＴＢ培地（アンピシリン５０μｇ／ｍＬを含む）に接種し、振とう数３１０ｒｐｍ、３０℃で１８時間培養した。培養終了時の濁度（Ａｂｓ６６０ｎｍ）は、１５．０、１６．２、１５．５であった。続いて、得られた菌体を遠心分離にて集菌し、２５ｍＭリン酸緩衝溶液（ｐＨ７．０）に懸濁し、氷冷下で超音波細胞破砕装置を用いて破砕した。その後、６５℃で１時間、熱処理を行い、その後、一般的な方法で脱塩処理を行い、アミノ酸合成用の酵素サンプル（Ｌ１８２Ｅ：２７．９ｍｇ／ｍＬ、５．２Ｕ／ｍＬ、Ｌ１８２Ｐ：７．３ｍｇ／ｍＬ、１．８Ｕ／ｍＬ、Ｒ３０５Ｎ：２８．５ｍｇ／ｍＬ、４．３Ｕ／ｍＬ）を調製した。なお、酵素活性の基質には、Ｎ－サクシニル－Ｄ－トリプトファンを用いた。 Example 4 N-succinyl-DL-tryptophan to D-tryptophan, N-succinyl-DL-phenylalanine to D-phenylalanine and N-succinyl-DL-, using modified P4DSA and N-succinyl amino acid racemase from Chloroflexus aurantiacus Synthesis of D-biphenylalanine from biphenylalanine (1)
Preparation of Modified P4DSA (L182E, L182P, R305N) A colony of E. coli transformants expressing the modified P4DSA (L182E, L182P, R305N) obtained in Example 1 was placed in 5 mL of LB medium ( Inoculating ampicillin (containing 50 μg / mL), and culturing for 16 hours at 180 ° C. and 30 ° C. to obtain a seed culture solution. This seed culture was inoculated into 60 mL of TB medium (containing 50 μg / mL of ampicillin) contained in a 500 mL Sakaguchi flask, and cultured at a shaking speed of 310 rpm and 30 ° C. for 18 hours. Turbidity (Abs 660 nm) at the end of the culture was 15.0, 16.2, and 15.5. Subsequently, the obtained cells were collected by centrifugation, suspended in a 25 mM phosphate buffer solution (pH 7.0), and crushed using an ultrasonic cell crusher under ice cooling. Thereafter, heat treatment is performed at 65 ° C. for 1 hour, and then desalting is performed by a general method, and an enzyme sample for amino acid synthesis (L182E: 27.9 mg / mL, 5.2 U / mL, L182P: 7. 3 mg / mL, 1.8 U / mL, R305N: 28.5 mg / mL, 4.3 U / mL). Note that N-succinyl-D-tryptophan was used as the enzyme activity substrate.
（２）各種Ｎ－サクシニルアミノ酸との反応及び分析
　５％Ｎ－サクシニル－ＤＬ－トリプトファン水溶液（ｐＨ７．５）５ｍＬ、５％Ｎ－サクシニル－ＤＬ－フェニルアラニン水溶液（ｐＨ７．５）５ｍＬ、５％Ｎ－サクシニル－ＤＬ－ビフェニルアラニン水溶液（ｐＨ７．５）５ｍＬをそれぞれ基質として用い、塩化コバルト水溶液を終濃度１ｍＭとなるようにそれぞれ添加後、上記（１）で調製した改変型ＤＳＡ（Ｌ１８２Ｅ、Ｌ１８２Ｐ、Ｒ３０５Ｎ）の酵素液（０．１６Ｕ、０．２Ｕ、０．０８Ｕ）をそれぞれの基質に加え、さらに参考例５で調製したＣｈｌｏｒｏｆｌｅｘｕｓ　ａｕｒａｎｔｉａｃｕｓ株由来のＮ－サクシニルアミノ酸ラセマーゼ溶液２５ｍｇ（６４００Ｕ）をそれぞれに対して加え、４５℃で３日間反応させた。また、一方でコントロールとして、ＰＣＴ／ＪＰ２０１１／０６４９４３の実施例に記載の方法で調製した野生型ＤＳＡ酵素液を用い、同様に反応させ、変換率及びＤ－アミノ酸の光学純度を算出した。変換率は、上記記載のジーエルサイエンス社製「Ｉｎｅｒｔｓｉｌ　ＯＤＳ－３」（５μｍ、４．６×１００ｍｍ）を利用した高速液体クロマトグラフィーで酵素反応前と反応後の基質のピーク面積値を測定することによって算出した。光学純度は、上記記載のダイセル化学工業株式会社製光学分割カラム「ＣＲＯＷＮＰＡＫ　ＣＲ（＋）」（５μｍ、４．０×１５０ｍｍ）で生成した遊離体のアミノ酸の光学純度を測定することによって算出した。結果を表５～７に示す。 (2) Reaction with various N-succinyl amino acids and analysis 5% N-succinyl-DL-tryptophan aqueous solution (pH 7.5) 5 mL, 5% N-succinyl-DL-phenylalanine aqueous solution (pH 7.5) 5 mL, 5% N -Modified DSA (L182E, L182P, L182E, L182P, prepared in (1) above after adding 5 mL each of succinyl-DL-biphenylalanine aqueous solution (pH 7.5) as a substrate and adding an aqueous cobalt chloride solution to a final concentration of 1 mM. R305N) enzyme solution (0.16 U, 0.2 U, 0.08 U) was added to each substrate, and 25 mg (6400 U) of an N-succinyl amino acid racemase solution derived from Chloroflexus aurantiacus strain prepared in Reference Example 5 was added to each substrate. And reacted at 45 ° C. for 3 days. On the other hand, as a control, a wild-type DSA enzyme solution prepared by the method described in the examples of PCT / JP2011 / 064943 was used, and reacted in the same manner to calculate the conversion rate and the optical purity of D-amino acid. The conversion rate is determined by measuring the peak area value of the substrate before and after the enzyme reaction by high performance liquid chromatography using “Inertsil ODS-3” (5 μm, 4.6 × 100 mm) manufactured by GL Sciences, Inc. Calculated by The optical purity was calculated by measuring the optical purity of the free amino acid produced by the above-described optical resolution column “CROWNPAK CR (+)” (5 μm, 4.0 × 150 mm) manufactured by Daicel Chemical Industries, Ltd. The results are shown in Tables 5-7.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　表５～７に示すように、Ｄ－トリプトファン合成における反応３日目の変換率は、野生型ＤＳＡ、Ｌ１８２Ｅ，Ｌ１８２Ｐ、Ｒ３０５Ｎの順に、８３％、８７％、８６％、８９％であり、Ｄ－フェニルアラニン合成における反応３日目の変換率は、野生型ＤＳＡ、Ｌ１８２Ｅ，Ｌ１８２Ｐ、Ｒ３０５Ｎの順に、８１％、８４％、８３％、８４％であり、Ｄ－ビフェニルアラニン合成における反応３日目の変換率は、野生型ＤＳＡ、Ｌ１８２Ｅ，Ｌ１８２Ｐ、Ｒ３０５Ｎの順に、９６％、９１％、９６％、９１％であり、いずれも問題なく反応が進んだ。また、表５～７に示すように、いずれの改変型ＤＳＡにおいても、芳香族アミノ酸によって構成されるＮ－サクシニル－ＤＬ－アミノ酸を基質とした場合、野生型ＤＳＡを使用する場合と比較して、高純度の芳香族Ｄ－アミノ酸を製造できることを確認した。特に、Ｌ１８２Ｅを使用して、Ｄ－トリプトファン、Ｄ－ビフェニルアラニンを製造した場合にそれぞれ９９．６％ｅｅ、９９．２％ｅｅ、Ｌ１８２Ｐを使用して、Ｄ－ビフェニルアラニンを製造した場合に９９．３％ｅｅ、Ｒ３０５Ｎを使用して、Ｄ－トリプトファンを製造した場合に、９９．６％ｅｅという高い光学純度に達した。また、Ｌ１８２Ｐは、実施例２で挙げられている改変型ＤＳＡの中では、Ｎ－サクシニルトリプトファンに対するＬ／Ｄ比が０．１３と比較的大きい値を示しているが、本実施例が示すようにＬ１８２Ｐを使用した場合のＤ－トリプトファンの光学純度が９８．６％ｅｅに達したということは、表３に記載のその他の改変型ＤＳＡも、それ相当の改変効果があることを示唆する。 As shown in Tables 5 to 7, the conversion rate on the third day of the reaction in D-tryptophan synthesis was 83%, 87%, 86%, and 89% in the order of wild-type DSA, L182E, L182P, and R305N. The conversion rate on the third day of the reaction in the synthesis of phenylalanine is 81%, 84%, 83%, 84% in the order of wild-type DSA, L182E, L182P, and R305N, and the third day of the reaction in the synthesis of D-biphenylalanine. The conversion rates were 96%, 91%, 96%, and 91% in the order of wild type DSA, L182E, L182P, and R305N, and the reaction proceeded without any problem. Further, as shown in Tables 5 to 7, in any modified DSA, when N-succinyl-DL-amino acid constituted by an aromatic amino acid is used as a substrate, compared to the case where wild-type DSA is used. It was confirmed that a highly pure aromatic D-amino acid could be produced. In particular, when L-182E is used to produce D-tryptophan and D-biphenylalanine, 99.6% ee, 99.2% ee, and L182P are used to produce D-biphenylalanine, respectively. When D-tryptophan was produced using .3% ee, R305N, a high optical purity of 99.6% ee was achieved. In addition, L182P has a relatively large L / D ratio of 0.13 with respect to N-succinyltryptophan among the modified DSAs listed in Example 2, but this example shows that The fact that the optical purity of D-tryptophan reached 98.6% ee when L182P was used in the sample suggests that other modified DSAs described in Table 3 also have a corresponding modification effect.
実施例５　二重変異改変型Ｐ４ＤＳＡ（Ｌ１８２Ｅ＋Ｌ３４８Ｉ）並びにＣｈｌｏｒｏｆｌｅｘｕｓ　ａｕｒａｎｔｉａｃｕｓ由来のＮ－サクシニルアミノ酸ラセマーゼを用いたＮ－サクシニル－ＤＬ－トリプトファンからのＤ－トリプトファン、Ｎ－サクシニル－ＤＬ－フェニルアラニンからのＤ－フェニルアラニン及びＮ－サクシニル－ＤＬ－ビフェニルアラニンからのＤ－ビフェニルアラニンの合成
（１）二重変異改変型プラスミドの構築
　二重変異改変体を発現するプラスミドを作製するために、実施例１で得られた改変型Ｐ４ＤＳＡ（Ｌ１８２Ｅ）を含む発現プラスミドを鋳型とし、上記に記載の方法でＰＣＲ反応を実施し、１８２番目のロイシンがグルタミン酸に置換され、かつ、３４８番目のロイシンがイソロイシンに置換された二重変異改変型Ｐ４ＤＳＡ（Ｌ１８２Ｅ＋Ｌ３４８Ｉ）を含む発現プラスミドを構築した。 Example 5 D-Tryptophan from N-succinyl-DL-tryptophan using N-succinyl-DL-tryptophan using double mutant modified P4DSA (L182E + L348I) and N-succinyl amino acid racemase from Chloroflexus aurantiacus D-phenylalanine from N-succinyl-DL-phenylalanine And D-biphenylalanine synthesis from N-succinyl-DL-biphenylalanine (1) Construction of double-mutant modified plasmids To obtain a plasmid that expresses double-mutant variants, they were obtained in Example 1. Using an expression plasmid containing the modified P4DSA (L182E) as a template, PCR was carried out by the method described above. The 182nd leucine was replaced with glutamic acid, and the 348th leucine was replaced with isoleucine. The expression plasmid containing the heavy variant modified P4DSA (L182E + L348I) was constructed.
（２）二重変異改変型Ｐ４ＤＳＡ（Ｌ１８２Ｅ＋Ｌ３４８Ｉ）の調製
（１）で得られた二重変異改変型Ｐ４ＤＳＡ（Ｌ１８２Ｅ＋Ｌ３４８I）を発現する大腸菌形質転換体のコロニーを、試験管に入った５ｍＬのＬＢ培地（アンピシリン５０μｇ／ｍＬを含む）に植菌し、振とう数１８０ｒｐｍ、３０℃で１６時間培養し、種培養液とした。この種培養液を、５００ｍＬの坂口フラスコに入った６０ｍＬのＴＢ培地（アンピシリン５０μｇ／ｍＬを含む）に接種し、振とう数３１０ｒｐｍ、３０℃で１８時間培養した。培養終了時の濁度（Ａｂｓ６６０ｎｍ）は１５．６であった。続いて、得られた菌体を遠心分離で集菌し、２５ｍＭリン酸緩衝溶液（ｐＨ７．５）に懸濁し、氷冷下で超音波細胞破砕装置を用いて破砕した。その後、５５℃で３時間、熱処理を行い、その後、０．４５飽和になるように硫酸アンモニウムを徐々に添加し、目的のタンパク質を沈殿させた。その後、上清を取り除き、２５ｍＭリン酸緩衝溶液（ｐＨ７．５）を加えて、沈殿を再溶解させ、一般的な脱塩処理を行った後、２５ｍＭリン酸緩衝溶液（ｐＨ７．５）で平衡化した５ｍＬのＤＥＡＥセファロースＦａｓｔ　Ｆｌｏｗ（ＧＥヘルスケア製）カラムにかけ、素通り画分を回収した。このようにして、アミノ酸合成用の酵素サンプル（Ｌ１８２Ｅ＋Ｌ３４８Ｉ：７．０ｍｇ／ｍＬ、１１．４Ｕ／ｍＬ）を調製した。なお、酵素活性の基質には、Ｎ－サクシニル－Ｄ－トリプトファンを用いた。 (2) Preparation of double-mutant modified P4DSA (L182E + L348I) Preparation of colony of E. coli transformant expressing double-mutated modified P4DSA (L182E + L348I) obtained in (1) in 5 mL of LB medium in a test tube (Including 50 μg / mL of ampicillin) and inoculated for 16 hours at 180 ° C. and 30 ° C., and used as a seed culture solution. This seed culture was inoculated into 60 mL of TB medium (containing 50 μg / mL of ampicillin) contained in a 500 mL Sakaguchi flask, and cultured at a shaking speed of 310 rpm and 30 ° C. for 18 hours. Turbidity (Abs 660 nm) at the end of the culture was 15.6. Subsequently, the obtained cells were collected by centrifugation, suspended in a 25 mM phosphate buffer solution (pH 7.5), and crushed using an ultrasonic cell crusher under ice cooling. Thereafter, heat treatment was performed at 55 ° C. for 3 hours, and then ammonium sulfate was gradually added to reach 0.45 saturation to precipitate the target protein. Thereafter, the supernatant is removed, 25 mM phosphate buffer solution (pH 7.5) is added, the precipitate is re-dissolved, subjected to general desalting treatment, and then equilibrated with 25 mM phosphate buffer solution (pH 7.5). The applied fraction was applied to a 5 mL DEAE Sepharose Fast Flow column (manufactured by GE Healthcare) and collected. In this way, an enzyme sample for amino acid synthesis (L182E + L348I: 7.0 mg / mL, 11.4 U / mL) was prepared. Note that N-succinyl-D-tryptophan was used as the enzyme activity substrate.
（３）Ｎ－サクシニル－ＤＬ－トリプトファンからのＤ－トリプトファンの調製
　５％Ｎ－サクシニル－ＤＬ－トリプトファン水溶液（ｐＨ７．５）５ｍＬを基質として用い、塩化コバルト水溶液を終濃度１ｍＭとなるように添加後、上記（２）で調製した二重変異改変型ＤＳＡ（Ｌ１８２Ｅ＋Ｌ３４８Ｉ）の酵素液０．０５ｍｇと参考例５で調製したＣｈｌｏｒｏｆｌｅｘｕｓ　ａｕｒａｎｔｉａｃｕｓ株由来のＮ－サクシニルアミノ酸ラセマーゼ溶液２．５ｍｇを添加し、４５℃で３日間反応させた。また、一方でコントロールとして、ＰＣＴ／ＪＰ２０１１／０６４９４３の実施例に記載の方法で調製した野生型ＤＳＡ酵素液０．０２５ｍｇと参考例５で調製したＣｈｌｏｒｏｆｌｅｘｕｓ　ａｕｒａｎｔｉａｃｕｓ株由来のＮ－サクシニルアミノ酸ラセマーゼ溶液２．５ｍｇを添加し、４５℃で３日間反応させ、変換率及びＤ－アミノ酸の光学純度を算出した。変換率は、上記記載のジーエルサイエンス社製「Ｉｎｅｒｔｓｉｌ　ＯＤＳ－３」（５μｍ、４．６×１００ｍｍ）を利用した高速液体クロマトグラフィーで酵素反応前と反応後の基質のピーク面積値を測定することによって算出した。光学純度は、上記記載のダイセル化学工業株式会社製光学分割カラム「ＣＲＯＷＮＰＡＫ　ＣＲ（＋）」（５μｍ、４．０×１５０ｍｍ）で生成した遊離体のアミノ酸の光学純度を測定することによって算出した。結果を表８に示す。なお、表８には、比較のため、表５のＬ１８２Ｅの単変異のデータを併記している。 (3) Preparation of D-tryptophan from N-succinyl-DL-tryptophan Using 5 mL of 5% N-succinyl-DL-tryptophan aqueous solution (pH 7.5) as a substrate, add cobalt chloride aqueous solution to a final concentration of 1 mM. Thereafter, 0.05 mg of the enzyme solution of double mutation-modified DSA (L182E + L348I) prepared in (2) above and 2.5 mg of N-succinyl amino acid racemase solution derived from Chloroflexus aurantiacus strain prepared in Reference Example 5 were added. The reaction was carried out at 3 ° C. for 3 days. On the other hand, as a control, 0.025 mg of a wild-type DSA enzyme solution prepared by the method described in the Examples of PCT / JP2011 / 064943 and an N-succinyl amino acid racemase solution derived from the Chloroflexus aurantiacus strain prepared in Reference Example 5. 5 mg was added and reacted at 45 ° C. for 3 days, and the conversion rate and optical purity of D-amino acid were calculated. The conversion rate is determined by measuring the peak area value of the substrate before and after the enzyme reaction by high performance liquid chromatography using “Inertsil ODS-3” (5 μm, 4.6 × 100 mm) manufactured by GL Sciences, Inc. Calculated by The optical purity was calculated by measuring the optical purity of the free amino acid produced by the above-described optical resolution column “CROWNPAK CR (+)” (5 μm, 4.0 × 150 mm) manufactured by Daicel Chemical Industries, Ltd. The results are shown in Table 8. In Table 8, for comparison, L182E single mutation data in Table 5 is also shown.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　表８に示すように、反応３日目にはいずれも８０％以上に達した。また、反応３日目のＤ－トリプトファンの光学純度は、野生型は９６．１％ｅｅであるのに対し、Ｌ１８２Ｅの単変異改変型は９９．６％ｅｅであり、Ｌ１８２Ｅ＋Ｌ３４８Ｉの二重変異改変型は９９．７％ｅｅであり、いずれの改変型も光学純度が野生型と比較して顕著に向上しており、特に二重変異改変型は、単変異改変型よりさらに光学純度が向上していた。本発明のＤ－サクシニラーゼは、工業規模でのＤ－アミノ酸の製造に使用されるため、製造されるＤ－アミノ酸の光学純度がわずかでも向上するということは、製造コストの低下をもたらす顕著な向上であるといえる。また、データには示さないが、Ｌ３４８Ｉの単変異のみでは、野生型と比較して選択性向上の効果は見られず、Ｌ１８２Ｅと組み合わせることで、相乗的な立体選択性向上への寄与が確認された。 As shown in Table 8, both reached 80% or more on the third day of the reaction. The optical purity of D-tryptophan on the third day of the reaction is 96.1% ee for the wild type, whereas it is 99.6% ee for the L182E single mutation, and double mutation modification of L182E + L348I. The type is 99.7% ee, and the optical purity of each modified type is markedly improved compared to the wild type. In particular, the double mutant modified type has a further improved optical purity than the single mutant modified type. It was. Since the D-succinylase of the present invention is used for the production of D-amino acids on an industrial scale, the slight improvement in the optical purity of the produced D-amino acids means a significant improvement resulting in a reduction in production costs. You can say that. In addition, although not shown in the data, only the single mutation of L348I does not show an effect of improving the selectivity compared to the wild type, and it is confirmed that the combination with L182E contributes to a synergistic improvement of stereoselectivity. It was done.
（４）Ｎ－サクシニル－ＤＬ－フェニルアラニンからのＤ－フェニルアラニンの調製
　５％Ｎ－サクシニル－ＤＬ－フェニルアラニン水溶液（ｐＨ７．５）５ｍＬを基質として用い、塩化コバルト水溶液を終濃度１ｍＭとなるように添加後、上記（２）で調製した二重変異改変型ＤＳＡ（Ｌ１８２Ｅ＋Ｌ３４８Ｉ）の酵素液０．０７５ｍｇと参考例５で調製したＣｈｌｏｒｏｆｌｅｘｕｓ　ａｕｒａｎｔｉａｃｕｓ株由来のＮ－サクシニルアミノ酸ラセマーゼ溶液２．５ｍｇを添加し、４５℃で３日間反応させた。また、一方でコントロールとして、ＰＣＴ／ＪＰ２０１１／０６４９４３の実施例に記載の方法で調製した野生型ＤＳＡ酵素液０．０２５ｍｇと参考例５で調製したＣｈｌｏｒｏｆｌｅｘｕｓ　ａｕｒａｎｔｉａｃｕｓ株由来のＮ－サクシニルアミノ酸ラセマーゼ溶液２．５ｍｇを添加し、４５℃で３日間反応させ、変換率及びＤ－アミノ酸の光学純度を算出した。変換率は、上記記載のジーエルサイエンス社製「Ｉｎｅｒｔｓｉｌ　ＯＤＳ－３」（５μｍ、４．６×１００ｍｍ）を利用した高速液体クロマトグラフィーで酵素反応前と反応後の基質のピーク面積値を測定することによって算出した。光学純度は、上記記載のダイセル化学工業株式会社製光学分割カラム「ＣＲＯＷＮＰＡＫ　ＣＲ（＋）」（５μｍ、４．０×１５０ｍｍ）で生成した遊離体のアミノ酸の光学純度を測定することによって算出した。結果を表９に示す。なお、表９には、比較のため、表６のＬ１８２Ｅの単変異のデータを併記している。 (4) Preparation of D-phenylalanine from N-succinyl-DL-phenylalanine Using 5 mL of 5% N-succinyl-DL-phenylalanine aqueous solution (pH 7.5) as a substrate, and adding cobalt chloride aqueous solution to a final concentration of 1 mM Thereafter, 0.075 mg of the enzyme solution of double mutation-modified DSA (L182E + L348I) prepared in (2) above and 2.5 mg of N-succinyl amino acid racemase solution derived from Chloroflexus aurantiacus strain prepared in Reference Example 5 were added. The reaction was carried out at 3 ° C. for 3 days. On the other hand, as a control, 0.025 mg of a wild-type DSA enzyme solution prepared by the method described in the Examples of PCT / JP2011 / 064943 and an N-succinyl amino acid racemase solution derived from the Chloroflexus aurantiacus strain prepared in Reference Example 5. 5 mg was added and reacted at 45 ° C. for 3 days, and the conversion rate and optical purity of D-amino acid were calculated. The conversion rate is determined by measuring the peak area value of the substrate before and after the enzyme reaction by high performance liquid chromatography using “Inertsil ODS-3” (5 μm, 4.6 × 100 mm) manufactured by GL Sciences, Inc. Calculated by The optical purity was calculated by measuring the optical purity of the free amino acid produced by the above-described optical resolution column “CROWNPAK CR (+)” (5 μm, 4.0 × 150 mm) manufactured by Daicel Chemical Industries, Ltd. The results are shown in Table 9. In Table 9, for comparison, the single-mutation data of L182E in Table 6 are also shown.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
　表９に示すように、反応３日目にはいずれも８０％以上に達した。また、反応３日目のＤ－フェニルアラニンの光学純度は、野生型は９３．６％ｅｅであるのに対し、Ｌ１８２Ｅの単変異改変型は９８．８％ｅｅであり、Ｌ１８２Ｅ＋Ｌ３４８Ｉの二重変異改変型は９９．４％ｅｅであり、いずれの改変型も光学純度が野生型と比較して顕著に向上しており、特に二重変異改変型は、単変異改変型よりさらに光学純度が向上していた。本発明のＤ－サクシニラーゼは、工業規模でのＤ－アミノ酸の製造に使用されるため、製造されるＤ－アミノ酸の光学純度がわずかでも向上するということは、製造コストの低下をもたらす顕著な向上であるといえる。また、データには示さないが、Ｌ３４８Ｉの単変異のみでは、野生型と比較して選択性向上の効果は見られず、Ｌ１８２Ｅと組み合わせることで、相乗的な立体選択性向上への寄与が確認された。 As shown in Table 9, all reached 80% or more on the third day of the reaction. The optical purity of D-phenylalanine on the third day of the reaction is 93.6% ee for the wild type, whereas it is 98.8% ee for the L182E single mutation, and double mutation modification for L182E + L348I. The type is 99.4% ee, and the optical purity of each modified type is remarkably improved as compared to the wild type. In particular, the double mutant modified type has further improved optical purity than the single mutant modified type. It was. Since the D-succinylase of the present invention is used for the production of D-amino acids on an industrial scale, the slight improvement in the optical purity of the produced D-amino acids means a significant improvement resulting in a reduction in production costs. You can say that. In addition, although not shown in the data, only the single mutation of L348I does not show an effect of improving the selectivity compared to the wild type, and it is confirmed that the combination with L182E contributes to a synergistic improvement of stereoselectivity. It was done.
（５）Ｎ－サクシニル－ＤＬ－ビフェニルアラニンからのＤ－ビフェニルアラニンの調製
　５％Ｎ－サクシニル－ＤＬ－ビフェニルアラニン水溶液（ｐＨ７．５）５ｍＬを基質として用い、塩化コバルト水溶液を終濃度１ｍＭとなるように添加後、上記（２）で調製した二重変異改変型ＤＳＡ（Ｌ１８２Ｅ＋Ｌ３４８Ｉ）の酵素液０．０５ｍｇと参考例５で調製したＣｈｌｏｒｏｆｌｅｘｕｓ　ａｕｒａｎｔｉａｃｕｓ株由来のＮ－サクシニルアミノ酸ラセマーゼ溶液２５ｍｇを添加し、４５℃で３日間反応させた。また、一方でコントロールとして、ＰＣＴ／ＪＰ２０１１／０６４９４３の実施例に記載の方法で調製した野生型ＤＳＡ酵素液０．０２５ｍｇと参考例５で調製したＣｈｌｏｒｏｆｌｅｘｕｓ　ａｕｒａｎｔｉａｃｕｓ株由来のＮ－サクシニルアミノ酸ラセマーゼ溶液２５ｍｇを添加し、４５℃で３日間反応させ、変換率及びＤ－アミノ酸の光学純度を算出した。変換率は、上記記載のジーエルサイエンス社製「Ｉｎｅｒｔｓｉｌ　ＯＤＳ－３」（５μｍ、４．６×１００ｍｍ）を利用した高速液体クロマトグラフィーで酵素反応前と反応後の基質のピーク面積値を測定することによって算出した。光学純度は、上記記載のダイセル化学工業株式会社製光学分割カラム「ＣＲＯＷＮＰＡＫ　ＣＲ（＋）」（５μｍ、４．０×１５０ｍｍ）で生成した遊離体のアミノ酸の光学純度を測定することによって算出した。結果を表１０に示す。なお、表１０には、比較のため、表７のＬ１８２Ｅの単変異のデータを併記している。 (5) Preparation of D-biphenylalanine from N-succinyl-DL-biphenylalanine Using 5 mL of 5% N-succinyl-DL-biphenylalanine aqueous solution (pH 7.5) as a substrate, the aqueous solution of cobalt chloride has a final concentration of 1 mM. After the addition, 0.05 mg of the double mutant DSA (L182E + L348I) enzyme solution prepared in (2) above and 25 mg of the N-succinyl amino acid racemase solution derived from Chloroflexus aurantiacus strain prepared in Reference Example 5 were added, The reaction was carried out at 45 ° C. for 3 days. On the other hand, as a control, 0.025 mg of a wild-type DSA enzyme solution prepared by the method described in the examples of PCT / JP2011 / 064943 and 25 mg of an N-succinylamino acid racemase solution derived from Chloroflexus aurantiacus strain prepared in Reference Example 5 were used. The mixture was added and reacted at 45 ° C. for 3 days, and the conversion rate and optical purity of D-amino acid were calculated. The conversion rate is determined by measuring the peak area value of the substrate before and after the enzyme reaction by high performance liquid chromatography using “Inertsil ODS-3” (5 μm, 4.6 × 100 mm) manufactured by GL Sciences, Inc. Calculated by The optical purity was calculated by measuring the optical purity of the free amino acid produced by the above-described optical resolution column “CROWNPAK CR (+)” (5 μm, 4.0 × 150 mm) manufactured by Daicel Chemical Industries, Ltd. The results are shown in Table 10. In Table 10, for comparison, L182E single mutation data in Table 7 is also shown.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　表１０に示すように、反応３日目にはいずれも９０％以上に達した。また、反応３日目のＤ－ビフェニルアラニンの光学純度は、野生型は８８．３％ｅｅであるのに対し、Ｌ１８２Ｅの単変異改変型は９９．２％ｅｅであり、Ｌ１８２Ｅ＋Ｌ３４８Ｉの二重変異改変型は９９．４％ｅｅであり、いずれの改変型も光学純度が野生型と比較して顕著に向上しており、特に二重変異改変型は、単変異改変型よりさらに光学純度が向上していた。本発明のＤ－サクシニラーゼは、工業規模でのＤ－アミノ酸の製造に使用されるため、製造されるＤ－アミノ酸の光学純度がわずかでも向上するということは、製造コストの低下をもたらす顕著な向上であるといえる。また、データには示さないが、Ｌ３４８Ｉの単変異のみでは、野生型と比較して選択性向上の効果は見られず、Ｌ１８２Ｅと組み合わせることで、相乗的な立体選択性向上への寄与が確認された。 As shown in Table 10, all reached 90% or more on the third day of the reaction. The optical purity of D-biphenylalanine on the third day of the reaction is 88.3% ee for the wild type, whereas it is 99.2% ee for the single mutation type of L182E, and the double mutation of L182E + L348I. The modified type is 99.4% ee, and the optical purity of each modified type is markedly improved compared to the wild type. In particular, the double mutant modified type has a further improved optical purity than the single mutant modified type. Was. Since the D-succinylase of the present invention is used for the production of D-amino acids on an industrial scale, the slight improvement in the optical purity of the produced D-amino acids means a significant improvement resulting in a reduction in production costs. You can say that. In addition, although not shown in the data, only the single mutation of L348I does not show an effect of improving the selectivity compared to the wild type, and it is confirmed that the combination with L182E contributes to a synergistic improvement of stereoselectivity. It was done.
実施例６　Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株由来Ｄ－サクシニラーゼと近縁種のＤ－サクシニラーゼにおけるアミノ酸配列の相同性比較
　Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株由来のＤ－サクシニラーゼにおいて明らかとなった立体選択性に関与するアミノ酸残基が、その他のＣｕｐｒｉａｖｉｄｕｓ属細菌のＤ－サクシニラーゼにおいて、保存されているかどうかを調べるために、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ、Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓ、Ｃｕｐｒｉａｖｉｄｕｓ　ｎｅｃａｔｏｒ及びＣｕｐｒｉａｖｉｄｕｓ　ｔａｉｗａｎｅｎｓｉｓのＤ－サクシニラーゼのアミノ酸配列の相同性比較を行った。なお、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－ＣとＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓについては、参考例１及び２で明らかにしたアミノ酸配列を、Ｃｕｐｒｉａｖｉｄｕｓ　ｎｅｃａｔｏｒとＣｕｐｒｉａｖｉｄｕｓ　ｔａｉｗａｎｅｎｓｉｓについては、ＮＣＢＩ（ｈｔｔｐ：／／ｂｌａｓｔ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ）に公開されているアミノ酸配列を用いた。詳しくはＣｕｐｒｉａｖｉｄｕｓ　ｎｅｃａｔｏｒについては、Ｐｅｎｉｃｉｌｌｉｎ　Ｇ　ａｃｙｌａｓｅ（ＡＣＣＥＳＳＩＯＮ　Ｎｏ．ＹＰ＿８４２０１１）として登録されているアミノ酸配列を、Ｃｕｐｒｉａｖｉｄｕｓ　ｔａｉｗａｎｅｎｓｉｓについては、Ｐｅｎｉｃｉｌｌｉｎ　Ｇ　ａｍｉｄａｓｅｐｒｅｃｕｒｓｏｒ（ＡＣＣＥＳＳＩＯＮ　Ｎｏ．ＹＰ＿００２００８８４３）として登録されているアミノ酸配列を用いた。また、アミノ酸配列解析ソフトは、ＧＥＮＥＴＹＸ　ＣＯＲＰＯＲＡＴＩＯＮから販売されているＧＥＮＥＴＹＸ　ＷＩＮ　Ｖｅｒｓｉｏｎ　６．１のものを使用した。結果を図３に示す。その結果、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株由来のＤ－サクシニラーゼで明らかになった立体選択性に関与しているアミノ酸残基の全てが、比較した全てのＣｕｐｒｉａｖｉｄｕｓ属細菌のＤ－サクシニラーゼで保存されていることが明らかになった。なお、これらの４種のＣｕｐｒｉａｖｉｄｕｓ属細菌のＤ－サクシニラーゼの相同性は、以下の表１１に示すように７５％～９３％であった。 Example 6 Cupriavidus sp. Comparison of homology of amino acid sequences between D-succinylase derived from P4-10-C strain and D-succinylase of related species Cupriavidus sp. In order to examine whether amino acid residues involved in stereoselectivity revealed in D-succinylase derived from P4-10-C strain are conserved in other D. succinylases of the genus Cupriavidus, Cupriavidus sp . A homology comparison of the amino acid sequences of D4 succinylases of P4-10-C, Cupriavidus metallidrans, Cupriavidus necator and Cupriavidus taiwanensis was performed. Cupriavidus sp. For P4-10-C and Cupriavidus metallidurans, the amino acid sequences revealed in Reference Examples 1 and 2 were used, and for Cupriavidus necator and Cupriavidus Taiwananesis, NCBI (http: //blast.ncbi.nlm.n. The amino acid sequence used was used. Specifically, the amino acid sequence registered as Penicillin G acylase (ACCESSION No. YP — 842011) is used for Cupriavidus necator, and Penicillin Gamidasepresor is used as the amino acid sequence registered as Penicillin G amidaseprecursor. In addition, the amino acid sequence analysis software of GENETYX WIN Version 6.1 sold by GENETYX CORPORATION was used. The results are shown in FIG. As a result, Cupriavidus sp. It is clear that all amino acid residues involved in stereoselectivity revealed by D-succinylase derived from P4-10-C strain are conserved by all D-succinylases of Cupriavidus bacteria compared. Became. The homology of D-succinylase of these four types of Cupriavidus bacteria was 75% to 93% as shown in Table 11 below.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
実施例７　改変型Ｃｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓ由来Ｄ－サクシニラーゼ（以下、ＣｍＤＳＡ遺伝子と称する）の調製
　そこで、実施例６のアミノ酸配列のアライメント結果に基づき、近縁種においても、Ｐ４ＤＳＡの立体選択性に関与する７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位、５３９位に同等な部位をアミノ酸置換することにより、Ｎ－サクシニル－ＤＬ－アミノ酸に対して、Ｄ体選択的に作用するようになるということを示すために、近縁種の一種であるＣｕｐｒｉａｖｉｄｕｓ　ｍｅｔａｌｌｉｄｕｒａｎｓの改変型ＣｍＤＳＡを作成し、立体選択性を評価することにした。
（１）改変型ＣｍＤＳＡ遺伝子発現プラスミドの構築
　実施例１（３）に記載した方法と同様の方法で改変型ＣｍＤＳＡ遺伝子発現プラスミドを構築した。作製した改変型酵素と変異導入に使用した合成オリゴＤＮＡプライマーの配列を表１２に示す。 Example 7 Preparation of Modified Cupriavidus metallidurans-derived D-succinylase (hereinafter referred to as CmDSA gene) Based on the alignment results of the amino acid sequence of Example 6, the related species are also involved in the stereoselectivity of P4DSA. Substituents, 181 to 185, 305, 348, 351, 461, 539, and D-form selective action on N-succinyl-DL-amino acid by amino acid substitution at equivalent sites In order to show that this is the case, it was decided to create a modified CmDSA of Cupriavidus metallidurans, a kind of related species, and evaluate the stereoselectivity.
(1) Construction of modified CmDSA gene expression plasmid A modified CmDSA gene expression plasmid was constructed in the same manner as described in Example 1 (3). Table 12 shows the sequences of the prepared modified enzymes and the synthetic oligo DNA primers used for introducing the mutation.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
（２）ＣｍＤＳＡ及び改変型ＣｍＤＳＡの調製
　実施例１（４）に記載した方法と同様の方法で、ＣｍＤＳＡ及び改変型ＣｍＤＳＡの粗酵素液を調製した。 (2) Preparation of CmDSA and modified CmDSA Crude enzyme solutions of CmDSA and modified CmDSA were prepared in the same manner as described in Example 1 (4).
実施例８　改変型ＣｍＤＳＡを用いたＮ－サクシニルトリプトファンに対する立体選択性の評価
　実施例７で調製した粗酵素液を用いて、実施例１と同様の条件及び手順で、Ｎ－サクシニルトリプトファンに対する立体選択性の評価を行った。その結果を表１３に示す。 Example 8 Evaluation of stereoselectivity for N-succinyltryptophan using modified CmDSA Stereoselection for N-succinyltryptophan using the crude enzyme solution prepared in Example 7 under the same conditions and procedures as in Example 1. Sexuality was evaluated. The results are shown in Table 13.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
　その結果、全ての改変型ＣｍＤＳＡにおいて、Ｄ体選択的に作用していることが確認された。すなわち、Ｃｕｐｒｉａｖｉｄｕｓ　ｓｐ．Ｐ４－１０－Ｃ株由来Ｄ－サクシニラーゼの立体選択性に関与する７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位及び５３９位は、本菌株のみならず、近縁種のＤ－サクシニラーゼにおいても、それぞれの部位に同等な部位をアミノ酸置換することにより、Ｄ体選択性を向上させることができる部位であることが示唆された。 As a result, it was confirmed that all modified CmDSAs acted selectively in the D form. That is, Cupriavidus sp. Positions 72, 181 to 185, 305, 348, 351, 461 and 539, which are involved in the stereoselectivity of P4-10-C-derived D-succinylase, are not only this strain but also related species It was suggested that the D-succinylase can also be improved in D-form selectivity by amino acid substitution of equivalent sites in the respective sites.
　本発明の改変型Ｄ－サクシニラーゼは、野生型Ｄ－サクシニラーゼと比較してＤ体選択性が著しく向上しているので、医薬品等の中間体の原料として有用なＤ－アミノ酸を効率良く製造するために有用である。 The modified D-succinylase of the present invention has a significantly improved D-form selectivity compared to the wild-type D-succinylase, so that it can efficiently produce a D-amino acid useful as a raw material for intermediates such as pharmaceuticals. Useful for.
　配列番号５～５４は、実施例に記載した設計ポリヌクレオチドの配列である。 SEQ ID NOs: 5 to 54 are the sequences of the designed polynucleotides described in the examples.

Claims (13)

  1. 　下記（Ａ）又は（Ｂ）のアミノ酸配列からなることを特徴とするタンパク質。
    （Ａ）配列番号２に示すアミノ酸配列において、下記（ａ）～（ｋ）から選択される少なくとも１個のアミノ酸残基の置換を有するアミノ酸配列
    （ａ）７２位のグルタミン残基のアルギニン残基への置換
    （ｂ）１８１位のグリシン残基のトリプトファン残基、リジン残基、アルギニン残基、アスパラギン酸又はグルタミン酸残基への置換
    （ｃ）１８２位のロイシン残基のトリプトファン残基、セリン残基、システイン残基、チロシン残基、リジン残基、アルギニン残基、アスパラギン酸残基、グルタミン酸残基又はプロリン残基への置換
    （ｄ）１８３位のスレオニン残基のプロリン残基、ロイシン残基又はアスパラギン残基への置換
    （ｅ）１８４位のロイシン残基のプロリン残基への置換
    （ｆ）１８５位のアスパラギン残基のプロリン残基、フェニルアラニン残基、セリン残基又はアスパラギン酸残基への置換
    （ｇ）３０５位のアルギニン残基のスレオニン残基、アラニン残基、グリシン残基、ヒスチジン残基、グルタミン残基、セリン残基、アスパラギン残基又はバリン残基への置換
    （ｈ）３４８位のロイシン残基のグルタミン酸残基、プロリン残基、メチオニン残基、トリプトファン残基、セリン残基、スレオニン残基、システイン残基、リジン残基、ヒスチジン残基又はグルタミン残基への置換
    （ｉ）３５１位のフェニルアラニン残基のロイシン残基、イソロイシン残基、メチオニン残基、アスパラギン残基又はグルタミン残基への置換
    （ｊ）４６１位のアスパラギン残基のイソロイシン残基、フェニルアラニン残基、スレオニン残基、リジン残基又はアルギニン残基への置換
    （ｋ）５３９位のグリシン残基のプロリン残基、バリン残基、メチオニン残基、スレオニン残基又はアスパラギン残基への置換
    （Ｂ）上記（Ａ）のアミノ酸配列において、７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位、及び５３９位以外の箇所に、１若しくは数個のアミノ酸残基の置換、欠失、挿入、付加および／または逆位を有するアミノ酸配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードするアミノ酸配列。     A protein comprising the following amino acid sequence (A) or (B):
    (A) an amino acid sequence having a substitution of at least one amino acid residue selected from the following (a) to (k) in the amino acid sequence shown in SEQ ID NO: 2 (a) an arginine residue of glutamine residue at position 72 (B) substitution of glycine residue at position 181 with tryptophan residue, lysine residue, arginine residue, aspartic acid or glutamic acid residue (c) tryptophan residue at position 182, tryptophan residue, serine residue Group, cysteine residue, tyrosine residue, lysine residue, arginine residue, aspartic acid residue, glutamic acid residue or proline residue (d) threonine residue at position 183, proline residue, leucine residue Or substitution to asparagine residue (e) substitution of leucine residue at position 184 to proline residue (f) proline residue of asparagine residue at position 185, Substituting with a phenylalanine residue, a serine residue or an aspartic acid residue (g) a threonine residue of the arginine residue at position 305, an alanine residue, a glycine residue, a histidine residue, a glutamine residue, a serine residue, Substitution to asparagine residue or valine residue (h) glutamic acid residue, proline residue, methionine residue, tryptophan residue, serine residue, threonine residue, cysteine residue, lysine residue of 348th leucine residue Group, substitution to histidine residue or glutamine residue (i) substitution of phenylalanine residue at position 351 to leucine residue, isoleucine residue, methionine residue, asparagine residue or glutamine residue (j) position 461 Placement of asparagine residues on isoleucine, phenylalanine, threonine, lysine or arginine residues (K) Replacement of the glycine residue at position 539 with a proline residue, valine residue, methionine residue, threonine residue or asparagine residue (B) position 72, 181 to 185 in the amino acid sequence of (A) above An amino acid sequence having substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues at positions other than positions 305, 348, 351, 461 and 539 An amino acid sequence encoding a protein having an activity of producing a D-amino acid by selectively acting on D-form to N-succinyl-DL-amino acid.
  2. 　請求項１の（Ａ）のアミノ酸配列が、配列番号２に示すアミノ酸配列において、１８２位のロイシン残基のグルタミン酸残基への置換、及び３４８位のロイシン残基のイソロイシン残基への置換を有するアミノ酸配列であることを特徴とする請求項１に記載のタンパク質。 In the amino acid sequence of (A) of claim 1, in the amino acid sequence shown in SEQ ID NO: 2, substitution of a leucine residue at position 182 with a glutamic acid residue and substitution of a leucine residue at position 348 with an isoleucine residue The protein according to claim 1, wherein the protein has an amino acid sequence.
  3. 　下記（Ａ）又は（Ｂ）の塩基配列からなることを特徴とする遺伝子。
    （Ａ）配列番号１に示す塩基配列において、下記（ａ）～（ｋ）から選択される少なくとも１個の塩基配列の置換を有する塩基配列
    （ａ）２１４～２１６位の塩基配列ｃａａの、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ又はａｇｇへの置換
    （ｂ）５４１～５４３位の塩基配列ｇｇｃの、ｔｇｇ、ａａａ、ａａｇ、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ、ａｇｇ、ｇａｔ、ｇａｃ、ｇａａ又はｇａｇへの置換
    （ｃ）５４４～５４６位の塩基配列ｃｔｇの、ｔｇｇ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ｔｇｔ、ｔｇｃ、ｔａｔ、ｔａｃ、ａａａ、ａａｇ、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ、ａｇｇ、ｇａｔ、ｇａｃ、ｇａａ、ｇａｇ、ｃｃｔ、ｃｃｃ、ｃｃａ又はｃｃｇへの置換
    （ｄ）５４７～５４９位の塩基配列ａｃｇの、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ｔｔａ、ｔｔｇ、ｃｔｔ、ｃｔｃ、ｃｔａ、ｃｔｇ、ａａｔ又はａａｃへの置換
    （ｅ）５５０～５５２位の塩基配列ｃｔｇの、ｃｃｔ、ｃｃｃ、ｃｃａ又はｃｃｇへの置換
    （ｆ）５５３～５５５位の塩基配列ａａｔの、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ｔｔｔ、ｔｔｃ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ｇａｔ又はｇａｃへの置換
    （ｇ）９１３～９１５位の塩基配列ｃｇｇの、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ｇｃｔ、ｇｃｃ、ｇｃａ、ｇｃｇ、ｇｇｔ、ｇｇｃ、ｇｇａ、ｇｇｇ、ｃａｔ、ｃａｃ、ｃａａ、ｃａｇ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ａａｔ、ａａｃ、ｇｔｔ、ｇｔｃ、ｇｔａ又はｇｔｇへの置換
    （ｈ）１０４２～１０４４位の塩基配列ｃｔｇの、ｇａａ、ｇａｇ、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ａｔｇ、ｔｇｇ、ｔｃｔ、ｔｃｃ、ｔｃａ、ｔｃｇ、ａｇｔ、ａｇｃ、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ｔｇｔ、ｔｇｃ、ａａａ、ａａｇ、ｃａｔ、ｃａｃ、ｃａａ又はｃａｇへの置換
    （ｉ）１０５１～１０５３位の塩基配列ｔｔｃの、ｔｔａ、ｔｔｇ、ｃｔｔ、ｃｔｃ、ｃｔａ、ｃｔｇ、ａｔｔ、ａｔｃ、ａｔａ、ａｔｇ、ａａｔ、ａａｃ、ｃａａ又はｃａｇへの置換
    （ｊ）１３８１～１３８３位の塩基配列ａａｃの、ａｔｔ、ａｔｃ、ａｔａ、ｔｔｔ、ｔｔｃ、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ａａａ、ａａｇ、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ又はａｇｇへの置換
    （ｋ）１６１５～１６１７位の塩基配列ｇｇｃの、ｃｃｔ、ｃｃｃ、ｃｃａ、ｃｃｇ、ｇｔｔ、ｇｔｃ、ｇｔａ、ｇｔｇ、ａｔｇ、ａｃｔ、ａｃｃ、ａｃａ、ａｃｇ、ａａｔ又はａａｃへの置換
    （Ｂ）上記（Ａ）の塩基配列とストリンジェントな条件でハイブリダイズする塩基配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードする塩基配列。     A gene comprising the following base sequence (A) or (B):
    (A) a base sequence having the substitution of at least one base sequence selected from the following (a) to (k) in the base sequence shown in SEQ ID NO: 1 (a) cgt of the base sequence caa at positions 214 to 216 (B) Substitution to cgg, cga, cgg, aga or agg (b) tgg, aaa, aag, cgt, cgg, cga, cgg, aga, agg, gat, gac, gaa or the base sequence ggg at positions 541 to 543 Substitution to gag (c) tgg, tct, tcc, tca, tcg, agt, agc, tgt, tgc, tat, tac, aaa, aag, cgt, cgc, cga, cgg of nucleotide sequence ctg at positions 544 to 546 , Aga, agg, gat, gac, gaa, gag, cct, ccc, cca or ccg substitution (d) nucleotide sequence from position 547 to 549 Substitution of acg to cct, ccc, cca, ccg, tta, ttg, ctt, ctc, cta, ctg, aat or aac (e) cct, ccc, cca or ccg of nucleotide sequence ctg at positions 550 to 552 (F) Substitution of base sequence aat at positions 553 to 555 to cct, ccc, cca, ccg, ttt, ttt, tct, tcc, tca, tcg, agt, agc, gat or gac (g) 913 to The base sequence cgg at position 915, act, acc, aca, acg, gct, gcc, gca, gcg, ggt, ggg, gga, ggg, cat, cac, caa, cag, tct, tcc, tca, tcg, agt, Substitution to agc, aat, aac, gtt, gtt, gta or gtg (h) bases at positions 1042 to 1044 Ctg, gaa, gag, cct, ccc, cca, ccg, atg, tgg, tct, tcc, tca, tcg, agt, agc, act, acc, aca, acg, tgt, tgc, aaa, aag, cat, Substitution to cac, caa or cag (i) substitution of base sequence ttc at positions 1051 to 1053 to tta, ttg, ctt, ctc, cta, ctg, att, atc, ata, atg, aat, aac, caa or cag Substitution (j) Substitution of nucleotide sequence aac at positions 1381 to 1383 to att, atc, ata, ttt, ttc, act, acc, aca, acg, aaa, aag, cgt, cgg, cga, cgg, aga or agg (K) cct, ccc, cca, ccg, gt of the base sequence ggc at positions 1615 to 1617 Substitution to t, gtc, gta, gtg, atg, act, acc, aca, acg, aat or aac (B) A nucleotide sequence that hybridizes with the nucleotide sequence of (A) above under stringent conditions, and N A base sequence encoding a protein having an activity of producing a D-amino acid by selectively acting on D-form to succinyl-DL-amino acid.
  4. 　請求項３の（Ａ）の塩基配列が、配列番号１に示す塩基配列において、５４４～５４６位の塩基配列ｃｔｇの、ｇａａ又はｇａｇへの置換、及び１０４２～１０４４位の塩基配列ｃｔｇの、ａｔｔ，ａｔｃ又はａｔａへの置換を有する塩基配列であることを特徴とする請求項３に記載の遺伝子。 The base sequence of (A) of claim 3 is an att of the base sequence ctg at positions 544 to 546 to gaa or gag in the base sequence shown in SEQ ID NO: 1, and the base sequence ctg at positions 1042 to 1044 The gene according to claim 3, wherein the gene has a substitution to atc or ata.
  5. 　下記（Ａ）又は（Ｂ）のアミノ酸配列からなることを特徴とするタンパク質。
    （Ａ）配列番号４に示すアミノ酸配列において、下記（ａ）～（ｅ）から選択される少なくとも１個のアミノ酸残基の置換を有するアミノ酸配列
    （ａ）１７７位のロイシン残基のアルギニン残基への置換
    （ｂ）１８０位のアスパラギン残基のアスパラギン酸残基への置換
    （ｃ）３４４位のロイシン残基のプロリン残基への置換
    （ｄ）３４７位のフェニルアラニン残基のイソロイシン残基への置換
    （ｅ）４５７位のアスパラギン残基のイソロイシン残基への置換
    （Ｂ）上記（Ａ）のアミノ酸配列において、１７７位、１８０位、３４４位、３４７位、及び４５７位以外の箇所に、１若しくは数個のアミノ酸残基の置換、欠失、挿入、付加および／または逆位を有するアミノ酸配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードするアミノ酸配列。     A protein comprising the following amino acid sequence (A) or (B):
    (A) an amino acid sequence having a substitution of at least one amino acid residue selected from the following (a) to (e) in the amino acid sequence shown in SEQ ID NO: 4 (a) an arginine residue of a leucine residue at position 177 (B) Substitution of asparagine residue at position 180 to aspartic acid residue (c) Substitution of leucine residue at position 344 to proline residue (d) To isoleucine residue of phenylalanine residue at position 347 (E) Substitution of asparagine residue at position 457 to isoleucine residue (B) In the amino acid sequence of (A) above, at positions other than positions 177, 180, 344, 347, and 457, An amino acid sequence having substitutions, deletions, insertions, additions and / or inversions of one or several amino acid residues, which is D-form selective for N-succinyl-DL-amino acids Amino acid sequence encoding a protein having an activity to produce the effect to D- amino acids.
  6. 　下記（Ａ）又は（Ｂ）の塩基配列からなることを特徴とする遺伝子。
    （Ａ）配列番号３に示す塩基配列において、下記（ａ）～（ｅ）から選択される少なくとも１個の塩基配列の置換を有する塩基配列
    （ａ）５２９～５３１位の塩基配列ｃｔｇの、ｃｇｔ、ｃｇｃ、ｃｇａ、ｃｇｇ、ａｇａ又はａｇｇへの置換
    （ｂ）５３８～５４０位の塩基配列ａａｃの、ｇａｔ又はｇａｃへの置換
    （ｃ）１０３０～１０３２位の塩基配列ｃｔｇの、ｃｃｔ、ｃｃｃ、ｃｃａ又はｃｃｇへの置換
    （ｄ）１０３９～１０４１位の塩基配列ｔｔｃの、ａｔｔ、ａｔｃ又はａｔａへの置換
    （ｅ）１３６９～１３７１位の塩基配列ａａｔの、ａｔｔ、ａｔｃ又はａｔａへの置換
    （Ｂ）上記（Ａ）の塩基配列とストリンジェントな条件でハイブリダイズする塩基配列であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有するタンパク質をコードする塩基配列。     A gene comprising the following base sequence (A) or (B):
    (A) a base sequence having a substitution of at least one base sequence selected from the following (a) to (e) in the base sequence shown in SEQ ID NO: 3 (a) cgt of the base sequence ctg at positions 529 to 531 , Cgc, cga, cgg, aga or agg substitution (b) substitution of base sequence aac at positions 538 to 540 to gat or gac (c) cct, ccc, cca of base sequence ctg at positions 1030 to 1032 Or substitution with ccg (d) substitution of base sequence ttc at positions 1039 to 1041 with att, atc or ata (e) substitution of base sequence aat at positions 1369 to 1371 with att, atc or ata (B) A base sequence that hybridizes with the base sequence of (A) above under stringent conditions and selectively acts on D-forms against N-succinyl-DL-amino acids. Nucleotide sequence encoding a protein having the activity to form D- amino Te.
  7. 　配列番号２と７０％以上の相同性を有するアミノ酸配列において、配列番号２の７２位、１８１～１８５位、３０５位、３４８位、３５１位、４６１位、及び５３９位のうちのいずれかと同等な位置のアミノ酸残基が、請求項１の（Ａ）に示すアミノ酸残基に置換されているアミノ酸配列からなるタンパク質であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有することを特徴とするタンパク質。 In the amino acid sequence having 70% or more homology with SEQ ID NO: 2, it is equivalent to any of positions 72, 181-185, 305, 348, 351, 461, and 539 of SEQ ID NO: 2. A protein comprising an amino acid sequence in which the amino acid residue at the position is substituted with the amino acid residue shown in (A) of claim 1, which acts in a D-form selective manner on N-succinyl-DL-amino acids. A protein having an activity of producing a D-amino acid.
  8. 　請求項７に記載のタンパク質をコードすることを特徴とする遺伝子。 A gene encoding the protein according to claim 7.
  9. 　配列番号４と７０％以上の相同性を有するアミノ酸配列において、配列番号４の１７７位、１８０位、３４４位、３４７位、及び４５７位のうちのいずれかと同等な位置のアミノ酸残基が、請求項３の（Ａ）に示すアミノ酸残基に置換されているアミノ酸配列からなるタンパク質であって、Ｎ－サクシニル－ＤＬ－アミノ酸に対してＤ体選択的に作用してＤ－アミノ酸を生成する活性を有することを特徴とするタンパク質。 In the amino acid sequence having 70% or more homology with SEQ ID NO: 4, an amino acid residue at a position equivalent to any of positions 177, 180, 344, 347, and 457 of SEQ ID NO: 4 is claimed A protein comprising an amino acid sequence substituted with the amino acid residue shown in (A) of Item 3, wherein the D-amino acid acts selectively on N-succinyl-DL-amino acid to produce D-amino acid A protein characterized by comprising:
  10. 　請求項９に記載のタンパク質をコードすることを特徴とする遺伝子。 A gene encoding the protein according to claim 9.
  11. 　請求項３，４，６，８又は１０に記載の遺伝子をベクターに挿入して組換えベクターを調製し、この組換えベクターで宿主細胞を形質転換して形質転換体を調製し、この形質転換体を培養する工程を含むことを特徴とする請求項１，２，５，７又は９に記載のタンパク質の製造方法。 A recombinant vector is prepared by inserting the gene according to claim 3, 4, 6, 8 or 10 into a vector, a host cell is transformed with the recombinant vector, and a transformant is prepared. The method for producing a protein according to claim 1, 2, 5, 7, or 9, further comprising a step of culturing the body.
  12. 　請求項１，２，５，７又は９に記載のタンパク質を用いてＮ－サクシニル－ＤＬ－アミノ酸中のＮ－サクシニル－Ｄ－アミノ酸を特異的に加水分解する工程を含むことを特徴とするＤ－アミノ酸の製造方法。 A step of specifically hydrolyzing N-succinyl-D-amino acid in N-succinyl-DL-amino acid using the protein according to claim 1, 2, 5, 7, or 9. -A method for producing amino acids.
  13. 　Ｎ－サクシニルアミノ酸ラセマーゼを用いてＮ－サクシニル－Ｌ－アミノ酸をラセミ化してＮ－サクシニル－Ｄ－アミノ酸を生成させる工程をさらに含むことを特徴とする請求項１２に記載の方法。 The method according to claim 12, further comprising the step of racemizing the N-succinyl-L-amino acid with an N-succinyl amino acid racemase to produce an N-succinyl-D-amino acid.
PCT/JP2012/055409 2011-03-14 2012-03-02 Modified d-succinylase having improved d-form selectivity for n-succinyl-dl-amino acid WO2012124513A1 (en)

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Publication number Priority date Publication date Assignee Title
US9700599B2 (en) 2012-11-13 2017-07-11 Adocia Rapid-acting insulin formulation comprising a substituted anionic compound
US10583175B2 (en) 2012-11-13 2020-03-10 Adocia Rapid-acting insulin formulation comprising a substituted anionic compound
US10646551B2 (en) 2012-11-13 2020-05-12 Adocia Rapid-acting insulin formulation comprising a substituted anionic compound
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
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