WO2008029921A1 - Function-modified phenylalanine dehydrogenase, and method for analysis of amino acid in biological sample using the enzyme - Google Patents

Abstract

A modified enzyme having such modification of at least three amino acid residues that the modified enzyme can have improved substrate specificity of phenylalanine dehydrogenase (EC 1.4.1.20) for methionine; DNA encoding the modified enzyme; a method for the preparation of a modified enzyme which has such modification of at least three amino acid residues that the modified enzyme can have improved substrate specificity of phenylalanine dehydrogenase (EC 1.4.1.20) for methionine, comprising the steps of culturing a transformant transformed with a vector carrying the DNA, and collecting the modified enzyme from the culture; and a method for the analysis of L-methionine contained in a sample by using the modified enzyme or protein. A phenylalanine dehydrogenase having a substrate specificity for L-methionine can be produced by an evolutionary molecular engineering approach to provide a function-modified amino acid dehydrogenase suitable for enzymatic fluorometry of methionine. It becomes possible to provide a method for the analysis of L-methionine contained in a sample (a blood sample) by using the function-modified amino acid dehydrogenase.

Description

 Specification

 Functionally modified phenylalanine dehydrogenase and method for analyzing amino acids in biological samples using this enzyme

 Technical field

 [0001] The present invention relates to a modified enzyme in which at least three amino acids are modified so as to improve the substrate specificity of phenylalanine dehydrogenase (EC 1.4.1.20) for methionine. Furthermore, the present invention relates to a method for analyzing amino acids such as L-methionine contained in a biological sample using this modified enzyme.

[0002] According to the present invention, particularly in diseases such as homocystinuria, phenylketonuria, and maple syrup urine disease, which are inborn errors of metabolism, newborn mass screening for early detection, or Provide available testing methods for the patient's regular health checkup. Furthermore, the analysis method of the present invention can be used for detection and quantification of L-methionine, L-phenylalanine, L-leucine, L-isoleucine, L-valine and the like contained in food or the environment.

 Background art

 [0003] The global spread of neonatal mass screening for the early detection of congenital metabolic disorders is the discovery of treatments for phenylketonuria (PKU) and the L in dry filter paper blood. -A semi-quantitative method for phenylalanin (L-Phe) has been developed by Guthrie et al. (Pediatrics, Vol. 32, p. 3 38-343 (1963), Non-Patent Document 1). About 500 cases of inborn errors of metabolism have been reported so far, and in particular, 7 to 22 cases of diseases for which normal growth is expected when treatment is established and appropriate treatment is performed. About mass screening for newborns is being conducted nationwide.

[0004] For screening of phenylketonuria, enzyme method (Screening, Vol. 1,. 63 (1992), Non-patent document 2, Medicine and pharmacy, Vol. 31, p. 1237 (1994), Non-patent Reference 3) or the enzyme reaction called microplate fluorescence method (Clinical Chemistry, Vol. 35, p. 1962 (1989), non-patent document 4) and subsequent fluorescence reaction are performed on the microplate. -A kit for the determination of phenylalanin was developed (Medicine and Pharmacy, Vol. 37, p. 1211 (1997), non-patent document 5). In addition, for the screening of maple syrup urine disease, a microplate fluorescence method using an L-leucine-specific dehydrogenase was developed in the same manner as the screening method for phenylketonuria.

[0005] On the other hand, as a screening method for homocystinuria, development of a mass screening method for homocystinuria using methionine γ-lyase (Research on evaluation method of mass screening system by the Ministry of Health and Welfare in 1993) pp. 237-240 (1993) and non-patent document 6) have been reported. In this method, ammonia (NH) released from L-methionine by the enzymatic reaction of L-methionine γ-lyase is converted into 0-phthalaldehyde (OPA) and 2-methanine.

 Three

 This is a measurement method using specific fluorescence in the neutral region of lucaptoethanol (2ME).

[0006] In addition to the high sample throughput, these microplate fluorescence quantification methods have the advantage that quantification and recording, which are objective judgments of test results that have been difficult with conventional methods, can be easily performed. is doing. In addition, it is possible to obtain test results in about 3 hours including sample pretreatment, which is a significant improvement over conventional methods in terms of speed, convenience, and work efficiency.

 [0007] In mass screening by enzyme fluorescence using microplates for important inborn errors of metabolism other than homocystinuria (eg, phenylketonuria, galactosemia, and maple syrup urine disease) It uses a dehydrogenase specific to the measurement item and is common in the measurement procedure, reagent composition, detection fluorescence wavelength, etc., and is a system capable of simultaneous measurement of multiple samples. On the other hand, in the screening method for homocystinuria, that is, the indirect blood methionine quantification method by the fluorescence quantification of ammonia using L-methionine γ-lyase, the measurement procedure, reagent composition, detection fluorescence wavelength, etc. This is very different from the method of measuring inborn errors of metabolism. In addition, this method is sensitive to contamination of the sample by ammonia in the environment, and it was necessary to pay close attention. For this reason, specimens must be sorted for each diagnostic item, and there was a need to improve work efficiency during screening.

[0008] In the present invention, an L-methionine-specific dehydrogenase is produced by an evolutionary molecular engineering technique as in the case of important inborn errors of metabolism other than homocystinuria, and the concentration of L-methionine in a blood sample is determined. Can be quantified by enzymatic fluorescence, and the measurement procedure and reagent set The purpose is to provide a significant improvement in screening efficiency by using the same specifications as other diagnostic kits for inborn errors of metabolism in terms of the wavelength of detection and detection fluorescence.

[0009] More specifically, the object of the present invention is to produce a phenylalanine dehydrogenase having a substrate specificity for L-methionine by an evolutionary molecular engineering technique, and to an enzyme fluorescence determination method for methionine. It is to provide a suitable function-modified amino acid dehydrogenase. Another object of the present invention is to provide a method for analyzing L-methionine contained in a test sample (blood sample) using the produced functionally modified amino acid dehydrogenase.

 [0010] Further, in the present invention, by producing amino acid dehydrogenase having a wide substrate specificity for L-phenylalanine, L-leucine, L-valine, L-isoleucine and L-methionine, The present invention provides an appropriate means for detecting or performing primary screening for inborn errors of metabolism in which abnormal blood concentrations have been confirmed and diseases in which abnormal blood concentrations of amino acids can be confirmed.

 [0011] Disclosure of the Invention

 The present invention for solving the above problems and achieving the object of the present invention is as follows.

 [1] A modified enzyme in which at least three amino acids have been modified to improve the substrate specificity of L-methionine for phenylalanine dehydrogenase (EC 1.4 · 1.20).

 [2] Sequential arrangement of threonine (Thr) dalysin (Gly) in the amino acid sequence of phenylalanin dehydrogenase IJ (wherein glycine (Gly) is in the 114th to 125th range) The modified enzyme according to [1], wherein glycine (Gly) is substituted with serine (Ser).

 [3] The modification is a continuous sequence of glutamine (Gin) valine (Val) in the amino acid sequence of phenylalanine dehydrogenase (however, the palin (Val) is in the range of 297 to 310) The modified enzyme according to [1] or [2], which is the substitution of palin (Val) with proline (Pro).

 [4] The modification is performed on a continuous sequence of alanine (Ala) leucine (Leu) in the amino acid sequence of phenylalanine dehydrogenase (wherein leucine (Leu) is in the 54th to 67th range). The modified enzyme according to any one of [1] to [3], wherein the leucine (Leu) is replaced with cysteine (Cys).

[5] The modifying power Asparagine (Asn) in the amino acid sequence of phenylalanine dehydrogenase , Norin (Val), Alanine (Ala) or Phenylalanin (Phe) and Glycine (Gly) IJ (provided that Asparagine (Asn), Norin (Val), Alanine (Ala) or Phoenix Luaranin (Phe) is a substitution of asparagine (Asn), valine (Val), alanine (Ala) or phenylalanine (Phe) with methionine (Met) in the 135th to 146th range [1] [4] V, the modified enzyme according to any one of the above.

 [6] Glycine (Gly) / Serine (Ser), Isoleucine (lie), Tryptophan (Trp) / Leucine (Leu) / Valine (Cystin) in the amino acid sequence of phenylalanine dehydrogenase (Cys), phenylalanine (Phe) / tyrosine (Tyr), alanine (Ala), proline (Pro), aspartic acid (Asp) consecutive sequences (provided that leucine (Leu), tryptophan (Tr Ρ), Substitution of leucine (Leu), tryptophan (Trp), norrin (Val) or cysteine (Cys) to asparagine (A sn) in valine (Val) or cysteine (Cys) is in the range 281-295. The modified enzyme according to any one of [1] to [5]!

 [7] Phenylalanine dehydrogenase power Bacillus badius IAM11059, Bacillus sphaericus R 79a, Sporosarcina ureae R04, Bacillus halodurans, Geobacillus kaustophilus, Ocean obacillus iheyensis, Rhodococcus sp. M_4 or fhermoactinomyces intermed1 The modified enzyme according to any one of the above.

 [8] The 65th amino acid residue of the amino acid sequence of phenylalanin dehydrogenase derived from Bacillus badius IAM11059 (described in SEQ ID NO: 2 in the sequence table) is the cysteine (Cys), and the 123rd amino acid residue is Serine (Ser), 144th amino acid residue is replaced with methionine (Met), 294th amino acid residue is replaced with asparagine (Asn), and 309th amino acid residue is replaced with proline (Pro) enzyme.

 [9] Amino acid configuration of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a IJ (described in SEQ ID NO: 4 in the sequence listing) is the 66th amino acid residue, Cys, and the 124th amino acid residue. Is substituted with serine (Ser), 145th amino acid residue with metionin (Met), 295th amino acid residue with asparagine (Asn), and 310th amino acid residue with proline (Pro) enzyme.

[10] Cysteine (Cys) at amino acid position IJ (described in SEQ ID No. 6 in the sequence listing), amino acid 125th amino acid sequence of phenylalanin dehydrogenase derived from Sporosarcina ureae R04 Replace the acid residue with serine (Ser), the 146th amino acid residue with methionine (Met), the 293rd amino acid residue with asparagine (Asn), and the 308th amino acid residue with proline (Pro). Replaced modified enzyme.

 [1] The amino acid sequence of phenylalanine dehydrogenase derived from UBacillus halodurans IJ (described in SEQ ID NO: 8 in the sequence listing) is the cysteine (Cys) as the 64th amino acid residue and the serine (Ser ), A modified enzyme in which the amino acid residue at position 143 is substituted with methionine (Met), the amino acid residue at position 293 is replaced with asparagine (Asn), and the amino acid residue at position 308 is replaced with proline (Pro).

 [12] Cysteine (Cys) is the 65th amino acid residue and Serine (Ser) is the 65th amino acid residue of the amino acid sequence of phenylalanin dehydrogenase from Geobacillus kaustophilus (described in SEQ ID NO: 10 in the sequence table). ), A modified enzyme in which the 144th amino acid residue is substituted with methionine (Met), the 294th amino acid residue is substituted with isparagine (Asn), and the 309th amino acid residue is substituted with proline (Pr0).

 [13] The amino acid sequence of phenylalanin dehydrogenase derived from Oceanobacillus iheyensis (described in SEQ ID NO: 12 in the sequence table) is cysteine (Cys) as the 64th amino acid residue and serine as the 122nd amino acid residue (Ser ), A modified enzyme in which the amino acid residue at position 143 is substituted with methionine (Met), the amino acid residue at position 293 is replaced with isparagine (Asn), and the amino acid residue at position 308 is replaced with proline (Pr 0).

 [14] The amino acid sequence of phenylalanin dehydrogenase derived from Rhodococcus sp. M_4 (described in SEQ ID NO: 14 in the Sequence Listing) is the 54th amino acid residue, Cys, and the 117th amino acid residue. Serine (Ser), a modified enzyme in which the 138th amino acid residue is substituted with methionine (Met) and the 281st amino acid residue is substituted with asparagine (Asn).

 [15] Cysteine (Cys) as the 56th amino acid residue and Serine (the 114th amino acid residue) of the amino acid ligand IJ (described in SEQ ID NO: 16 of the Sequence Listing) of the phenolinolanine dehydrogenase from Thermoactinomyces intermedius Ser), a modified enzyme in which the 135th amino acid residue is replaced with methionine (Met), the 282nd amino acid residue is replaced with wasparagine (Asn), and the 297th amino acid residue is replaced with porin (Pro).

[16] In the amino acid sequence of the modified enzyme according to any one of [1] to [15], 1 to several amino acids An amino acid sequence having deletion, substitution and / or addition of nonacid (provided that the amino acid to be deleted and / or substituted is threonine (Thr) serine (Ser ) And a continuous sequence of serine (Ser), and serine (Ser) linking (A) and (B) are not included) and have improved substrate specificity for methionine and have improved phenylalanin dehydrogenase activity. A modified enzyme having an amino acid sequence.

 [17] The modified enzyme according to any one of [1] to [16], wherein the activity relative to phenylalanin is 100 or less when the relative activity relative to methionine is 100.

 [18] The modified enzyme according to any one of [1] to [16], wherein the relative activity with respect to methionine is 100, and the activity with respect to leucine is S100 or less.

 [19] The modified enzyme according to any one of [1] to [16], wherein the relative activity to methionine is 100 or less when the relative activity to methionine is 100.

 [20] The modified enzyme according to any one of [1] to [16], wherein the activity is S 100 or less for any of phenylalanin, leucine and valine when the relative activity to methionine is 100.

 [21] A protein in which an oligopeptide (histidine.tag) comprising 1 to 12 histidines is fused to the N-terminal side of the enzyme according to any one of [1] to [20].

 [22] A DNA having a base sequence encoding the enzyme according to any one of [1] to [20] or the fusion protein according to claim 21.

[23] Below!

 (1) DNA having the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing, wherein the 193 to 195th ttg force gt or tgc, the 367 to 369th ggt force gt, agc, tct, tcc, tea or tcg 430-432th gtt is atg, 880-882th ctg is aat! /, Is aac, and 925-927th gta force, cct, ccc, cca

 (2) DNA having the base sequence described in SEQ ID NO: 3 in the sequence listing, wherein 196 to 198th ctg is tgt or tgc, and 370 to 372rd ggt is agt, agc, tct, tcc, tea or tcg The 433th to 435th aac is atg, the 883th to 885th cta is aat! /, The aac, and the 928th to 930th gtt force cct, ccc, ccafc (the ccg DNA,

(3) DNA having the base sequence set forth in SEQ ID NO: 5 in the sequence listing, wherein the 199th to 201st positions tta is tgt or tgc, 373 to 375th ggt force gt, agc, tct, tcc, tea or tcg, 436 to 438th gtg is atg, 877 to 879th ttg is aat or aac, and 922 to 924th Gtg force, cct, ccc, cca mei (or ccg DNA,

 (4) DNA having the base sequence described in SEQ ID NO: 7 in the sequence listing, wherein 190 to 192nd ctt is tgt or tgc, and 364 to 366th ggt is agt, agc, tct, tcc, tea or tcg DNA whose 427 to 429th gtt is atg, 877 to 879th tgg is aat or aac, and 922 to 924th gta force, cct, ccc, cca mei i ccg,

 (5) DNA having the base sequence set forth in SEQ ID NO: 9 in the sequence listing, wherein the 193 to 195th etc gt or tgc and the 367 to 369th ggc are agt, agc, tct, tcc, tea! /, Tcg, 430

˜432rd gtc is atg, 880th to 882th gtg is aat or aac, and 925th to 927th gtg force, cct, ccc, cca mei (or ccg DNA,

 (6) DNA having the base sequence set forth in SEQ ID NO: 11 in the sequence listing, wherein ttg from 190 to 192 is tgt or tgc, and ggt from 364 to 366 is agt, agc, tct, tcc, tea or tcg 42, DNA whose 7th to 429th aat is atg, 877th to 879th tta is aat or aac, and 922th to 924th gtt is cct, ccc, cca or ccg,

 (7) DNA having the base sequence set forth in SEQ ID NO: 13 in the sequence listing, the 160-162th etc. force gt or tgc, the 349-351st gga force gt, agc, tct, tcc, tea or tcg 41 DNA whose ttc from 2nd to 414th is atg, and ctg from 841 to 843th is aat or aac,

 (8) DNA having the base sequence set forth in SEQ ID NO: 15 in the sequence listing, wherein ttg at 166 to 168 is tgt or tgc, and gga at 340 to 342 is agt, agc, tct, tcc, tea or tcg 40 3rd to 405th gcc is atg, 844th to 846th tgt is aat or aac, and 889th to 891th gtg force ct, ccc ゝ ccafc (or ccg DNA ゝ

 (9) A phenylalanine dehydration having a base sequence having a deletion, substitution and / or addition of one to several bases and improved substrate specificity for methionine in the base sequences of (1) to (8) DNA having a base sequence encoding elementary enzyme,

 (10) DNA having a base sequence complementary to the DNA of (1) to (9).

[24] Below!

(11) The amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing, The 123rd amino acid is serine (Ser),

 The 309th amino acid is proline (Pro),

 The 65th amino acid is cysteine (Cys),

 The 144th amino acid force is S-methionine (Met), and

 294th amino acid is asparagine (Asn),

Any one of the following, DNA encoding an amino acid sequence satisfying three,

(12) The amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing, wherein the 124th amino acid is serine (Ser),

 The 310th amino acid is proline (Pro),

 The 66th amino acid is cystine (Cys),

 The 145th amino acid force is S-methionine (Met), and

 295th amino acid is asparagine (Asn),

Any one of the following, DNA encoding an amino acid sequence satisfying three,

(13) The amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing, wherein the 125th amino acid is serine (Ser),

 308th amino acid is proline (Pro),

 The 67th amino acid is cysteine (Cys),

 The 146th amino acid force is S-methionine (Met), and

 The 293rd amino acid is asparagine (Asn),

Any one of the following, DNA encoding an amino acid sequence satisfying three,

(14) The amino acid sequence set forth in SEQ ID NO: 8 in the sequence listing, wherein the 122nd amino acid is serine (Ser),

 308th amino acid is proline (Pro),

 The 64th amino acid is cystine (Cys),

 The 143rd amino acid force is S-methionine (Met), and

 The 293rd amino acid is asparagine (Asn),

Any one of the following, DNA encoding an amino acid sequence satisfying three,

(15) The amino acid sequence set forth in SEQ ID NO: 10 in the sequence listing, The 123rd amino acid is serine (Ser),

The 309th amino acid is proline (Pro),

The 65th amino acid is cysteine (Cys),

The 144th amino acid force is S-methionine (Met), and

294th amino acid is asparagine (Asn),

Any one of the following, DNA encoding an amino acid sequence satisfying three,

(16) the amino acid sequence set forth in SEQ ID NO: 12 in the sequence listing,

The 122nd amino acid is serine (Ser),

308th amino acid is proline (Pro),

The 64th amino acid is cystine (Cys),

The 143rd amino acid force is S-methionine (Met), and

The 293rd amino acid is asparagine (Asn),

Any one of the following, DNA encoding an amino acid sequence satisfying three,

 (17) The amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing,

The 117th amino acid is serine (Ser),

The 54th amino acid is cystine (Cys),

The 138th amino acid force is S-methionine (Met), and

281st amino acid is asparagine (Asn),

Any one of the following, DNA encoding an amino acid sequence satisfying three,

 (18) the amino acid sequence set forth in SEQ ID NO: 16 in the sequence listing,

114th amino acid is serine (Ser),

 The 297th amino acid is proline (Pro),

 The 56th amino acid is cysteine (Cys),

 The 135th amino acid force is S-methionine (Met), and

 The 282nd amino acid is asparagine (Asn),

Any one of the following, DNA encoding an amino acid sequence satisfying three,

(19) The amino acid sequence according to (11) to (18), which has an amino acid sequence having one to several amino acid deletions, substitutions and / or additions, and has improved substrate specificity for methionine. DNA having a base sequence encoding an amino acid sequence having dilauranine dehydrogenase activity, and

 (20) DNA having a base sequence complementary to the DNA of (11) to (19).

 [25] A vector comprising the DNA of [23] or [24].

 [26] The vector according to [25], further comprising DNA encoding an oligopeptide consisting of 1 to 12 histidines at either end of the DNA according to [23] or [24].

 [27] A transformant obtained by transforming a host with the vector according to [25] or [26].

 [28] [27] The transformant according to [27] is cultured, and at least three amino acids are added from the culture so as to improve the substrate specificity of phenalanin dehydrogenase (EC 1.4.1.20) to methionine. A method for preparing a modified enzyme, which comprises collecting a modified modified enzyme.

 [29] The preparation method according to [28], wherein the modified enzyme to be collected is the enzyme according to any one of [2] to [20] or the fusion protein according to [21].

 [30] A method for analyzing L-methionine contained in a test sample using the enzyme according to any one of [1] to [20] or the fusion protein according to [21].

 [31] The method according to [30], wherein at least one of L-leucine, L-isoleucine, L-valine and L-phenylalanin is analyzed in combination.

 [32] Resazurin, diaphorase and nicotinamide adenine dinucleotide

The method according to [30] or [31], comprising mixing with (NAD ⁺ ) and detecting color development.

 [33] Test sample and resazurin, diaphorase and nicotinamide adenine dinucleotide

The method according to [32], wherein the mixing with (NAD ⁺ ) is carried out in a microplate well.

 [34] The method according to any one of [30] to [33], wherein the test sample is a blood sample.

 [0012] According to the present invention, a modified enzyme of phenylalanine dehydrogenase specific to L-methionine can be provided, and the modified enzyme according to the present invention can be used for a test sample. The contained L-methionine can be analyzed by an enzyme fluorescence method using resazurin, diaphorase and NAD.

 BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a modified enzyme in which at least three amino acids have been modified to improve the substrate specificity of phenalanin dehydrogenase (EC 1.4.1.20) for methionine. In the present invention, the phenylalanine dehydrogenase (EC 1.4.1.20), which is the object of modification, is an enzyme that specifically catalyzes an oxidative deamination reaction with respect to the amino group of L-phenylalanine. . Examples of phenylalanine dehydrogenases include, for example, Bacillus badius IAM 11059, Bacillus sphaericus R79a, Sporosarcina ureae R04, Bacillus halodurans, eo bacillus kaustophilus, Oceanobacillus iheyensis, Rhodococcus sp. M_4, or Thermo actinomyces intermedius Can mention dehydrogenase

[0014] Phenylalanine dehydrogenase derived from Bacillus badius IAM11059 is an optimal enzyme for the determination of L-phenylalanine concentration in the blood of phenylketonuria with extremely high substrate specificity for L-phenylalanine. It is used as an enzyme agent for enzyme fluorescence determination using microplates in mass screening for the disease. In addition, this enzyme requires nicotinamide adenine dinucleotide (oxidized form; NAD ⁺ ) as a coenzyme. The oxidation reaction by this enzyme is reversible, and it catalyzes the reductive amination reaction in the presence of ammonium ion and nicotinamide adenine dinucleotide (reduced form; NADH) in the neutral to weakly alkaline pH range. . The nucleotide sequence and amino acid sequence of phenylalanine dehydrogenase derived from Bacillus badius IAM11059 are from Yamada A, Dairi T, Ohn ο, riuang XL and Asano Y. Nucleotide sequencing of Phenyloredafunine dehydroge nase gene from Bacillus badius IAM 11059 Biosci. Biotechnol. Biochem. 59 (10). 199 4-1995 (1995), described as SEQ ID NOS: 1 and 2.

[0015] Phenylalanine dehydrogenase (EC 1.4.1.20) derived from Bacillus sphaericus R79a specifically catalyzes oxidative deamination reaction to the amino group of L-phenylalanine or L-tyrosine It is an enzyme. This enzyme is used in the enzymatic synthesis of L-amino acids that have high substrate specificity for L-tyrosine as well as L-phenylalanine. In addition, this enzyme requires nicotinamide adenine dinucleotide (NAD ⁺ ) as a coenzyme, as in the case of the phenylalanine dehydrogenase derived from B. badius IAM11059. The oxidation reaction by this enzyme is reversible, and ammonium ion and nicotinamide adenine dinucleotide (reduced type; in the pH range from near neutral to weakly alkaline as in the case of phenylalanine dehydrogenase derived from Bacillus sbadius IAM11059) Reductive amination in the presence of (NADH) It is an enzyme that also catalyzes the reaction. The nucleotide sequence and amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a are N. Okazaki, Y. Hibino, Y. Asano, M. Ohmor i, N. Numao and. Ondo. Cloning and nucleotide sequencing of Rarefunin dehydrogenase gene of Bacillus sphaericus. Gene. 63. 337-341 (1988), described as SEQ ID NOS: 3 and 4.

 [0016] The base sequence and amino acid sequence of phenylalanin dehydrogenase derived from Sporosarcina ureae R04 are described in DDBJ / EMBL / GenBank databases (Accession No. AB001031), and are described as SEQ ID NOs: 5 and 6.

 [0017] The base sequence and amino acid coordination IJ of phenylalanine dehydrogenase derived from Bacillus halodurans are listed in GenBank Database (Accession No. NC # 002570) and NCBI Protein Da tabase (Accession No. NP # 241084). And are listed as SEQ ID NOS: 7 and 8.

 [0018] The nucleotide sequence and amino acid sequence of phenylalanine dehydrogenase derived from Geobacillus kaustophilus are described in GenBank Database (Accession No. BA000043.1) and NCBI Protein Database (Accession No. BAD76316). Listed as numbers 9 and 10.

 [0019] The nucleotide sequence and amino acid sequence of phenylalanine dehydrogenase derived from Oceanobacillus iheyensis are described in GenBank (Accession No. BA000028) and NCBI Protein Database (Accession No. BAC14834), SEQ ID NO: 11 and Described as 12.

 [0020] The base sequence and amino acid sequence of phenylalanin dehydrogenase derived from Rhodococcus sp. M_4 are described in the following literature, and are described as SEQ ID NOS: 13 and 14. Brunhuber N.., Banerjee Α ·, Jacobs vV. R. Jr., Blanchard JS and lomng, sequencin g, and expression of Rhodococcus L -Fuenore, Junin dehydrogenase. Chem., 269. 16203-16211 (1994)

[0021] The base sequence and amino acid sequence of phenylalanin dehydrogenase derived from Thermoactinomyces intermedius are described in the following literature, and are described as SEQ ID NOS: 15 and 16.

Takada Η ·, Yosnimura Τ ·, Ohshima Τ ·, J ^ saki Ν ·, Soaa Κ. Thermostable Fueninore, Junin dehydrogenase of Thermoactinomyces intermedius; cloning, expression, and s equencing of its gene. J. Biochem., 109. 371-376 (1991)

 [0022] Wild-type phenylalanin dehydrogenase has a very high substrate specificity for L-phenylalanine, and L_phenol in enzymes derived from Bacillus badius IAM 11059, Bacillus sphaericus R79a or Sporo sarcina ureae R04. When the relative activity with respect to lulanin is 100, the relative activity with respect to substrates of natural amino acids such as L-methionine, L-leucine, L-isoleucine or L-valine is 10 or less.

 [0023] In the present invention, as described above, it is a mutant enzyme in which the substrate specificity for L-methionine is remarkably improved by modifying the phenylalanine dehydrogenase having extremely low substrate specificity for L-methionine. . Specifically, it is a modified enzyme obtained by modifying at least three amino acids of wild-type phenylalanine dehydrogenase so that the substrate specificity for L-methionine is improved. For example, when the relative activity for L-methionine is 100, the relative activity for L-phenylalanine is preferably S 100 or less, more preferably 60 or less. Most preferably, it is 40 or less. Furthermore, for example, when the relative activity to L-methionine is 100, the relative activity to leucine is preferably 100 or less, more preferably 60 or less, and most preferably 40 or less. preferable. Furthermore, for example, when the relative activity with respect to L-methionine is 100, the relative activity against valine is preferably 60 or less, more preferably 40 or less. Furthermore, for example, when the relative activity with respect to L-methionine is defined as 100, the relative activity S 100 or less is preferred for any of phenylalanine, leucine and parin, preferably 60 or less. The most preferable value is 40 or less.

[0024] Modifications that improve substrate specificity for methionine specifically include a sequence of threonine (Thr) glycine (Gly) in the 114th to 125th range of the amino acid sequence of wild-type phenylalanine dehydrogenase. Substitution of glycine (Gly) with serine (Ser) in the sequence (provided that the glycine (Gly) force is in the range of S114 to 125). The position of glycine (Gly) in the contiguous sequence of threonine (Thr) glycine (Gly) in the amino acid sequence of wild-type phenylalanine dehydrogenase (glycine (Gly) is in the 114th to 125th range) is It varies depending on the origin and is in the position shown in the table below. [0025] [Table 1]

 * Position of glycine (Gly) in contiguous sequence of threonine (Thr) glycine (Gly)

 ** Sequence of wild-type enzyme

 [0026] Modifications that further improve the substrate specificity for methionine are specifically those of glutamine (G1 n) valine (Val) in the 297-310th range of the amino acid sequence of wild-type phenylalanine dehydrogenase. Substitution of valine (Val) with arginine (Arg), threonine (Thr), glycine (Gly) or proline (Pro) in a contiguous sequence (however, the palin (Val) force is in the range of ¾97 to 310). is there. The position of valine (Val) in the contiguous sequence of glutamine (Gin) and phosphorus (Val) in the amino acid sequence of wild-type phenylalanine dehydrogenase (in the range of valine (Val) forces ¾97 to 310) is It depends on the origin of the enzyme and is shown in the table below.

 [0027] [Table 2] Origin of phenylalanine dehydrogenase Val position * SEQ ID NO:

 Base sequence Amino acid sequence

Bacillus badius IAM11059 309th 1 2

 Bacillus sphaericus R79a 310th 3 4

 Sporosarcina ureae R04 308th 5 6

 Bacillus halodurans 308th 7 8

 Geobacillus ka ustophilus 309th 9 10

 Oceanobacillus iheyensis ^ 308th 11 12

Thermoactinomyces intermedins 297th 15 16 * Position of palin (Val) in the contiguous sequence of glutamine (Gin) valine (Val) ** Sequence of wild-type enzyme

 [0028] Modifications that further improve the substrate specificity for methionine specifically include isoleucine (II e) and norin (Val) in the range 135 to 146 of the amino acid sequence of wild-type phenylalanine dehydrogenase. ) / Vasparagine (Asn) and glycine (Gly) continuous sequence (however, valine (Val) / asparagine (Asn) is in the 135th to 146th range) Are alanine (Alal) and leucine (Leu)! /, Are substitutions for methionine (Met). In the amino acid sequence of wild-type phenylalanine dehydrogenase, a continuous sequence of iso-oralin (lie), norin (Val) / asparagine (Asn) and glycine (Gly) (valin (Val) / asparagine (Asn) The position of valine (Val) / asparagine (Asn) in the range 135 to 146) depends on the origin of the enzyme and is in the position shown in the table below

[0029] [Table 3]

 * Position of valine (Val) / asparagine (Asn) in contiguous sequence of isoleucine (lie), norin (Val) / asparagine (Asn) and glycine (Gly)

 ** Sequence of wild-type enzyme

[0030] The modified enzyme of the present invention is a modified enzyme obtained by modifying at least three amino acids of wild-type phenylalanin dehydrogenase so that the substrate specificity for L-methionine is improved. Is preferably an amino acid shown in Tables 1 to 3 above. Tables 4 and 5 below further describe the amino acids of the wild type phenylalanine dehydrogenase to be modified. These amino acids may not require modification, depending on the origin of wild-type phenylalanine dehydrogenase. Tables 4 and 5 show the origins of wild-type phenylalanine dehydrogenases that are preferably modified.

 [0031] Modifications that further improve the substrate specificity for methionine specifically include glycine (Gly), isoleucine (lie), and leucine (lie) in the 281 to 295th range of the amino acid sequence of wild-type phenylalanine dehydrogenase. A series of leucine (Leu) / tryptophan (Trp) / valine (Val) / cysteine (Cys), tyrosine (Tyr) / phenylalanine (Phe), alanine (Ala), proline (Peo) and aspartic acid (Asp) Leucine (Leu) / tryptophan (Trp) / valine (Val) in the above sequence (however, leucine (Leu) / tryptophan (Trp) / valine (Val) / cystine (Cys) is in the range of 281 to 295) / Substitution of cystine (Cys) to asparagine (Asn). Glycine (Gly), isoleucine (lie), mouth isine (Leu) / tryptophan (Trp) / valine (Val) / cystine (Cys), tyrosine (Tyr) / in the amino acid sequence of wild-type phenylalanine dehydrogenase Continuous arrangement of phenylalanine (Phe), alanine (Ala), proline (Peo) and aspartate (Asp) IJ (leucine (Leu) / tryptophan (Trp) / valine (Val) / cystine (Cys) The position of leucine (Leu) in the 281 to 295th range depends on the origin of the enzyme and is in the position shown in the table below.

 [0032] [Table 4]

* Glycine (Gly), Isoleucine (lie), Leucine (Leu) / Tryptophan (Trp) / Valine (Val) / Cystin (Cys), Tyrosine (Tyr) / Phenyralalanin (Phe), Alanine (Ala), Proline Position of leucine (Leu) in the contiguous sequence of (Peo) and aspartic acid (Asp) ** Sequence of wild-type enzyme

 [0033] Modifications that improve the substrate specificity for methionine are specifically a sequence of alanine (Ala) and leucine (Leu) in the 54th to 67th amino acid sequence of wild-type phenylalanine dehydrogenase. Substitution of leucine (Leu) with cysteine in the sequence (provided that the leucine (Leu) is in the 54th to 67th range). The position of leucine (Leu) in the contiguous sequence of alanine (Ala) and leucine (Leu) in the amino acid sequence of wild type phenylalanine dehydrogenase (leucine (Leu) is in the 54th to 67th range) is the enzyme Depending on the origin, it is in the position shown in the table below.

 [0034] [Table 5]

 * Position of leucine (Leu) in consecutive sequences of alanine (Ala) and leucine (Leu) ** Sequence of wild-type enzyme

 [0035] Furthermore, the present invention provides a peptide having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence of the modified enzyme of the present invention, and improved substrate specificity for methionine. It includes a modified enzyme having an amino acid sequence having dilauranine dehydrogenase activity. However, the amino acid to be deleted and / or substituted does not include serine (Ser) having a continuous sequence of threonine (Thr) serine (Ser) having serine (Ser) in the 114th to 125th range. The modified enzyme further having substitution, insertion or deletion has improved substrate specificity for methionine as compared to the wild-type enzyme, and the improvement of substrate specificity is the same as described above.

[0036] That is, for example, when the relative activity with respect to L-methionine is defined as 100, Relative activity power with respect to hydrogen is preferably 60 or less, more preferably 40 or less. Furthermore, for example, when the relative activity with respect to L-methionine is 100, the relative activity with respect to leucine S is preferably 100 or less, more preferably 60 or less, and most preferably 40 or less. preferable. Furthermore, for example, when the relative activity with respect to L-methionine is 100, the relative activity with respect to palin is S 100 or less, preferably S, and more preferably 60 or less, most preferably 40 or less. Preferred. Furthermore, for example, when the relative activity to L-methionine is 100, the relative activity S 100 or less is preferred for any of phenylalanine, leucine and valine, preferably 60 or less. The most preferable is 40 or less.

[0037] The range of "1 to several" in "base sequence IJ having deletion, substitution and / or addition of one to several bases" as used herein is not particularly limited. Means from 1 to 40, preferably from 1 to 30, more preferably from 1 to 20, more preferably from 1 to 10, even more preferably from 1 to 5, particularly preferably from 1 to 3. The same applies to the following

[0038] In the following, as specific examples (shown in Examples) of the modified enzymes of the present invention, Bacillus sphaericus R79a-derived enzyme is taken as an example, and the relative activities to phenylalanin when the relative activity to methionine is taken as 100 are listed. Shown below.

[0039] [Table 6]

Mutant enzyme Content P e

BS124S145M66C29 124th Serine, 145th Methien 93

5N (Met), 66th to Siswo-In (Cys), 295

 Replace eyes with asparagine (Asn)

 BS124S145M66C29 124th serine, 145th methionine 57

5N310R (Met), 66th to Siswo-In (Cys), No.295

 Eye Asparagine (Asn), 310th Argu

(Ar _g )

 BS124S145M66C29 124th for serine (Ser), 145th for metionin 43

5N310G (Met), 66th to Siswo-In (Cys), 295

 Eye Asparagine (Asn), 310th Glycine

 Replace with (Gly)

 BS124S145M66C29 124th serine, 145th metionin 20

5N310P (Met), 66th to Siswo-In (Cys), 295

 Eye Asno, "Lagin (Asn), 310th Proline

 Replace with (Pro)

 BS124S145L310T 124th S for Serine, 145th S for Leucine 33

 (Leu) replaces 310 with threonine (Thr)

[0040] The method for obtaining the modified enzyme (protein) of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis! / Or a recombinant protein produced by a gene recombination technique. When producing a modified enzyme (recombinant protein), first, as will be described later, a gene (DNA) encoding the modified enzyme (protein) is obtained. By introducing this DNA into an appropriate expression system, the modified enzyme of the present invention can be produced. The expression of the protein in the expression system will be described later in this specification.

 [0041] The method for obtaining the DNA of the present invention is not particularly limited. Appropriate probes and primers are prepared based on the amino acid sequence and base sequence information described in SEQ ID NOs: 1 to 16 in the sequence listing in the present specification, and the above-described phenylalanine dehydrogenase is used using them. It is possible to isolate the gene of the present invention by screening a cDNA library of the bacteria containing it. A cDNA library can be prepared by a conventional method from a bacterium expressing the gene of the present invention.

[0042] The gene of the present invention can also be obtained by PCR. Using the DNA library or cDNA library derived from the above bacteria as a saddle type, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 1 Perform PCR using a pair of primers designed to amplify the nucleotide sequence described in 3 or 15. PCR reaction conditions can be set as appropriate. For example, a reaction system consisting of 94 ° C for 30 seconds (denaturation), 55 ° C for 30 seconds to 1 minute (ayling), and 72 ° C for 2 minutes (extension). One cycle, for example, after 30 cycles, can be listed as a condition of reacting at 72 ° C for 7 minutes. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

[0043] Operations such as the preparation of the probe or primer, construction of the cDNA library, screening of the cDNA library, and cloning of the gene of interest are well known to those skilled in the art. For example, Molecular Cloning: A laboratory Mannual , 2 ⁿ Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as Molecular Cloning), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (Hereinafter, abbreviated as “Current Protocols” in “Molecular” “Biology”) and the like.

 [0044] Further, in the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 in the sequence listing! /, Deletion, substitution and / or deletion of 1 to several amino acids A gene having an amino acid sequence having an addition and encoding a protein having glycosylation enzyme activity; and deletion, substitution and / or deletion of 1 to several bases in the base sequence described in SEQ ID NO: 5 or 6 in the sequence listing Alternatively, for a gene having a base sequence having an addition and having a base sequence encoding a glycosylase (hereinafter, these genes are referred to as mutant genes), the amino acid sequences described in SEQ ID NOs: 1 to 16; Based on the information of the base sequence, it can be prepared by any method known to those skilled in the art, such as chemical synthesis, genetic engineering, or mutagenesis.

[0045] For example, a method in which a DNA having the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 in the sequence listing is brought into contact with a mutagen agent, or irradiated with ultraviolet rays. This method can be carried out using methods such as genetic engineering techniques. Site-directed mutagenesis, which is one of the genetic engineering techniques, is a technique that can introduce a specific mutation at a specific position, and is useful. Molecular cloning ^2nd edition, Current Protocols It can be performed according to the method described in “Molekyura Ichi” biology and the like.

[0046] [Vector of the present invention] The present invention relates to a vector containing any of the DNAs of the present invention. There are no particular restrictions on the vector carrying any of the DNAs of the present invention, but it may be, for example, a self-replicating vector (such as a plasmid), or it may be introduced into the host cell genome when introduced into the host cell. It may be integrated and replicated with the integrated chromosome. Preferably, the vector used in the present invention is an expression vector. In the expression vector, the gene of the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like). A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected according to the type of host.

[0047] Promoters operable in bacterial cells include Bacillus stearothermophilus maltogenic amylase ge, Bacillus stearothermophilus maltogenic amylase ge ne, Bacillus stearothermophilus maltogenic amylase ge ne, Bacillus licheniformis alpha—amylas e gene), Bacillus amyloliquef aciens BAN amylase gene, Bacillus 'Subtilis alkaline protease gene' or Notinoles 'pminores' xylosidase gene (Bacillus pumilus xylosldase gene) promoter or phage 'lambda P

 R

 P promoter, Escherichia coli lac, trp or tac promoter.

 L

[0048] Examples of promoters that can operate in mammalian cells include the SV40 promoter, the MT-1 (metamouth thionein gene) promoter, or the adenovirus 2 major late promoter. Examples of promoters that can operate in insect cells include the polyhedrin promoter, P10 promoter, autographer 'California 2' polyhedrosis basic protein promoter, Bakiurovirus immediate early gene 1 promoter, or Bakiurovirus 39K. There are delayed early gene promoters. Examples of a promoter operable in a yeast host cell include a promoter derived from a yeast glycolytic gene, an alcohol dehydrogenase gene promoter, a TPI1 promoter, an ADH2-4c promoter, and the like. Examples of promoters which are operative in filamentous fungal cells is the force ^s like ADH3 promoter or tpiA promoter.

[0049] Furthermore, the DNA of the present invention may be functionally bound to an appropriate terminator as necessary. The recombinant vector of the present invention further comprises a polyadenylation signal (eg SV4 0 or derived from the adenovirus 5Elb region), or a transcription enhancer sequence (eg, SV40 enhancer)! /, May!

 The recombinant vector of the present invention may further comprise a DNA sequence that allows the vector to replicate in the host cell, an example of which is the SV40 origin of replication (when the host cell is a mammalian cell). It is done.

 [0050] The recombinant vector of the present invention may further contain a selection marker. Selectable markers include, for example, dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI genes whose complement is lacking in the host cell, such as ampicillin, kanamycin, tetracycline, chloramphenicol And drug resistance genes such as neomycin or hygromycin. Methods for ligating the gene, promoter, and, if desired, terminator and / or secretory signal sequence of the present invention and inserting them into an appropriate vector are well known to those skilled in the art.

 [0051] [Production of transformant of the present invention and modified enzyme using the same]

 A transformant can be prepared by introducing the DNA (gene) or recombinant vector of the present invention into an appropriate host. Examples of host cells into which the gene or recombinant vector of the present invention is introduced include bacteria, yeasts, fungi, and higher eukaryotic cells that can be used in any cell as long as the gene of the present invention can be expressed.

 [0052] Examples of bacterial cells include Gram-positive bacteria such as Bacillus or Streptomyces, or Gram-negative bacteria such as Escherichia coli. Transformation of these bacteria may be carried out by using a competent cell by a protoplast method or a known method.

 Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells or CHO cells. Methods for transforming mammalian cells and expressing the DNA sequences introduced into the cells are also known, and for example, the electopore method, the calcium phosphate method, the lipofuxion method and the like can be used.

[0053] Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, for example, Saccharomyces cerevislae or Saccharomyces kluyveri. Examples of methods for introducing a recombinant vector into a yeast host include the electoral position method and the Examples thereof include a blast method and a lithium acetate method.

 [0054] Examples of other fungal cells are cells belonging to filamentous fungi, such as Aspergillus, Neurospora, Fusarium, or Trichoderma. When filamentous fungi are used as host cells, transformation can be achieved by integrating the DNA construct into the host chromosome to obtain recombinant host cells. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination.

 [0055] When an insect cell is used as a host, a recombinant gene transfer vector and a baculovirus are co-introduced into the insect cell to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further used. Can infect insect cells and express proteins (eg, Baculovirus Expression Vectors, A Laboratory Manual; and Current 'Protocols' in' molecular. Biology, Bio / Technology, 6, 47 (1988), etc.) )

Yes

 [0056] As a baculovirus, for example, Autographa californica nuclear polyhedrosis virus can be used, such as Autographa californica nuclear polyhedrosis virus. .

Insect cells include Spodoptera frugiperda's ovarian cells Sf9, Sf21 (Baculo Inores 'Expression' Vectors, Laboratories, Manuel Yuanole, W. H. Freeman and Kannon Niichi ( _w . Company, New York, (1992)], and using HiFive (manufactured by Invitrogen), which is an ovary cell of Trichoplusia ni.

 As a method for co-introducing a recombinant gene introduction vector into insect cells and the above baculovirus for preparing a recombinant virus, for example, a calcium phosphate method or a lipofusion method can be mentioned.

[0057] More specifically, for example, it was amplified by PCR using a site-specific mutation primer with a phenylalanine dehydrogenase gene (pdh) derived from Bacillus badius IAM11059 or Bacillus sphaericus R79a as a saddle type. A site-directed mutagenesis and / or its complementary sequence is prepared, and this site-specific mutagenesis and / or its complementary strand is transformed into, for example, Escherichia coli or other recombination-capable lodging. It can be expressed in main cells (eg, animal cells, plant cells, insect cells, etc.) to obtain the modified enzyme of the present invention.

 [0058] In the present invention, the transformant of the present invention is cultured, and at least three amino acids are modified from the culture so as to improve the substrate specificity of phenylalanine dehydrogenase (EC 1.4 to 20) to methionine. The modified enzyme preparation method for collecting the modified enzyme is included.

 [0059] The transformant is cultured in an appropriate nutrient medium under conditions that allow expression of the introduced DNA. In order to isolate and purify the modified enzyme of the present invention from the culture of the transformant, a usual method for isolating and purifying the modified enzyme may be used.

 For example, when the modified enzyme of the present invention is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then disrupted by an ultrasonic disrupter or the like. A cell-free extract is obtained. From the supernatant obtained by centrifuging the cell-free extract, a normal protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as Jetylaminoethyl (DEAE) Sepharose, Cation exchange chromatography using a resin such as SS sign harose FF (manufactured by Pharmacia), Butyl Sepharose, Phenyl Sepharose There are independent methods such as hydrophobic chromatography using resins such as resins, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing! Use a combination of / and S to obtain a purified sample.

 [0060] [L-methionine analysis method]

 The present invention relates to a method for analyzing L-methionine contained in a test sample using the modified enzyme or protein of the present invention.

[0061] Specifically, in the L-methionine analysis method of the present invention, a test sample is mixed with resazurin, diphorase, and nicotinamide adenine dinucleotide (NAD ⁺ ) to detect color development, preferably fluorescence color development. Including that. Mixing the test sample with resazurin, diaphorase and nicotinamide adenine dinucleotide (NAD ⁺ ) can be performed, for example, in a microplate well.

[0062] Further, the L-methionine analysis method of the present invention comprises (1) a test sample and nicotinamide adenine dinu Mixing leotide (NAD ⁺ ) and measuring the increase in absorbance of reduced NADH, (2) Test sample and nicotinamide adenine dinucleotide (NAD ⁺ ) and phenazine methosulfate (PMS), etc. The test sample is mixed with a reducing chromogenic reagent, INT (INT), using the electronic carrier as described above, and formazan-generated color is detected, or (3) the test sample and nicotinamide adenine dinucleotide (NAD ⁺ ) are detected. Metal ions (such as Co ³⁺ ) are reduced through an electronic carrier such as 1-methoxy-phenazine methosulfate (1-Methox y PMS), and reacted with 5-Br-PAPS, which is a chelate indicator, It can also be carried out by a method such as measuring and detecting the color absorption value. In the methods (1) to (3) above, the reagent can be mixed in the well of the microplate.

[0063] In the present invention, the test sample can be, for example, a blood sample.

[0064] In the present invention, in parallel with the L-methionine analysis, at least one of L-leucine, L-isoleucine, L-noline and L-phenylalanine may be analyzed together. it can. This is because the modified enzyme or protein of the present invention used in the method of the present invention is not only L-methionine but also at least one of L-leucine, L-isoleucine, L-valine and L-phenylalanine. This is because it has substrate specificity.

[0065] Examples

 Example 1

 [Molecular modeling of phenylalanine dehydrogenase]

Modeling the homology of proteins using the integrated computational chemistry system program (MOE) manufactured by CCG, Canada, using the amino acid sequence of Swiss-Protein Accession No. P23307 as the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a. went. Homologous proteins were searched and several amino acid dehydrogenase proteins were extracted. Multiple sequence alignment was performed based on the amino acid sequence obtained above. Preserving the structure of the leucine dehydrogenase derived from Bacillus sphaericus (PDB accession No. 1LEH, homology: 47.2%) that is the most homologous value among amino acid dehydrogenases with known three-dimensional structures. The region analysis was carried out using the same program as above, and the predicted three-dimensional structure of the phenylalanine dehydrogenase from Bacillus sphaericus R79a was constructed. Based on the AMBER '89 / '94, CHARMM22, and Engh-Huber force field theories included in the MOE program The three-dimensional structure was optimized by calculating energy minimization.

 [0066] [Coordinate extraction and transcription of coenzyme NAD]

Crystal structure analysis of phenylalanine dehydrogenase has been reported for an enzyme derived from Rhodococcus sp. M4 (Biochemistry, 38. 2326-2339 (1999); Non-patent document 3, Biochemistry, 39. 9174-9187 (2000). Non-patent document 4). Rhodococcus sp. M4 derived phenylalanine dehydrogenase has been analyzed for the three-dimensional structure of the complex of enzyme protein, coenzyme (NAD ⁺ ) and reaction product (phenylrubivic acid) (PDB accession). No. 1 BW9). The coordinates of NAD ⁺ were extracted from the PDB data and transferred to the predicted three-dimensional structure data. As described in the above reference, the amino acid residue that interacts with NAD ⁺ and protein is searched for alignment with the amino acid sequence of phenylalanin dehydrogenase derived from B. sphaericus. Valine (Val), 214th aspartic acid (Asp), 254th alanine (Ala) and 277th asparagine (Asn) were extracted. Using MOE program, said amino acid residue and NAD ⁺ is multiplied by constraining the distance between atoms interacting performs energy minimization calculations using the same as above program, you to predict conformation! /, Te NA D ⁺ Derived the most stable spatial coordinates.

 [0067] [Coordinate extraction and transcription of substrate L-phenylalanine]

In the same manner as NAD ⁺ , the coordinates of the substrate L-phenylalanine were extracted from the three-dimensional structure coordinates (PDB accession No. 1C1D) of Rhodococcus sp. M4, and B. sphaericus R79a-derived phenylhydrogenan dehydrogenation constructed above was constructed. Transcribed onto the coordinates of the predicted 3D structure data of the enzyme. According to the same procedure as above, the amino acid residues that the enzyme protein and L-Phe interact with are 53th glycine, 79th lysine, 91st lysine, 126th aspartic acid and substrate L-phenylalanine We performed energy minimization calculations with constraints on the distance, and derived the spatial coordinates where L-Phe is most stable in the predicted 3D structure.

 [0068] [Extraction of mutant amino acid residue candidates]

D sphaericus derived felanulalanin dehydrogenase protein constructed as described above. NA D ⁺ 'L-phenylalanine complex predicted 3D structure is output in PDB data format, and molecular model display software (PyMOL, DeLano Scientific LLC, South San Francisco, CA, USA). Substrate (L-phenylalanine) ligand and protein surface can be contacted The amino acid residues were extracted while confirming the drawn three-dimensional structure. Furthermore, the amino acid residue that Rhodococcus sp. M4 derived phenylalanine dehydrogenase (Non-patent Documents 1 and 2) interacts with the substrate (L-phenylalanine) is changed to the amino acid residue of B. sphaericus derived phenylalanine dehydrogenase. The amino acid sequence was extracted from the no acid sequence. By the above operation, a total of 28 amino acid residues were selected as candidates. Selected amino acid residues are shown in Table 7 below.

[Table 7]

Table 7 Amino acid residues and plies = m = _L

 Ma HX PT

 Amino acids

 SEQ ID NO: amino acid. Lymer

 Residue number

 21 51 Leu 5 '-ctaggccccgctnnsggtggaactcgc-3'

 22 52 Gly 5 '-cgccccgctttannsggaactcgcatg-3'

 23 53 Gly 5 '-cccgctttaggtnnsactcgcatgtatccc-3'

 24 66 Leu 5 '-gtggatgaagctnnsgaagatgtgcttcgc-3'

 25 76 Met 5 '-cgcctgtcagaaggannsacgtataaatgcgcag'cc-3'

26 79 Lys 5 '-gaatgacgtatnnntgcgcagccgcc-3'

 27 91 Lys 5 '-ttc ggcggcgggnnsgcggtcattatc-3'

 28 122 Tyr 5 '-aatggacgatttnnsacaggtactgac-3'

 29 123 Thr 5 '-ggacgattttacnnsggtactgacatg-3'

 30 124 Gly 5 '-cgattttacacannnactgacatgggg-3'

 31 125 Thr 5 '-ttttacacaggtnnsgacatggggacc- 3'

 32 126 Asp 5 '-tacacaggtactnnsatggggaccacg-3'

 33 127 Met 5 'acaggtactgacnnsgggaccacgatg-3'

 34 143 Phe 5 '-gagacgaatnnsattaacggaattcctgag-3'

 35 145 Asn 5 '-gagacgaatttcattnnsggaattcctgag-3'

 36 146 Gly 5 '-aatttcattaacnnsattcctgagcag-3'

 37 153 Gly 5 '-gagcagtatggtnnsagcggcgactcg-3'

 38 157 Ser 5 '-ggaagcggcgacnnstcgattccgacc-3'

 39 172 Thr 5 '-ctgaaggctnnsaaccagtatttatttggaagc-3'

40 277 Asn 5 '-gttgtgggatcagccnnsaaccagctcaaagac-3 ¹

41 295 Leu 5 '-gaaaagggaattnnstatgcacccgattatatc-3'

42 306 Gly 5 '-cgtcaatgccggcnnsttgatccaggttg-3'

 43 307 Leu 5 '-gtcaatgccggcggcnnsatccaggttgctgac-3'

44 309 Gin 5 '-gccggcggcttgatcnnsgttgctgacgaactt-3'

45 310 Val 5 '-ggcttgatccagnnsgctgacgaactttatggg- 3'

46 311 Ala 5 '-ggcttgatccaggttnnsgacgaactttatggg-3'

47 322 Val 5 '-aataaagagcggnnsttgctcaaaacg-3'

48 346 Thr 5 '-cttgactgcatcnnsacagtggaggcc-3' [0070] (1; Preparation of the p-type pdh gene)

 Escherichia coli JM109 / pPD H-DBL (gene, vol. 63. 337-341 (Escherichia coli JM109 / pPD H-DBL) incorporating the phenylalanine dehydrogenase gene (pdh) derived from Bacillus sphaericus R79a and its complementary sequence. 1988)), plasmid DNA (pPD H-DBL) was extracted by the alkaline SDS method. The pdh gene sequence and amino acid sequence derived from Bacillus sphaericus R79a are shown in SEQ ID NOs: 3 and 4, respectively. RNase solution (manufactured by SIGMA) adjusted to 5 mg / ml was added to 0.1 ml of the obtained plasmid DNA for treatment. The treated reaction solution was treated with phenol / chloroform according to a conventional method. The treated reaction solution was subjected to a polyethylene glycol precipitation treatment to obtain a purified vertical plasmid DNA solution (pPDH-DBL).

 [0071] [Screening by saturation mutation]

 For the 28 amino acid residue candidates, primers were prepared to saturate 20 amino acids each (see Table 1), and QuikChange (Multi Site-Directed Mutagenesis Kit manufactured by Stratagene) was used according to the company's protocol. Mutations were introduced by the PCR method In this example, PCR was performed using the primer combinations shown in Table 8 and the B. sphaericus R7 9a-derived phenylalanine dehydrogenase gene as a saddle type. The reaction solution was transformed into E. coli JM109 according to a standard method, applied to an LB agar medium containing 50 (g / ml ampicillin), and cultured at 37 ° C for 10 hours. The resulting colonies were 96-well deep plate (NUNC, 80 (LB medium containing g / ml ampicillin; 300 (L) was inoculated and cultured at 1000 rpm at 37 ° C for 12 to 18 hours.

[0072] [Table 8]

Table 8 Mutagenesis primer combinations Primer SEQ ID NO

 Set (1) 2 8, 3 5, 41

 Set (2) 3 0, 3 3, 36, 3 8

 Set (3) 2 4, 3 5, 41, 4 2

 Set (4) 2 3, 2 9, 37, 3 8, 40

 Set (5) 2 3, 2 4, 35, 3 7, 46

 Set (6) 2 2, 2 4, 34, 3 5, 41

 Set (7) 3 1, 3, 9, 45, 4 • 7, 48

 Set 1, (8) 2 4, 3 0, 35, 4 1, 45 [Primary Screening-Formazan Colorimetric Method]

 The transformed E. coli JM109 colonies were inoculated into a 96-well deep well plate that had been pre-dispensed with 300 L of LB medium containing 50 µg / ml ampicillin at 37 ° C per minute. , Shaking culture at 000 rpm for 16 hours or more. 150 L of the obtained culture broth was collected into a 96-well round bottom microphone mouthplate and collected by centrifugation. The centrifugation supernatant was removed by decantation. Add 1011 L of lysozyme solution (0.1 M phosphate buffer containing 10 mM EDTA, adjusted to pH 7.0 to a concentration of 10 mg / ml) and suspend it at 37 ° Hold at C for 1 hour. The treated plate was frozen in a deep freezer at -80 ° C for 30 minutes, and then freeze-thawed for 1 hour in a 37 ° C incubator was repeated twice.

 Dispense 100 μL of 10 mM phosphate buffer containing 5 mM magnesium chloride, pH 7.0, precooled into each well of the treated plate, and stir for 10 minutes on a shaker to extract the cell-free extract. Went. The supernatant obtained by centrifugation was used as the cell-free extract for the primary screening.

Formazan colorimetric method was used for the primary screening of enzyme activity. That is, 10 L of the cell-free extract obtained above was dispensed into each well of a 384-well plate, and 20 μL of reaction solution (0.1 μGlycine-KCH OH buffer, pH 10.4, 2.5 mM NAD, 0.1 mM) 1-Methox By adding y-PMS (or PMS), 0.1 mM INT and 10 mM substrate (phenylalanine, leucine or methionine)), react at 37 ° C and visually observe the red purple color. Judged. In the primary screening, recombinants with good color development on the methionine substrate were selected. As a result, 5 strains could be obtained (see Table 9 (Bacillus sphaericus R79a-derived enzyme)).

[Table 9]

[Secondary screening-NAD absorption-]

The recombinant obtained in the first screening was added to 3 mL LB test tube medium (50 g / ml (Including ampicillin) was cultured with shaking at 37 ° C for 12 hours, and the cells were disrupted by ultrasonic disruption. The cell-free extract obtained by centrifugation was used for rate assessment by a double beam spectrophotometer. The reaction volume was 1 ml, and the enzyme activity was calculated from the increase in NAD absorbance at 340 nm. The enzyme activities of L-phenylalanine, L-methionine, and L-leucine were measured, and the relative activities when the enzyme activity for L-phenylalanine was 100 were calculated as L-methionine and L- Calculated for leucine! / (See Table 9). As a result, the strains with improved substrate specificity for methionine were five strains of mutant enzyme names BS1 24S145M66C295N, BS124S145M66C295N310R, BS124S145M66C295N310G, BS12 4S145M66C295N310P and BS124S145L310T. Among the five strains, BS124S145 M66C295N310P was a high specific activity strain with good substrate specificity for L-methionine.

[0076] [Preparation of Mutant Phenylalanine Dehydrogenase]

 LB liquid medium containing 50 μg / ml ampicillin of host E. coli (Escheric hia coli JM109) introduced with a plasmid containing a mutant enzyme gene modified from L-methionine reaction of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a After culturing at 37 ° C for 12 hours, isopropyl-β-D-thiogalatatoviranoside (IPTG) was added to a final concentration of 0.5 mM, followed by further culturing at 30 ° C for 12 hours. The culture broth was centrifuged (8,000 × g, 10 minutes, 4 ° C.), and the cells were collected and washed with physiological saline. The washed cells were stored at -30 ° C until use.

 [0077] 5 mM volume of 20 mM Tris-HCl buffer (pH 8.0, 0.3 M NaCl, 20 mM)

It was suspended in imidazole and 5 mM 2-mercaptoethanol) and crushed with an ultrasonic crusher (UBOTA INSONATOR model 201M, manufactured by Kubota) for 20 minutes. The supernatant (cell-free extract) was obtained by centrifugation (28,400 X g, 20 minutes, 4 ° C). The cell-free extract was added to a metal chelate affinity resin (Chelating-S mark ha rose FF: 2 mL) pre-filled with nickel and equilibrated with the same buffer as above. After washing the resin with the same buffer containing 50 mM imidazole, the active fraction was eluted with the same buffer containing 500 mM imidazole. The obtained active fraction was dialyzed with 20 mM Tris-HCl buffer (pH 8.0, containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol) to prepare an enzyme preparation. [0078] [Substrate specificity]

Measurement of the activity of phenylalanine dehydrogenase was performed using a double beam spectrophotometer (PharmaSpec UV_1700, manufactured by Shimadzu Corporation) according to the method of Asano et al. (Eur. J. Biochem. (1987) 168 (1), 153-159). Measured with a PMMA cuvette (BRAND) with an optical path length of 1 cm. The composition of the reaction solution was as follows. 0.1 ml of 1 M Glycine-NaCl-NaOH buffer, pH 10.4, 0.1 ml of 25 mM NAD ⁺ (Oriental Yeast) solution, 0.1 M L-phenylalanine (Nippon Rika Chemicals) or L-leucine ( 0.1 ml of an aqueous solution such as Nippon Rika Chemicals Co., Ltd.) or L-methionine (manufactured by Nippon Rika Chemicals Co., Ltd.) (the substrate specificity varies depending on the nature of the enzyme, so select an appropriate substrate each time) To make the total reaction volume 1.0 ml. Coenzymes NAD ⁺ molecular extinction coefficient of the (() is 6,220 ¹ · mol- 1 'cm- 1 and then, the absorbance change rate per unit time from the increase in absorbance (min) in 340 nm ((340 nm / min ) In the present invention, 1 unit (U) of enzyme activity was defined as the amount of enzyme that produces 1 mol of NADH per minute. Specific activity (UI mg) is the enzyme per 1 mg of protein. Defined as activity (U).

 [0079] [Substrate Specificity of Five-point Mutant Phenylalanine Dehydrogenase]

B. sphaericus derived PheDH derived amino acid substitution residue of the mutant enzyme substituted with 5 amino acid residues was extracted from the predicted three-dimensional structure constructed by MOE, and the conformation (red! /, Amino acid residue displayed by Ball & stick) Figure 1 shows the basis. Predicted enzyme of phenylalanine dehydrogenase Saturation mutation of amino acid residues extracted from protein, substrate, NAD ⁺ complex three-dimensional structure results in activity against phenylalanine and leucine (when measuring 10 mM concentration) Mutants that were suppressed compared to S-methionine were screened. As a result, Gly_124, Asn-145, Val-310, Leu-295 and Leu-66 were found to have 5 amino acid substitutions (BS12 4S145M66C295N310P) _o Gly_124, Asn-145 and Va 卜 310 3 residues Conforms near the benzene ring of the substrate phenylalanine! /. Leu-295 is located away from the substrate, and the predicted interaction with the substrate appears to be related to NAD ⁺ rather than the substrate. At this time, the side chain of Leu-295 was exposed on the subunit surface. On the other hand, the Leu-66 residue is located on the molecular surface of the N-terminal domain of the subunit, and direct interaction with the substrate and NAD ⁺ is unlikely. [0080] Table 10 shows the substrate specificity of the enzyme for aromatic amino acids and aliphatic amino acids. This mutant enzyme catalyzed the best oxidative deamination reaction for L-norleucine. Subsequently, it reacted with L-methionine, and L-phenylalanine, L-leucine, and L-valine were suppressed to 50% or less when L-methionine was used as a reference. When quantifying amino acids in blood samples, there is almost no L-norleucine or L-force revalin, so there is no effect on fluorescence quantification by enzymatic reactions. That is, if branched-chain amino acids (L-leucine, L-isoleucine and L-valine) in blood samples can be appropriately treated, fluorescence quantification using this enzyme can be performed for L-methionine in filter paper blood. I can expect that.

 [0081] [Table 10]

 Substrate specificity of BS124S145M66C295N310P

 Activity

 Substrate Relative activity (%)

 (U / ral)

 L-Fennino Realan 0.27 20

 L-tyrosine 0

 L-Leucine 0.667 50

 L-Solo Ishin 0. 77 57

 L—Nonoleleucine 2. 06 152

 L-Valine 0. 39 29

 L—Norenoline 0. 89 66

 L-methionine 1. 35 100

L-Ethionine 0.776 56 The kinetic parameters of this enzyme against L-methionine and NAD ⁺ were examined (Table 11). The K value for L-methionine of this enzyme is 0.33 soil 0.014 mM.

 m

 The V straight of 1.73 soil was 0.18 U / mg. The molecular weight of this enzyme is 45,00 max according to SDS-PAGE.

0, 45, 150 calculated from the deduced amino acid sequence, and a molecular weight of approximately 400,000 calculated by gel filtration, suggesting that a homo-octamer structure is formed. Therefore, k of this enzyme is 1.4 S 0.15 s- ^{1 and} k for L-methionine

 cat

/ Became 4.1 soil 0.38 s— ¹ mM— ¹ . Meanwhile, NAD + vs cat m

The K value was 0.16 soil 0.012 mM when the L-methionine concentration was fixed at 2 mM. The K value for NAD ⁺ of the wild-type enzyme from B. sphaericus is determined using L-phenylalanine as the substrate and m

Under the fixed 1 mM concentration condition, 0.24 mM, B. badius-derived wild-type enzyme has a K value of 0.

 m

15 mM. The K value for NAD ⁺ of this mutant enzyme is m from PheDH from B. sphaericus

On the other hand, PheDH derived from に 対 す る · badius had a value close to that of NAD ⁺ . NAD ⁺ to m

On the other hand, the improvement in K value is thought to be due to the amino acid substitution of Leu-295.

 m

 [Table 11]

BS 124S 145M66C295N310P power energetics. Laminarity Substrate K mM) ¹ ) No C (s— 'mM') ¹ )

L-Fuel Layun 2.5 ± 0 16 0.27 ± 0.0043 0.11 ± 0

L-Leucine 0.045 ± 0.0008 1.0 ± 0.0082 22 ± 0.31

L-isoleucine 0.22 ± 0 ・ 021 0.67 ± 0.012 3.1 ± 0.26

L-norleucine 0.24 ± 0.0166 1.8 ± 0.030 7.4 ± 0.51

L-methionine 0.33 ± 0.014 1.4 ± 0.15 4.1 ± 0.38

L-ethionine 0.79 ± 0, 14 0.73 ± 0.017 0.95 ± 0.14

L-nonorenoline 0.34 ± 0.0044 0.74 ± 0.012 2.2 ± 0.0059

L-valine 0.75 ± 0.13 0.32 ± 0.023 0.43 ± 0.046 Example 2

 [Amino acid fluorescence determination of mutant enzyme]

Dispense 0.04 ml of L-phenylalanine and L-methionine at known concentrations into a 96-well black microplate well, add 0.08 ml of 50 mM Tris-HCl buffer (pH 8.9) containing 40 M resazurin, and stir Then add 0.04 ml of enzyme mixture solution (containing 10mU mutant type of ferroalanine dehydrogenase (BS124S145M66C295N310P), 4mM β _NAD +, 0.03mg / ml diaphorase (made by Oriental Yeast)) at room temperature. Incubated for hours. The obtained fluorescence was measured with a filter having an excitation wavelength of 545 nm and a fluorescence wavelength of 590 nm using a fluorescence / absorption / emission microplate reader (Genos, manufactured by Tecan). The obtained data was analyzed using LS-PATE manager2001 (manufactured by Wako Pure Chemical Industries, Ltd.). A calibration curve was created from the analysis results and shown in Figure 2. Furthermore, quantitative results were obtained from this calibration curve (Table 12). From the calibration curve created using BS124 S145M66C295N310P, the L-methionine concentration contained in the control could be quantified. At this time, it was difficult to quantify L-methionine due to the influence of L-phenylalanine concentration with conventional modified enzymes BS124S145M66C By using 295N310P enzyme, L-methionine could be quantified regardless of the concentration of L-phenylalanine.

[Table 12]

 Table 12 Enzyme fluorescence determination of methionine with BS124S145M66C295N310P mutant enzyme

L-Phe (μ. M) Calculated concentration (Μ) SD cv (%)

 30 30 27 1. 4 5. 2

 30 60 55 4. 0 7. 3

 —

 30 120 130 11. 7 9

 60 30 28 0. 9 3. 4

 60 60 53 4. 0 7. 6

 60 120 116 10. 9 9. 4

 120 30 29 1. 2 4. 2

 120 60 47 1. 9 4. 1

 120 120 94 10. 3 10. 9 Brief description of the drawings

[Fig. 1] Amino acid residues and putative conformations of five-point mutant phenylalanine dehydrogenase.

 [FIG. 2] Amino acid fluorescence quantitative calibration curve using a mutant enzyme (BS124S145M66C295N310P).

Claims

The scope of the claims  [1] A modified enzyme in which at least three amino acids have been modified to improve the substrate specificity of phenylalanine dehydrogenase (EC 1.4.1.20) for L-methionine.  [2] The modification is a continuous arrangement of threonine (Thr) glycine (Gly) in the amino acid sequence of phenylalanine dehydrogenase IJ (however, the glycine (Gly) is in the 114th to 125th range) The modified enzyme according to claim 1, wherein the glycine (Gly) is substituted with serine (Ser).  [3] The modification is a continuous sequence of glutamine (Gin) valine (Val) in the amino acid sequence of phenylalanine dehydrogenase (however, the parin (Val) is in the range of 297 to 310) The modified enzyme according to claim 1 or 2, which is a substitution of proline (Pro) for palin (Val).  [4] The modification is alanine (Ala) leucine in the amino acid sequence of phenylalanine dehydrogenase.  The substitution of leucine (Leu) with cystine (Cys) in a continuous sequence of (Leu) (wherein leucine (Leu) is in the 54th to 67th range) The modified enzyme according to item 1.  [5] The above-mentioned modification is a sequence of asparagine (Asn), norin (Val), alanine (Ala) or phenylalanine (Phe) and glycine (Gly) in the amino acid sequence of phenylalanine dehydrogenase (however, , Asparagine (Asn), valine (Val), alanine (Ala) or asparagine (Val), alanine (Ala) or asparagine (Val), alanine (Ala) The modified enzyme according to any one of claims 1 to 4, which is a substitution of phenylalanin (Phe) with methionine (Met).  [6] The modification is a glycine (Gly) / serine in the amino acid sequence of phenylalanine dehydrogenase.  (Ser), isoleucine (lie), tryptophan (Trp) / leucine (Leu) / valine (Val) / cystine (Cys), phenylalanine (Phe) / tyrosine (Tyr), alanine (Ala), proline (Pro ), Aspartic acid (Asp) in a continuous sequence (wherein leucine (Leu), tryptophan (Trp), valine (Val) or cystine (Cys) are in the range 281 to 295)) The modified enzyme according to any one of claims 1 to 5, which is a substitution of asparagine (Asn) from eu), tryptophan (Trp), Norin (Val) or cysteine (Cys). [7] Phenylalanine dehydrogenase power Bacillus badius IAM11059, Bacillus sphaericus R79 a, Sporosarcina ureae R04, Bacillus halodurans, Geobacillus kaustophilus, Oceanob 7. The modified enzyme according to any one of claims 1 to 6, which is derived from acillus iheyensis, Rhodococcus sp. M-4 or (also Thermoactinomyces intermedius). [8] The amino acid sequence of phenylalanine dehydrogenase derived from Bacillus badius IAM11059 (described in SEQ ID NO: 2 in the sequence listing) is the 65th amino acid residue as cystine (Cys) and the 123rd amino acid residue as serine ( Ser), a modified enzyme in which the 144th amino acid residue is replaced with methionine (Met), the 294th amino acid residue is replaced with asparagine (Asn), and the 309th amino acid residue is replaced with proline (Pro).  [9] Cysteine (Cys) as the 66th amino acid residue and amino acid residue as the 124th amino acid residue of IJ (described in SEQ ID NO: 4) of the phenylalanine dehydrogenase derived from Bacillus sphaericus R79a Serine (Ser), a modified enzyme in which the 145th amino acid residue is replaced with methionine (Met), the 295th amino acid residue is replaced with asparagine (Asn), and the 310th amino acid residue is replaced with proline (Pro) .  [10] Cystein (Cys) is the 67th amino acid residue and Serine is the 125th amino acid residue of the amino acid sequence IJ (described in SEQ ID NO: 6) of Sporosarcina ureae R04 (Ser), a modified enzyme in which the amino acid residue at position 146 is substituted with methionine (Met), the amino acid residue at position 293 is replaced with asparagine (Asn), and the amino acid residue at position 308 is replaced with proline (Pro).  [11] The amino acid sequence of phenylalanine dehydrogenase derived from Bacillus halodurans IJ (described in SEQ ID NO: 8 in the sequence listing) is the 64th amino acid residue (Cys) and the 122nd amino acid residue is serine. (Ser), a modified enzyme in which the 143rd amino acid residue is substituted with methionine (Met), the 293rd amino acid residue is replaced with wasparagine (Asn), and the 308th amino acid residue is replaced with proline (Pro).  [12] Cysteine (Cys) as the 65th amino acid residue and Serine as the 123rd amino acid residue of the amino acid coordination IJ (described in SEQ ID NO: 10 in the sequence listing) of the phenylalanine dehydrogenase derived from Geobacillus kaustophilus (Ser), a modified enzyme in which the 144th amino acid residue is substituted with methionine (Met), the 294th amino acid residue is substituted with asparagine (Asn), and the 309th amino acid residue is substituted with proline (Pro). [13] Amino acid coordination of phenylalanine dehydrogenase from Oceanobacillus iheyensis IJ (Sequence Listing) Cysteine (Cys), 122th amino acid residue is serine (Ser), 143rd amino acid residue is methionine (Met), and 293th amino acid residue. A modified enzyme in which the residue is substituted with asparagine (Asn) and the 308th amino acid residue is substituted with proline (Pro).  [14] Cysteine (Cys) is the 54th amino acid residue of the amino acid sequence IJ (described in SEQ ID NO: 14) of Rhodococcus sp. M_4. Serine (Ser), a modified enzyme in which the 138th amino acid residue is substituted with methionine (Met) and the 281st amino acid residue is substituted with asparagine (Asn).  [15] Cystein (Cys) is the 56th amino acid residue of serine (the 56th amino acid residue of the amino acid sequence lj (described in SEQ ID NO: 16 of the Sequence Listing) of the thermoalanine dehydrogenase derived from Thermoactinomyces intermedius. Ser), a modified enzyme in which the 135th amino acid residue is substituted with methionine (Met), the 282nd amino acid residue is substituted with wasparagine (Asn), and the 297th amino acid residue is substituted with proline (Pro).  [16] The amino acid sequence of the modified enzyme according to any one of claims 1 to 15, wherein the amino acid sequence has a deletion, substitution and / or addition of one to several amino acids (provided that the deletion and / or Is the serine (Ser) of the contiguous sequence of threonine (Thr) serine (Ser) with serine (Ser) in the 114th to 125th range, and serine that links (A) and (B). A modified enzyme having an amino acid sequence having phenylalanine dehydrogenase activity, wherein (Ser) is not included! /, And has improved substrate specificity for methionine.  [17] The modified enzyme according to any one of [1] to [16], wherein the activity with respect to phenylalanin is 100 or less when the relative activity with respect to methionine is 100.  [18] The modified enzyme according to any one of [1] to [16], wherein the relative activity with respect to methionine is 100 or less when the relative activity against leucine is S100.  [19] The modified enzyme according to any one of [1] to [16], wherein the relative activity with respect to methionine is 100 or less when the relative activity with respect to parin is S100. [20] The modified enzyme according to any one of [1] to [16], wherein the activity is 100 or less for any of phenylalanine, leucine and valine when the relative activity to methionine is 100. A protein in which 1 to 12 histidine forces and an oligopeptide (histidine tag) comprising the enzyme of claim 1 are fused to the N-terminal side of the enzyme according to claim 1. A DNA having a base sequence encoding the enzyme according to any one of claims 1 to 20 or the fusion protein according to claim 21. Any of the following DNA.  (1) DNA having the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing, wherein the 193 to 195th ttg force gt or tgc, the 367 to 369th ggt force gt, agc, tct, tcc, tea or tcg 430-432th gtt is atg, 880-882th ctg is aat! /, Is aac, and 925-927th gta force, cct, ccc, cca  (2) DNA having the base sequence described in SEQ ID NO: 3 in the sequence listing, wherein 196 to 198th ctg is tgt or tgc, and 370 to 372rd ggt is agt, agc, tct, tcc, tea or tcg The 433th to 435th aac is atg, the 883th to 885th cta is aat! /, The aac, and the 928th to 930th gtt force cct, ccc, ccafc (the ccg DNA,  (3) DNA having the base sequence set forth in SEQ ID NO: 5 in the sequence listing, wherein tta at positions 199 to 201 is tgt or tgc, ggt force at positions 373 to 375 gt, agc, tct, tcc, tea or tcg 436 to 438 th gtg is atg, 877 to 879 th ttg is aat or aac, and 922 to 924 th gtg force, cct, ccc, cca mei  (4) DNA having the base sequence described in SEQ ID NO: 7 in the sequence listing, wherein 190 to 192nd ctt is tgt or tgc, and 364 to 366th ggt is agt, agc, tct, tcc, tea or tcg DNA whose 427 to 429th gtt is atg, 877 to 879th tgg is aat or aac, and 922 to 924th gta force, cct, ccc, cca mei i ccg,  (5) DNA having the base sequence set forth in SEQ ID NO: 9 in the sequence listing, wherein the 193 to 195th etc gt or tgc and the 367 to 369th ggc are agt, agc, tct, tcc, tea! /, Tcg, 430 ˜432rd gtc is atg, 880th to 882th gtg is aat or aac, and 925th to 927th gtg force, cct, ccc, cca mei (or ccg DNA, (6) DNA having the base sequence set forth in SEQ ID NO: 11 in the sequence listing, wherein ttg from 190 to 192 is tgt or tgc, and ggt from 364 to 366 is agt, agc, tct, tcc, tea or tcg , 42 7th to 429th aat is atg, 877th to 879th tta is aat or aac, and 922th to 924th DNA whose gtt is cct, ccc, cca or ccg,  (7) DNA having the base sequence set forth in SEQ ID NO: 13 in the sequence listing, the 160-162th etc. force gt or tgc, the 349-351st gga force gt, agc, tct, tcc, tea or tcg 41 DNA whose ttc from 2nd to 414th is atg, and ctg from 841 to 843th is aat or aac,  (8) DNA having the base sequence set forth in SEQ ID NO: 15 in the sequence listing, wherein ttg at 166 to 168 is tgt or tgc, and gga at 340 to 342 is agt, agc, tct, tcc, tea or tcg 40 3rd to 405th gcc is atg, 844th to 846th tgt is aat or aac, and 889th to 891th gtg force ct, ccc ゝ ccafc (or ccg DNA ゝ  (9) A phenylalanine dehydration having a base sequence having a deletion, substitution and / or addition of one to several bases and improved substrate specificity for methionine in the base sequences of (1) to (8) DNA having a base sequence encoding elementary enzyme,  (10) DNA having a base sequence complementary to the DNA of (1) to (9). Any of the following DNA.  (11) The amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing, The 123rd amino acid is serine (Ser),  The 309th amino acid is proline (Pro),  The 65th amino acid is cysteine (Cys),  The 144th amino acid force is S-methionine (Met), and  294th amino acid is asparagine (Asn), Any one of the following, DNA encoding an amino acid sequence satisfying three,  (12) The amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing, The 124th amino acid is serine (Ser),  The 310th amino acid is proline (Pro),  The 66th amino acid is cystine (Cys),  The 145th amino acid force is S-methionine (Met), and  295th amino acid is asparagine (Asn), Any one of the following, DNA encoding an amino acid sequence satisfying three, (13) The amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing, The 125th amino acid is serine (Ser),  308th amino acid is proline (Pro),  The 67th amino acid is cysteine (Cys),  The 146th amino acid force is S-methionine (Met), and  The 293rd amino acid is asparagine (Asn), Any one of the following, DNA encoding an amino acid sequence satisfying three, (14) The amino acid sequence set forth in SEQ ID NO: 8 in the sequence listing, wherein the 122nd amino acid is serine (Ser),  308th amino acid is proline (Pro),  The 64th amino acid is cystine (Cys),  The 143rd amino acid force is S-methionine (Met), and  The 293rd amino acid is asparagine (Asn), Any one of the following, DNA encoding an amino acid sequence satisfying three, (15) The amino acid sequence set forth in SEQ ID NO: 10 in the sequence listing, wherein the 123rd amino acid is serine (Ser),  The 309th amino acid is proline (Pro), The 65th amino acid is cysteine (Cys), The 144th amino acid force is S-methionine (Met), and 294th amino acid is asparagine (Asn), DNA encoding an amino acid sequence satisfying three, (16) the amino acid sequence of SEQ ID NO: 12 in the sequence listing, wherein the 122nd amino acid is serine (Ser), 308th amino acid is proline (Pro), The 64th amino acid is cystine (Cys), The 143rd amino acid force is S-methionine (Met), and The 293rd amino acid is asparagine (Asn), DNA encoding an amino acid sequence satisfying three, (17) the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing, The 117th amino acid is serine (Ser),  The 54th amino acid is cystine (Cys),  The 138th amino acid force is S-methionine (Met), and  281st amino acid is asparagine (Asn),  Any one of the following, DNA encoding an amino acid sequence satisfying three,  (18) the amino acid sequence set forth in SEQ ID NO: 16 in the sequence listing,  114th amino acid is serine (Ser),  The 297th amino acid is proline (Pro),  The 56th amino acid is cysteine (Cys),  The 135th amino acid force is S-methionine (Met), and  The 282nd amino acid is asparagine (Asn),  Any one of the following, DNA encoding an amino acid sequence satisfying three,  (19) A phenylalanine having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids and improved substrate specificity for methionine in the amino acid sequence of (11) to (18) DNA having a base sequence encoding an amino acid sequence having dehydrogenase activity, and  (20) DNA having a base sequence complementary to the DNA of (11) to (19). [25] A vector comprising the DNA of claim 23 or 24.  [26] The vector according to claim 25, further comprising DNA encoding an oligopeptide consisting of 1 to 12 histidines at either end of the DNA according to claim 23 or 24.  [27] A transformant obtained by transforming a host with the vector according to claim 25 or 26.  [28] The transformant according to claim 27 is cultured, and at least three amino acids are improved from the culture so as to improve the substrate specificity of phenalanin dehydrogenase (EC 1.4 · 1.20) to methionine. A method for preparing a modified enzyme, which comprises collecting a modified enzyme modified with an acid.  [29] The preparation method according to claim 28, wherein the collected modified enzyme is the enzyme according to any one of claims 2 to 20 or the fusion protein according to claim 21. [30] A method for analyzing L-methionine contained in a test sample using the enzyme according to any one of claims 1 to 20 or the fusion protein according to claim 21. [31] The method according to claim 30, wherein at least one of L-leucine, L-isoleucine, L-valine and L-phenylalanin is analyzed in combination. [32] Test samples were resazurin, diaphorase and nicotinamide adenine dinucleotide (N 32. The method of claim 30 or 31, comprising mixing with AD ⁺ ) and detecting color development. [33] Test sample and resazurin, diaphorase and nicotinamide adenine dinucleotide (NA The method according to claim 32, wherein the mixing with D ⁺ ) is carried out in a microplate tool. 34. The method according to claim 30, wherein the test sample is a blood sample.