WO2012124513A1

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

Info

Publication number: WO2012124513A1
Application number: PCT/JP2012/055409
Authority: WO
Inventors: 洋輔角田; 幸夫岩井; 西矢　芳昭; 伸弥熊谷
Original assignee: 東洋紡績株式会社; 積水メディカル株式会社
Priority date: 2011-03-14
Filing date: 2012-03-02
Publication date: 2012-09-20
Also published as: JP5903298B2; JP2012205587A

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

Modified D-succinylase with improved D-form selectivity for N-succinyl-DL-amino acids

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.

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.

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).

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.

Japanese Patent Application No. 2010-148895

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.

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.

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.

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. 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.

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.

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.

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.

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.

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.

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). .

(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.

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.

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.

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.

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.

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.

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.

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.

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.

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. .

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. 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.

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

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.

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.

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.

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.

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.

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.

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.

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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

The reaction of racemizing N-succinyl-D-amino acid using N-succinyl amino acid racemase is performed by, for example, mixing in a reaction solution containing N-succinyl amino acid, N-succinyl amino acid racemase and a buffer under the following conditions: Do. The reaction temperature is not particularly limited as long as the N-succinyl amino acid racemase to be used is sufficiently effective, but is generally preferably 25 to 70 ° C, more preferably 37 to 60 ° C. The pH during the reaction is not particularly limited as long as the N-succinyl amino acid racemase to be used acts sufficiently, but in general, pH 5 to 9 is preferable, and pH 6 to 8 is more preferable. N-succinyl amino acid racemase is preferably used at a concentration of 50 to 15000 mg / L (5000 to 1500,000 U / L) in the reaction solution. The activity of N-succinyl amino acid racemase is significantly improved by adding a divalent metal ion at a final concentration of 0.1 mM to 1 M (preferably 0.1 to 1 mM). 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.

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.

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.

In this example, N-succinyltryptophan, N-succinylphenylalanine, and N-succinylbiphenylalanine were quantified under the following analysis conditions depending on the purpose unless otherwise specified.

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.

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

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

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

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.

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

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.

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

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

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

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 .

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.

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) 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) 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) 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.

(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.

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) 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.

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.

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. .

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.

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) 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) 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.

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.

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

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) 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.

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.

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.

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

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.

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.

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) 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.

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.

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) 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) 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.

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) 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.

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) 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.

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.

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.

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.

(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).

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.

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.

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.

Claims

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.
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.
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.
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.
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.
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.
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.
A gene encoding the protein according to claim 7.
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:
A gene encoding the protein according to claim 9.
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.
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.
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.