JP4228121B2 - Functionally modified phenylalanine dehydrogenase and method for analyzing amino acids in biological samples using this enzyme - Google Patents

Description

  The present invention relates to 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 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.

  According to the present invention, particularly in diseases such as homocystinuria, phenylketonuria and maple syrup urine disease, which are inborn errors of metabolism, neonatal mass screening for early detection, or periodic health checkup of the patient Can provide available inspection methods. Furthermore, the analysis method of the present invention can also 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.

  The worldwide spread of neonatal mass screening for the early detection of congenital metabolic disorders is the discovery of a cure for phenylketonuria (PKU) and L-phenylalanine (L-phenylalanine) in dry filter paper blood. Phe) was developed by Guthrie et al. (Pediatrics, Vol. 32, p. 338-343 (1963), Non-Patent Document 1). About 500 cases of inborn errors of metabolism have been reported so far, and in particular, there are 7 to 22 cases of diseases for which normal growth is expected by the establishment of treatment and appropriate treatment. Mass screening is conducted on newborns nationwide.

  For screening for phenylketonuria, an enzymatic method (Screening, Vol. 1, p. 63 (1992), Non-patent document 2, Medicine and pharmacy, Vol. 31, p. 1237 (1994), Non-patent document 3) Alternatively, an enzyme reaction called microplate fluorescence (Clinical Chemistry, Vol. 35, p. 1962 (1989), Non-Patent Document 4) followed by a fluorescence reaction is performed on the microplate, and L-phenylalanine in the sample is determined from the fluorescence intensity. A kit for quantification was developed (Medicine and Pharmacy, Vol. 37, p. 1211 (1997), Non-Patent Document 5). Further, 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 above-described screening method for phenylketonuria.

On the other hand, as a screening method for homocystinuria, the development of a mass screening method for homocystinuria using methionine γ-lyase (Research on evaluation method of mass screening system, Ministry of Health and Welfare, 1993) pp. 237- 240 (1993), Non-Patent Document 6) has been reported. In this method, ammonia (NH ₃ ) released from L-methionine by enzymatic reaction of L-methionine γ-lyase is detected in the neutral region of o-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). This is a method of measuring using fluorescence.

These microplate fluorescence quantification methods have an advantage that quantification and recording, which are objective determinations of test results, which have been difficult in the conventional method, can be easily performed in addition to high specimen processing ability. Furthermore, the test result can be obtained in about 3 hours including the pretreatment of the specimen, and the speed, convenience and work efficiency are greatly improved compared to the conventional method.
Pediatrics, vol. 32, p. 338 (1963) Screening, vol. 1, p. 63 (1992) Medicine and pharmacy, vol. 31, p. 1237 (1994) Clinical Chemistry, vol. 35, p. 1962 (1989) Medicine and pharmacy, vol. 37, p. 1211 (1997) 1993 Ministry of Health and Welfare Psychosomatic Research "Study on Evaluation Method of Mass Screening System" pp. 237-240 (1993)

  Mass screening by enzyme fluorescence using microplates for important inborn errors of metabolism other than homocystinuria (phenylketonuria, galactosemia, maple syrup urine disease, etc.) is specific to each measurement item. Using a dehydrogenase, the measurement procedure, reagent composition, detection fluorescence wavelength, etc. are common, and the system allows simultaneous measurement of multiple samples. On the other hand, in the screening method for homocystinuria, that is, the indirect blood methionine quantification method by fluorescence quantification of ammonia using L-methionine γ-lyase, other congenital methods such as measurement procedure, reagent composition and detection fluorescence wavelength are used. This is very different from the method of measuring metabolic disorders. In addition, this method is sensitive to contamination of the sample by ammonia in the environment, and it is necessary to pay close attention. For this reason, there is a need to improve the work efficiency when performing screening because the specimen must be sorted for each diagnostic item.

  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 by an enzyme fluorescence method. In addition to providing the same specifications as other diagnostic kits for inborn errors of metabolism in terms of measurement procedure, reagent composition and detection fluorescence wavelength, etc., it will provide a significant improvement in screening work efficiency. Objective.

  More specifically, an object of the present invention is to create a phenylalanine dehydrogenase having substrate specificity for L-methionine by an evolutionary molecular engineering technique, and to provide a functionally modified amino acid suitable for enzymatic fluorescence determination of methionine. It is to provide a 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 function-modified amino acid dehydrogenase.

  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, abnormal concentrations of these amino acids in blood can be increased. The present invention provides an appropriate means as a method for detection or primary screening of confirmed inborn errors of metabolism and diseases in which abnormal blood concentrations of the amino acids can be confirmed.

The present invention for solving the above problems and achieving the object of the present invention is as follows.
[1] The amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a (described in SEQ ID NO: 4 in the sequence listing), the 66th amino acid residue is cysteine (Cys), the 124th amino acid residue is serine (Ser), 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), arginine (Arg) or glycine (Gly). Replaced modified enzyme.
[2] The modified enzyme according to claim 1, wherein the 310th amino acid residue is substituted with proline (Pro).
[3] The amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a (described in SEQ ID NO: 4 in the sequence listing), the 66th amino acid residue is cysteine (Cys), the 124th amino acid residue is serine (Ser), A modified enzyme in which the 145th amino acid residue is replaced with methionine (Met) and the 295th amino acid residue is replaced with asparagine (Asn).
[4] The amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a (described in SEQ ID NO: 4 in the sequence listing) is the 124th amino acid residue as serine (Ser) and the 145th amino acid residue is as leucine (Lue). A modified enzyme in which the 310th amino acid residue is substituted with threonine (Thr).
[5] 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 [4].
[6] DNA having a base sequence encoding the enzyme according to any one of [1] to [4] or the fusion protein according to [5].
[7] Any of the following DNA.
(1) DNA having the base sequence set forth 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, agg, tct, tcc, tca or tcg 433-435th aac is atg, 883-885th cta is aat or aac, and 928-930th gtt is cct, ccc, cca or ccg,
(2) DNA having a base sequence complementary to the DNA of (1).
[8] Any of the following DNA.
(11) The amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing,
124 th amino acid Ri serine (Ser) Der,
Ri 310 amino acid proline (Pro) Der,
66th amino acid Ri cysteine (Cys) Der,
145 amino acid Ri methionine (Met) der, and
295th amino acid is asparagine (Asn),
DNA encoding the amino acid sequence,
(12) The amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing,
The 124th amino acid is serine (Ser),
The 66th amino acid is cysteine (Cys)
The 145th amino acid is methionine (Met); and
295th amino acid is asparagine (Asn),
DNA encoding the amino acid sequence,
(13) A DNA having a base sequence complementary to the DNA of any one of (11) and (12) .
[9] A vector comprising the DNA according to any one of [6] to [8].
[10] The vector according to [9], further comprising DNA encoding an oligopeptide consisting of 1 to 12 histidines on any terminal side of the DNA according to any of [6] to [8].
[11] A transformant obtained by transforming a host with the vector according to [9] or [10].
[12] Modification in which at least three amino acids are modified so that the substrate specificity of phenylalanine dehydrogenase (EC 1.4.1.20) to methionine is improved from the culture by culturing the transformant according to [11] A method for preparing a modified enzyme for collecting an enzyme.
[13] The preparation method according to [12], wherein the modified enzyme to be collected is the enzyme according to any one of [1] to [4] or the fusion protein according to [5].
[14] A method for analyzing L-methionine contained in a test sample using the enzyme according to any one of [1] to [4] or the fusion protein according to [5].
[15] The method according to [14], wherein at least one of L-leucine, L-isoleucine, L-valine and L-phenylalanine is analyzed in combination.
[16] The method according to [14] or [15], which comprises mixing a test sample with resazurin, diaphorase and nicotinamide adenine dinucleotide (NAD ⁺ ) and detecting color development.
[17] The method according to [16], wherein the test sample is mixed with resazurin, diaphorase, and nicotinamide adenine dinucleotide (NAD ⁺ ) in a well of a microplate.
[18] The method according to any one of [14] to [17], wherein the test sample is a blood sample.

  According to the present invention, a phenylalanine dehydrogenase-modified enzyme specific for L-methionine can be provided, and by using the modified enzyme according to the present invention, the L-methionine contained in a test sample The analysis can be performed by an enzymatic fluorescence method using resazurin, diaphorase and NAD.

  The present invention relates to 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 methionine. In the present invention, phenylalanine dehydrogenase (EC 1.4.1.20) to be modified 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, enzymes derived from Bacillus badius IAM11059, Bacillus sphaericus R79a, Sporosarcina ureae R04, Bacillus halodurans, Geobacillus kaustophilus, Oceanobacillus iheyensis, Rhodococcus sp. M-4 or Thermoactinomyces intermedius be able to.

Phenylalanine dehydrogenase derived from Bacillus badius IAM11059 has an extremely high substrate specificity for L-phenylalanine, and is an optimal enzyme for the quantification of blood L-phenylalanine concentration in phenylketonuria. It is used as an enzyme agent for the enzyme fluorescence quantification method used. 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 a reductive amination reaction in the presence of ammonium ion and nicotinamide adenine dinucleotide (reduced type: NADH) in the neutral to weakly alkaline pH range. Nucleotide sequencing of phenylalanine dehydrogenase gene from Bacillus badius IAM 11059 Biosci. Biotechnol. Biochem. 59 The nucleotide sequence and amino acid sequence of phenylalanine dehydrogenase derived from Bacillus badius IAM11059 are as follows. (10). It is described in 1994-1995 (1995) and described as SEQ ID NOs: 1 and 2.

Phenylalanine dehydrogenase (EC 1.4.1.20) derived from Bacillus sphaericus R79a is an enzyme that specifically catalyzes an oxidative deamination reaction to the amino group of L-phenylalanine or L-tyrosine. This enzyme has high substrate specificity for L-tyrosine as well as L-phenylalanine, and is used in the enzymatic synthesis reaction of L-amino acids. 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. Oxidation reaction by this enzyme is reversible, and ammonium ions and nicotinamide adenine dinucleotide (reduced form; NADH) are present in the pH range from near neutral to weakly alkaline as in the case of phenylalanine dehydrogenase derived from Bacillus badius IAM11059. An enzyme that also catalyzes a reductive amination reaction. The nucleotide sequence and amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a are N. Okazaki, Y. Hibino, Y. Asano, M. Ohmori, N. Numao and K. Kondo. Cloning and nucleotide sequencing of phenylalanine dehydrogenase gene of Bacillus sphaericus. Gene. 63. 337-341 (1988), described as SEQ ID NOs: 3 and 4.

  The nucleotide sequence and amino acid sequence of phenylalanine 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.

  The nucleotide sequence and amino acid sequence of phenylalanine dehydrogenase derived from Bacillus halodurans are described in GenBank Database (Accession No. NC_002570) and NCBI Protein Database (Accession No. NP_241084), and are described as SEQ ID NOs: 7 and 8.

  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), and are described as SEQ ID NOs: 9 and 10.

  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), and are described as SEQ ID NOS: 11 and 12.

The base sequence and amino acid sequence of phenylalanine dehydrogenase derived from Rhodococcus sp. M-4 are described in the following literature, and are described as SEQ ID NOS: 13 and 14.
Brunhuber NM, Banerjee A., Jacobs WR Jr., Blanchard JS Cloning, sequencing, and expression of Rhodococcus L-phenylalanine dehydrogenase. Sequence comparisons to amino-acid dehydrogenases. J. Biol. Chem., 269. 16203-16211 (1994)

The base sequence and amino acid sequence of phenylalanine dehydrogenase derived from Thermoactinomyces intermedius are described in the following literature, and are described as SEQ ID NOS: 15 and 16.
Takada H., Yoshimura T., Ohshima T., Esaki N., Soda K. Thermostable Phenylalanine dehydrogenase of Thermoactinomyces intermedius; cloning, expression, and sequencing of its gene.J. Biochem., 109. 371-376 (1991)

  Wild-type phenylalanine dehydrogenase has extremely high substrate specificity for L-phenylalanine, and L-phenylalanine relative to L-phenylalanine in an enzyme derived from Bacillus badius IAM 11059, Bacillus sphaericus R79a, or Sporosarcina ureae R04 is assumed to be L- The relative activity of a natural amino acid such as methionine, L-leucine, L-isoleucine or L-valine against a substrate is 10 or less.

  In the present invention, as described above, the mutant enzyme is a mutant enzyme in which phenylalanine dehydrogenase having a very low substrate specificity for L-methionine is modified to significantly improve the 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 to L-methionine is 100, the relative activity to L-phenylalanine is preferably 100 or less, more preferably 60 or less, and 40 or less. Is most preferred. Furthermore, for example, when the relative activity with respect to L-methionine is 100, the relative activity with respect to leucine is preferably 100 or less, more preferably 60 or less, and most preferably 40 or less. Furthermore, for example, when the relative activity to L-methionine is 100, the relative activity to valine is preferably 100 or less, more preferably 60 or less, and most preferably 40 or less. Furthermore, for example, when the relative activity with respect to L-methionine is 100, the relative activity is preferably 100 or less, more preferably 60 or less, and more preferably 40 or less for any of phenylalanine, leucine and valine. Most preferably.

  Modifications that improve substrate specificity for methionine specifically include a contiguous sequence of threonine (Thr) glycine (Gly) in the range 114 to 125 of the amino acid sequence of wild-type phenylalanine dehydrogenase (provided that Substitution of glycine (Gly) with serine (Ser) in glycine (Gly) is in the 114th to 125th range. The position of glycine (Gly) in the continuous 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) depends on the origin of the enzyme Differently, it is in the position shown in the following table.

  Furthermore, the modification which improves the substrate specificity for methionine specifically includes a continuous sequence of glutamine (Gln) valine (Val) in the range of 297 to 310 of the amino acid sequence of wild-type phenylalanine dehydrogenase (however, Substitution of valine (Val) to arginine (Arg), threonine (Thr), glycine (Gly) or proline (Pro) in the valine (Val) is in the 297-310th range. The position of valine (Val) in the continuous sequence of glutamine (Gln) valine (Val) in the amino acid sequence of wild-type phenylalanine dehydrogenase (valin (Val) is in the range of 297 to 310) depends on the origin of the enzyme. Differently, it is in the position shown in the following table.

  Modifications that further improve substrate specificity for methionine specifically include isoleucine (Ile), valine (Val) / asparagine (Asn) and amino acids 135 to 146 of the wild-type phenylalanine dehydrogenase amino acid sequence. Consecutive sequence of glycine (Gly) (wherein valine (Val) / asparagine (Asn) is in the 135th to 146th range) valine (Val) / asparagine (Asn) alanine (Alal), leucine (Leu) ) Or methionine (Met). Consecutive sequence of isoleucine (Ile), valine (Val) / asparagine (Asn) and glycine (Gly) in the amino acid sequence of wild-type phenylalanine dehydrogenase (valine (Val) / asparagine (Asn) is the 135th to 146th positions) The position of valine (Val) / asparagine (Asn) in the range) depends on the origin of the enzyme and is in the position shown in the table below.

  The modified enzyme of the present invention 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. The amino acid shown in 3 is preferred. Tables 4 and 5 below further show amino acids of wild-type phenylalanine dehydrogenase to be modified. These amino acids may not require modification depending on the origin of wild-type phenylalanine dehydrogenase. The origins of wild-type phenylalanine dehydrogenases that are preferably modified are shown in Tables 4 and 5.

  Modifications that further improve the substrate specificity for methionine are specifically glycine (Gly), isoleucine (Ile), leucine (Leu) in the 280 to 295th amino acid sequence of wild-type phenylalanine dehydrogenase, Asparagine of leucine (Leu) in a contiguous sequence of tyrosine (Tyr), alanine (Ala), proline (Peo) and aspartic acid (Asp) (wherein leucine (Leu) is in the 280-295th range) Asn). Sequence of glycine (Gly), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), alanine (Ala), proline (Peo) and aspartic acid (Asp) in the amino acid sequence of wild-type phenylalanine dehydrogenase The position of leucine (Leu) in leucine (Leu) is in the 280-295th range depends on the origin of the enzyme and is in the position shown in the following table.

  Modifications that improve the substrate specificity for methionine specifically include a sequence of alanine (Ala) and leucine (Leu) in the 53rd to 67th amino acid sequence of wild-type phenylalanine dehydrogenase (however, This is a substitution of leucine (Leu) with cysteine (Cys) in leucine (Leu) in the 53rd to 67th range. The position of leucine (Leu) in the sequence of alanine (Ala) and leucine (Leu) in the amino acid sequence of wild-type phenylalanine dehydrogenase (leucine (Leu) is in the 53rd to 67th range) is derived from the enzyme. Depending on the location, it is in the position shown in the table below.

  Furthermore, the present invention provides a phenylalanine dehydrogenase 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 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 compared to the wild-type enzyme, and the improvement of substrate specificity is the same as described above.

  That is, for example, when the relative activity for L-methionine is 100, the relative activity for L-phenylalanine is preferably 100 or less, more preferably 60 or less, and most 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 is preferably 100 or less, more preferably 60 or less, and most preferably 40 or less. Furthermore, for example, when the relative activity to L-methionine is 100, the relative activity to valine is preferably 100 or less, more preferably 60 or less, and most preferably 40 or less. Furthermore, for example, when the relative activity with respect to L-methionine is 100, the relative activity is preferably 100 or less, more preferably 60 or less, and more preferably 40 or less for any of phenylalanine, leucine and valine. Most preferably.

  The range of “1 to several” in the “base sequence having deletion, substitution and / or addition of 1 to several bases” as used herein is not particularly limited, but for example, 1 to 40, preferably Means 1 to 30, more preferably 1 to 20, more preferably 1 to 10, further preferably 1 to 5, and particularly preferably about 1 to 3. The same applies to the following.

  As specific examples (shown in Examples) of the modified enzyme of the present invention, Bacillus sphaericus R79a-derived enzyme is taken as an example, and the relative activity with respect to phenylalanine when the relative activity with respect to methionine is defined as 100 is shown as a list.

  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 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.

  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 sequences and base sequence information described in SEQ ID NOs: 1 to 16 in the sequence listing in the present specification, and the bacteria containing the phenylalanine dehydrogenase mentioned above are used by using them. The gene of the present invention can be isolated by screening the cDNA library. A cDNA library can be prepared by a conventional method from a bacterium expressing the gene of the present invention.

  The gene of the present invention can also be obtained by PCR. Using a DNA library or cDNA library derived from the bacteria described above as a template, a pair of designed to be able to amplify the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 PCR is performed using primers. PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) For example, after 30 cycles, a condition of reacting at 72 ° C. for 7 minutes can be exemplified. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

Preparation of the probes or primers, construction of cDNA library, screening of cDNA libraries, as well as operations such as cloning the gene of interest are known to those skilled in the art, for example, Molecular Cloning: A laboratory Mannual, 2 nd Ed,. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) Protocols in Molecular Biology) and the like.

  And having an amino acid sequence having a deletion, substitution and / or addition of 1 to several amino acids in the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 in the sequence listing , A gene encoding a protein having glucosidase activity; and a base sequence having a deletion, substitution and / or addition of 1 to several bases in the base sequence set forth in SEQ ID NO: 5 or 6 in the sequence listing As for genes having a nucleotide sequence encoding a glycosylase (hereinafter, these genes are referred to as mutant genes), chemical synthesis, genes based on the amino acid sequence and nucleotide sequence information described in SEQ ID NOs: 1 to 16 It can be made by any method known to those skilled in the art, such as engineering techniques or mutagenesis.

  For example, a method of contacting a DNA having the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 of the sequence listing with a drug that becomes a mutagen, a method of irradiating with ultraviolet rays, It can be performed using genetic engineering techniques. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular cloning 2nd edition, Current Protocols in Molecular. It can be performed according to the method described in biology and the like.

[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 on which any of the DNAs of the present invention is placed, but it may be, for example, a self-replicating vector (for example, a plasmid), or 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 depending on the type of host.

Promoters operable in bacterial cells include the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, and the Bacillus amyloliquefati. Enns-BAN amylase gene (Bacillus amyloliquefaciens BAN amylase gene), Bacillus subtilis alkaline protease gene (Bacillus subtilis alkaline protease gene) or promoters of the Bacillus pumilus-xylosidase gene (Bacillus pumilus xylosldase gene) or phage lambda, P _R or P _L promoters, lac of E.coli, such as trp or tac promoter and the like.

  Examples of promoters that can operate in mammalian cells include the SV40 promoter, the MT-1 (metallothionein gene) promoter, or the adenovirus 2 major late promoter. Examples of promoters operable in insect cells include polyhedrin promoter, P10 promoter, autographa caliornica polyhedrosic basic protein promoter, baculovirus immediate early gene 1 promoter, or baculovirus 39K delayed early gene. There are 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 that can operate in filamentous fungal cells include the ADH3 promoter or the tpiA promoter.

Moreover, the DNA of the present invention may be functionally bound to an appropriate terminator as necessary. The recombinant vector of the present invention may further have elements such as a polyadenylation signal (for example, derived from SV40 or adenovirus 5E1b region), a transcription enhancer sequence (for example, SV40 enhancer) and the like.
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). .

  The recombinant vector of the present invention may further contain a selection marker. Selectable markers include, for example, genes that lack their complement in host cells, such as dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI genes, or such as ampicillin, kanamycin, tetracycline, chloramphenicol, Mention may be made of 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.

[Transformant of the present invention and production of 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. The host cell into which the gene of the present invention or the recombinant vector is introduced may be any cell as long as it can express the gene of the present invention, and examples thereof include bacteria, yeast, fungi and higher eukaryotic cells.

Examples of bacterial cells include Gram positive bacteria such as Bacillus or Streptomyces or Gram negative bacteria such as E. coli. Transformation of these bacteria may be performed by using competent cells 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 DNA sequences introduced into the cells are also known, and for example, electroporation method, calcium phosphate method, lipofection method and the like can be used.

  Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, for example, Saccharomyces cerevis 1ae or Saccharomyces kluyveri. Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method.

  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 performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination.

  When insect cells are used as the host, the recombinant gene transfer vector and baculovirus are co-introduced into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected with the insect cells. And protein can be expressed (for example, as described in Baculovirus Expression Vectors, A Laboratory Manua1; and Current Protocols in Molecular Biology, Bio / Technology, 6, 47 (1988), etc.).

As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects Coleoptera insects, can be used.
Insect cells include Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company), New York, which is an ovarian cell of Spodoptera frugiperda (1992)], HiFive (manufactured by Invitrogen), which is an ovarian cell of Trichoplusia ni, can be used.
Examples of the method for co-introducing a recombinant gene introduction vector into insect cells and the baculovirus for preparing a recombinant virus include the calcium phosphate method and the lipofection method.

  More specifically, for example, a site-directed mutagenesis gene amplified by a PCR reaction using a site-directed mutagenesis primer using a phenylalanine dehydrogenase gene (pdh) derived from Bacillus badius IAM11059 or Bacillus sphaericus R79a as a template and / or Alternatively, a complementary sequence thereof is prepared, and this site-specific mutagenesis gene and / or its complementary strand is transferred to, for example, Escherichia coli or other recombinable host cells (eg, animal cells, plant cells, insect cells, etc.) ) To obtain the modified enzyme of the present invention.

  In the present invention, the transformant of the present invention is cultured, and a modified enzyme modified with at least three amino acids so as to improve the substrate specificity of phenylalanine dehydrogenase (EC 1.4.1.20) to methionine from the culture is obtained. It includes a method for preparing a modified enzyme to be collected.

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, an ordinary 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 diethylaminoethyl (DEAE) Sepharose, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), resin such as butyl sepharose and phenyl sepharose Use a hydrophobic chromatography method, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing etc. alone or in combination to obtain a purified preparation be able to.

[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.

The L-methionine analysis method of the present invention specifically includes mixing a test sample with resazurin, diaphorase and nicotinamide adenine dinucleotide (NAD ⁺ ) and detecting color development, preferably fluorescence color development. Mixing of a test sample with resazurin, diaphorase and nicotinamide adenine dinucleotide (NAD ⁺ ) can be performed, for example, in a well of a microplate.

Furthermore, the L-methionine analysis method of the present invention comprises (1) mixing a test sample with nicotinamide adenine dinucleotide (NAD ⁺ ) and measuring an increase in the absorbance value of reduced NADH, (2) test The sample and nicotinamide adenine dinucleotide (NAD ⁺ ) are mixed with the test sample and the reducing reagent, Into (INT), using an electron carrier such as phenazine methosulfate (PMS) as a medium to detect formazan-generated color development. Or (3) Metal ions (for example, Co ³⁺ etc.) to the test sample and nicotinamide adenine dinucleotide (NAD ⁺ ) via an electron carrier such as 1-methoxy-phenazine methosulfate (1-Methoxy PMS) It can also be carried out by a method such as reducing the amount of the compound, reacting with chelate indicator 5-Br-PAPS or the like, and measuring and detecting the colored absorbance value. Also in the methods (1) to (3) above, the reagent can be mixed in the well of the microplate.

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

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

Example 1
[Molecular modeling of phenylalanine dehydrogenase]
Using the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a (Swiss-Prot accession No. P23307) as a template, protein homology modeling was performed using the Canadian Computational Chemistry System Program (MOE). Homologous proteins were searched and some amino acid dehydrogenase proteins were extracted. Multiple sequence alignment was performed based on the amino acid sequence obtained above. Structure retention region analysis using the 3D structure coordinates (PDB accession No. 1LEH, homology: 47.2%) of leucine dehydrogenase from Bacillus sphaericus, which showed the highest homology among amino acid dehydrogenases with known 3D structures. The same program was used to construct a predicted three-dimensional structure of phenylalanine dehydrogenase from Bacillus sphaericus R79a. The predicted three-dimensional structure was optimized by calculating the energy minimization based on the AMBER '89 / '94, CHARMM22 and Engh-Huber force field theories included in the MOE program.

[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); Patent Document 4). In the three-dimensional structure analysis of phenylalanine dehydrogenase derived from Rhodococcus sp. M4, the coordinates of the complex of enzyme protein, coenzyme (NAD ⁺ ) and reaction product (phenylpyruvic acid) are analyzed (PDB accession No. 1BW9). ). The coordinates of NAD ⁺ were extracted from the PDB data and transferred to the predicted three-dimensional structure data. Amino acid residues interacting with NAD ⁺ and proteins described in the above references are searched from alignment with the amino acid sequence of phenylalanine dehydrogenase derived from B. sphaericus, and amino acid residue 195th valine (Val), 214 The aspartic acid (Asp), the 254th alanine (Ala) and the 277th asparagine (Asn) were extracted. The MOE program is used to constrain the interatomic distance where the amino acid residue interacts with NAD ⁺ , and the above program is used to perform energy minimization calculations to determine the spatial coordinates where NAD ⁺ is most stable in the predicted 3D structure. Derived.

[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 the predicted three-dimensional structure of phenylalanine dehydrogenase derived from B. sphaericus R79a constructed above Transferred onto the coordinates of the structure data. According to the same procedure as above, the amino acid residue that interacts with the enzyme protein and L-Phe is constrained to the interatomic distance between the 53rd glycine, 79th lysine, 91st lysine and 126th aspartic acid and the substrate L-phenylalanine. The energy minimization calculation was performed over time, and the spatial coordinates where L-Phe was most stable in the predicted 3D structure were derived.

[Extraction of mutated amino acid residue candidates]
The B. sphaericus-derived phenylalanine dehydrogenase protein / NAD ⁺ / L-phenylalanine complex predicted three-dimensional structure constructed as described above is output in PDB data format, and molecular model display software (PyMOL, DeLano Scientific LLC, South San Francisco, CA) , USA). Extraction was performed while confirming the drawn three-dimensional structure of amino acid residues that can contact the substrate (L-phenylalanine) ligand and the protein molecule surface. Furthermore, Rhodococcus sp. M4-derived phenylalanine dehydrogenase (Non-patent Documents 1 and 2) was subjected to amino acid sequence alignment from the amino acid sequence of B. sphaericus-derived phenylalanine dehydrogenase by interacting with the substrate (L-phenylalanine). Extracted. By the above operation, a total of 28 amino acid residues were selected as candidates. The selected amino acid residues are as shown in Table 7 below.

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

[Screening by saturation mutation]
For the 28 amino acid residue candidates, primers were prepared to saturate 20 amino acids each (see Table 1), and PCR was performed according to the protocol using QuikChange (Multi Site-Directed Mutagenesis Kit manufactured by Stratagene). In this example, PCR was performed using the combination of primers shown in Table 8 and the B. sphaericus R79a-derived phenylalanine dehydrogenase gene as a template. E. coli JM109 was transformed, applied to an LB agar medium containing 50 (g / ml ampicillin, and cultured at 37 ° C. for 10 hours. The obtained colony was 96-well deep plate (NUNC, 80 (g LB medium containing / ml ampicillin; inoculated into 300 (L) and cultured at 1000 rpm and 37 ° C. for 12 to 18 hours.

[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 μL of LB medium containing 50 μg / ml ampicillin at 37 ° C. and 1,000 rotations per minute. Shake culture for 16 hours or more. 150 μL of the obtained culture solution was collected on a 96-well round bottom microplate and collected by centrifugation. The centrifugal supernatant was removed by decantation. 10 μL of lysozyme solution (0.1 M phosphate buffer containing 10 mM EDTA, adjusted to pH 7.0 to 10 mg / ml) is added to the cells, and suspended at 37 ° C. Hold for 1 hour. The treated plate was frozen for 30 minutes in a -80 ° C deep freezer, and then freeze-thawed for 1 hour in a 37 ° C incubator was repeated twice.
To each well of the treatment plate, dispense 100 μL of 10 mM phosphate buffer, pH 7.0, containing 5 mM magnesium chloride, cooled in advance, and stir for 10 minutes on a shaker to extract the cell-free extract. It was. The supernatant obtained by centrifugation was used as a cell-free extract for primary screening.
Formazan colorimetric method was used for primary screening of enzyme activity. Specifically, 10 μL of the cell-free extract obtained above was dispensed into each well of a 384-well plate, and 20 μL of the reaction solution (0.1 M glycine-KCl-KOH buffer, pH 10.4, 2.5 mM NAD, 0.1 mM 1 -Methoxy-PMS (or PMS), 0.1 mM INT and 10 mM substrate (phenylalanine, leucine or methionine)) were added and reacted in the dark at 37 ° C., and red-purple color development was visually observed. 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)).

[Secondary screening-NAD absorption-]
The recombinant obtained in the primary screening was cultured with shaking in 3 mL of LB test tube medium (containing 50 μg / ml ampicillin) at 37 ° C. for 12 hours, and the cells were disrupted by ultrasonic disruption. A rate assay with a double beam spectrophotometer was performed using the cell-free extract obtained by centrifugation. The reaction solution was 1 ml, and the enzyme activity was calculated from the increase in absorbance of NAD at 340 nm. Enzyme activity for each substrate of L-phenylalanine, L-methionine and L-leucine was measured, and relative activity was calculated for L-methionine and L-leucine, respectively, when the enzyme activity for L-phenylalanine was 100 (Table). 9). As a result, the five strains with the improved substrate specificity for methionine were the mutant enzyme names BS124S145M66C295N, BS124S145M66C295N310R, BS124S145M66C295N310G, BS124S145M66C295N310P and BS124S145L310T. Among the five strains, BS124S145M66C295N310P was a strain with good substrate specificity for L-methionine and high specific activity.

[Preparation of mutant phenylalanine dehydrogenase]
Using Escherichia coli JM109, a plasmid containing a modified enzyme gene modified from the reaction of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a with L-methionine, using LB liquid medium containing 50 μg / ml ampicillin 37 After culturing at 12 ° C. for 12 hours, isopropyl-β-D-thiogalactopyranoside (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.

  Suspend in 20 mM Tris-HCl buffer (pH 8.0, 0.3 M NaCl, 20 mM imidazole and 5 mM 2-mercaptoethanol) 5 times the cell weight, and use an ultrasonic crusher (KUBOTA INSONATOR model 201M And manufactured by Kubota) for 20 minutes. A supernatant (cell-free extract) was obtained by centrifugation (28,400 × g, 20 minutes, 4 ° C.). The cell-free extract was added to a metal chelate affinity resin (Chelating-Sepharose FF: 2 mL) previously filled with nickel and equilibrated with the same buffer. 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 against 20 mM Tris-HCl buffer (pH 8.0, containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol) to prepare an enzyme preparation.

[Substrate specificity]
The phenylalanine dehydrogenase activity was measured 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). The measurement was performed with a PMMA cuvette (BRAND) having 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 Riken) or L-leucine (Nippon Riken) ) Or L-methionine (manufactured by Nihon Rikagaku Kabushiki Kaisha), etc. (The substrate specificity varies depending on the nature of the enzyme, so select the appropriate substrate each time) 0.1 ml and the appropriate amount of enzyme solution are added to react. The total liquid volume was 1.0 ml. The molecular extinction coefficient of coenzyme NAD ⁺ (() is 6,220 l · mol ^-1 · cm ^-1, and the rate of change in absorbance per unit time (min) from the increase in absorbance at 340 nm ((340 nm / min)) In the present invention, 1 unit (U) of enzyme activity is defined as the amount of enzyme that produces 1 mol of NADH per minute. Specific activity (U / mg) is the enzyme per mg of protein. Defined as activity (U).

[Substrate specificity of 5-point mutant phenylalanine dehydrogenase]
The amino acid substitution residues of the mutant enzyme in which 5 amino acid residues of B. sphaericus-derived PheDH were substituted were extracted from the predicted three-dimensional structure constructed by MOE, and the conformation (amino acid residues indicated by red Ball & stick) was It is shown in FIG. As a result of saturation mutation of amino acid residues extracted from the predicted three-dimensional structure of phenylalanine dehydrogenase enzyme protein / substrate / NAD ⁺ complex, the activity against phenylalanine and leucine (when measured at 10 mM concentration) is suppressed compared to methionine The mutants were screened. As a result, amino acid substitutions were observed in 5 residues of Gly-124, Asn-145, Val-310, Leu-295 and Leu-66 (BS124S145M66C295N310P). Three residues of Gly-124, Asn-145 and Val-310 were conformed near the benzene ring of the substrate phenylalanine. Leu-295 is conformed to a position remote 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.

  In addition, 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 against L-norleucine. Subsequently, it reacted with L-methionine, and with respect to L-phenylalanine, L-leucine and L-valine, each was suppressed to 50% or less when L-methionine was used as a reference. When quantifying amino acids in blood samples, L-norleucine and L-norvaline are scarcely contained, so there is no influence on fluorescence quantification by enzymatic reaction. 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.

The kinetic parameters of the enzyme for L-methionine and NAD ⁺ were examined (Table 11). The K _m value of this enzyme for L-methionine was 0.33 ± 0.014 mM, and the V _max value at this time was 1.73 ± 0.18 U / mg. The molecular weight of this enzyme was calculated to be 45,000 by SDS-PAGE, 45,150 from the deduced amino acid sequence, and calculated to be about 400,000 by gel filtration. Therefore, it was considered that a homo-octamer structure was formed. It is done. Therefore, k _cat of this enzyme was 1.4 ± 0.15 s ⁻¹ , and k _cat / K _m for L-methionine was 4.1 ± 0.38 s ⁻¹ mM ⁻¹ . On the other hand, the K _m value for NAD ⁺ was 0.16 ± 0.012 mM when the L-methionine concentration was fixed at 2 mM. The K _m value for NAD ^{+ of the} wild type enzyme derived from B. sphaericus is 0.24 mM under the fixed condition of 1 mM concentration using L-phenylalanine as a substrate, and the K _m value of the wild type enzyme derived from B. badius is 0.15 mM. K _m values for NAD ⁺ of the mutant enzyme, B. Had rather have a Km value and close to the values for NAD + of B. badius from PheDH than sphaericus derived PheDH. Improvement in K _m values for NAD ⁺ is considered to have influenced the amino acid substitutions of Leu-295.

Example 2
[Amino acid fluorescence determination of mutant enzyme]
Dispense 0.04 ml each 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 μM resazurin, and then add 0.04 ml An enzyme mixed solution (containing 10 mU mutant phenylalanine dehydrogenase (BS124S145M66C295N310P), 4 mM β-NAD +, 0.03 mg / ml diaphorase (manufactured by Oriental Yeast)) was added and incubated at room temperature for 1 hour. 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 / luminescence microplate reader (manufactured by Tecan, Genios). 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 FIG. Furthermore, quantitative results were obtained from this calibration curve (Table 12). From the calibration curve created using BS124S145M66C295N310P, the L-methionine concentration contained in the control could be quantified. At this time, quantification of L-methionine was difficult due to the effect of L-phenylalanine concentration with conventional modified enzymes, but by using the BS124S145M66C295N310P enzyme, L-methionine was used regardless of the concomitant concentration of L-phenylalanine. Could be quantified.

Amino acid residue and putative conformation of 5-point mutant phenylalanine dehydrogenase. A calibration curve of amino acid fluorescence using a mutant enzyme (BS124S145M66C295N310P).

Claims (18)

In the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a (described in SEQ ID NO: 4 in the sequence listing), the 66th amino acid residue is cysteine (Cys), the 124th amino acid residue is serine (Ser), the 145th amino acid residue Modification by replacing amino acid residue with methionine (Met), 295th amino acid residue with asparagine (Asn), and 310th amino acid residue with proline (Pro), arginine (Arg) or glycine (Gly) enzyme. 2. The modified enzyme according to claim 1, wherein the 310th amino acid residue is substituted with proline (Pro). In the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a (described in SEQ ID NO: 4 in the sequence listing), the 66th amino acid residue is cysteine (Cys), the 124th amino acid residue is serine (Ser), the 145th amino acid residue A modified enzyme in which the amino acid residue is replaced with methionine (Met) and the 295th amino acid residue is replaced with asparagine (Asn). In the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a (described in SEQ ID NO: 4 in the sequence listing), the 124th amino acid residue is replaced with serine (Ser), the 145th amino acid residue is replaced with leucine (Lue), A modified enzyme in which the 310th amino acid residue is replaced with threonine (Thr). 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 claims 1 to 4. A DNA having a base sequence encoding the enzyme according to any one of claims 1 to 4 or the fusion protein according to claim 5. Any of the following DNA.
(1) DNA having the base sequence set forth 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, agg, tct, tcc, tca or tcg 433-435th aac is atg, 883-885th cta is aat or aac, and 928-930th gtt is cct, ccc, cca or ccg,
(2) DNA having a base sequence complementary to the DNA of (1). Any of the following DNA.
(11) The amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing,
124 th amino acid Ri serine (Ser) Der,
Ri 310 amino acid proline (Pro) Der,
66th amino acid Ri cysteine (Cys) Der,
145 amino acid Ri methionine (Met) der, and
295th amino acid is asparagine (Asn),
DNA encoding the amino acid sequence,
(12) The amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing,
The 124th amino acid is serine (Ser),
The 66th amino acid is cysteine (Cys)
The 145th amino acid is methionine (Met); and
295th amino acid is asparagine (Asn),
DNA encoding the amino acid sequence,
(13) A DNA having a base sequence complementary to the DNA of any one of (11) and (12) . A vector comprising the DNA according to any one of claims 6 to 8. 14. The vector according to claim 13, further comprising DNA encoding an oligopeptide consisting of 1 to 12 histidines on any terminal side of the DNA according to any one of claims 6 to 8. A transformant obtained by transforming a host with the vector according to claim 9 or 10. 12. The transformant according to claim 11 is cultured, and a modified enzyme modified with at least three amino acids is collected from the culture so as to improve the substrate specificity of phenylalanine dehydrogenase (EC 1.4.1.20) to methionine. A method for preparing a modified enzyme. 13. The preparation method according to claim 12, wherein the modified enzyme to be collected is the enzyme according to any one of claims 1 to 4 or the fusion protein according to claim 5. A method for analyzing L-methionine contained in a test sample using the enzyme according to any one of claims 1 to 4 or the fusion protein according to claim 5. 15. The method according to claim 14, wherein at least one of L-leucine, L-isoleucine, L-valine and L-phenylalanine is analyzed in combination. 16. The method according to claim 14 or 15, comprising mixing a test sample with resazurin, diaphorase and nicotinamide adenine dinucleotide (NAD ⁺ ) and detecting color development. 17. The method according to claim 16, wherein the test sample is mixed with resazurin, diaphorase, and nicotinamide adenine dinucleotide (NAD ⁺ ) in a well of a microplate. The method according to any one of claims 14 to 17, wherein the test sample is a blood sample.