JP2008086312A - Method for analysing l-methionine in biological sample by using function-modified phenylalanine dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply analyzing, in a high sensitivity, L-methionine contained in a tested sample by using a function-modified phenylalanine dehydrogenase having substrate specificity to L-methionine and a method capable of reducing measurement errors caused by an amino acid other than L-methionine occurring in a blood sample. <P>SOLUTION: The method for the analysis of L-methionine contained in a tested sample includes the steps of subjecting the test sample to a treatment that enables the conversion of at least a part of a branched amino acid into an oxo acid, a step for incubating the treated sample together with a modified phenylalanine dehydrogenase and a reaction solution containing resazurin, diaphorase and an oxidation type nicotinamide adenine dinucleotide (NAD<SP>+</SP>) and a step for detecting the color developed in the reaction solution after the incubation. A method for reducing the amount of a branched amino acid in a tested sample includes converting at least a part of the branched amino acid that is often contained in the tested sample for the analysis of L-methionine into an oxo acid. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for selectively analyzing L-methionine contained in a biological sample using phenylalanine dehydrogenase (EC 1.4.1.20) modified to have substrate specificity for L-methionine. About.

  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 congenital metabolic disorders have been reported so far, and in particular, newborns nationwide regarding 6 types of diseases that are expected to grow normally when treatment is established and appropriate treatment is performed. Mass screening has been conducted on

  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 method (Clinical Chemistry, Vol. 35, p. 1962 (1989), Non-Patent Document 4) followed by a fluorescence reaction is performed on the microplate, and the L- A kit for quantifying phenylalanine has been 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 the 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)

  Specific for each measurement item in newborn mass screening by enzyme fluorescence using microplates for important inborn errors of metabolism other than homocystinuria (phenylketonuria, galactosemia, and maple syrup urine disease, etc.) A common dehydrogenase, the measurement procedure, the reagent composition, the detection fluorescence wavelength, and the like, 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 ammonia fluorimetry 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, it has been pointed out that this method reacts non-enzymatically with measurement workers and ammonia derived from the measurement environment and develops a fluorescent dye, and it is necessary to pay close attention to the analysis operation. This fact entails the risk of newborn false positives in newborn mass screening and concerns the mental and physical burden on the newborn and his parents.

  In any case, it is necessary to improve the work efficiency at the time of screening because samples must be sorted for each diagnosis item.

  Against this background, the present inventors determined that the L-methionine concentration in the blood sample was measured by enzyme fluorescence in the same measurement procedure, reagent composition, and detection fluorescence wavelength as in important inborn errors of metabolism other than homocystinuria. We attempted to create an L-methionine-specific dehydrogenase by an evolutionary molecular engineering method with the aim of making it possible to quantify by the method and greatly improving the screening work efficiency. As a result, a modified enzyme in which at least one amino acid was modified so as to improve the substrate specificity of phenylalanine dehydrogenase (EC 1.4.1.20) for methionine was obtained and studied to achieve the above-mentioned purpose. As a result, a modified enzyme having a reduced substrate specificity for L-phenylalanine and an improved substrate specificity for L-methionine was obtained and applied for a patent (Japanese Patent Application No. 2006-57547).

  However, the obtained modified enzyme with improved substrate specificity for L-methionine still has substrate specificity for substrates other than L-methionine, such as L-leucine, L-isoleucine, and L-valine. Therefore, the background may still be high for use in L-methionine microplate fluorimetry, and further improvement was required for practical use.

  Therefore, the present invention is a method capable of quantifying the L-methionine concentration in a blood sample using an amino acid dehydrogenase specific for L-methionine as described above, which is other than L-methionine present in the blood sample. An object to be solved is to provide a method capable of suppressing measurement errors due to amino acids.

  In particular, in the present invention, a highly sensitive L-methionine contained in a test sample using a modified phenylalanine dehydrogenase having substrate specificity for L-methionine as an amino acid dehydrogenase specific for L-methionine. Providing a simple analysis method is a problem to be solved.

  Furthermore, another object of the present invention is to provide a method that can suppress measurement errors caused by amino acids other than L-methionine present in a blood sample.

  As a result of studying to solve the above problems, the inventors of the present invention pre-treated a test sample (blood sample) using a branched-chain amino acid transaminase, and contained L- contained in the test sample (blood sample). The inventors have found that the above problems can be solved by reducing the concentration of branched chain amino acids such as leucine, L-isoleucine, and L-valine, and have completed the present invention.

The present invention for solving the above problems and achieving the object of the present invention is as follows.
[1] A step of subjecting a test sample to a treatment capable of converting at least a part of a branched chain amino acid into an oxo acid,
The test sample after the treatment is a reaction solution containing modified phenylalanine dehydrogenase and resazurin, diaphorase, and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ). A method for analyzing L-methionine contained in a test sample, comprising a step of incubation together with the step of detecting color development of a reaction solution after the incubation.
[2] The method according to [1], wherein the color development is a color development of a resazurin-derived fluorescent dye.
[3] A step of subjecting the test sample to a treatment capable of converting at least a part of the branched chain amino acid into an oxo acid,
A step of mixing a test sample after treatment with modified phenylalanine dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), and generated NADH Or the analysis method of L-methionine contained in a test sample including the process of measuring the absorption of NADPH.
[4] A step of subjecting the test sample to a treatment capable of converting at least a part of the branched chain amino acid into an oxo acid,
After the treatment, the test sample is treated with modified phenylalanine dehydrogenase, oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), electron carrier and reducing chromogenic reagent. Incubating with a reaction solution containing,
A method for analyzing L-methionine contained in a test sample, comprising a step of detecting formazan dye color development in a reaction solution after incubation.
[5] A step of subjecting the test sample to a treatment capable of converting at least a part of the branched chain amino acid into an oxo acid,
The test sample after the treatment is modified with phenylalanine dehydrogenase, oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), electron carrier, metal ion and chelate indicator Incubating with a reaction solution comprising:
A method for analyzing L-methionine contained in a test sample, comprising a step of detecting color development of a reaction solution after incubation.
[6] The treatment capable of converting at least a part of the branched chain amino acid into oxo acid is carried out by incubating the test sample with a reaction solution containing branched chain amino acid transaminase (EC 2.6.1.42), 2-oxoglutarate and pyridoxal phosphate. The method according to any one of [1] to [5], wherein
[7] The method according to [6], wherein the branched-chain amino acid transaminase (EC 2.6.1.42) is an enzyme derived from archaea, microorganisms or eukaryotes having substrate specificity for branched-chain amino acids.
[8] The method according to [7], wherein the branched chain amino acid is at least one selected from the group consisting of L-leucine, L-isoleucine and L-valine.
[9] The method according to any one of [1] to [8], wherein the modified phenylalanine dehydrogenase is an enzyme having substrate specificity for L-methionine.
[10] The method according to any one of [1] to [9], which is a method for quantifying L-methionine in a test sample.
[11] The method according to any one of [1] to [10], wherein the treatment step capable of converting at least a part of the branched chain amino acid into an oxo acid and the color development step are performed in a well of a microplate.
[12] The method according to any one of [1] to [11], wherein the test sample is a blood sample.
[13] A branched-chain amino acid serving as an inhibitor of L-methionine analysis, comprising converting at least a part of the branched-chain amino acid that may be contained in a test sample for analyzing L-methionine to oxo acid A method for reducing the amount of the test sample in the test sample.
[14] The conversion according to [13], wherein the conversion of at least a part of the branched chain amino acid into an oxo acid is performed using a branched chain amino acid transaminase (EC 2.6.1.42) in the presence of 2-oxoglutarate and pyridoxal phosphate. the method of.
[15] The method according to [14], wherein the branched chain amino acid transaminase (EC 2.6.1.42) is an enzyme derived from archaea, microorganisms or eukaryotes having substrate specificity for branched chain amino acids.
[16] The method according to [15], wherein the branched chain amino acid is at least one selected from the group consisting of L-leucine, L-isoleucine and L-valine.
[17] The method according to any one of [13] to [16], wherein the analysis of L-methionine is performed using a modified phenylalanine dehydrogenase.
[18] The method according to any one of [12] to [16], wherein the test sample is a blood sample.

According to the present invention, a branched chain amino acid transaminase having substrate specificity for branched chain amino acids such as L-leucine, L-isoleucine and L-valine is used to convert a branched chain amino acid contained in a test sample into glutamic acid. Analysis of L-methionine using phenylalanine dehydrogenase modified to have substrate specificity for L-methionine by introducing a pretreatment step to L-leucine, L-isoleucine and L- This can be carried out while reducing the influence of coexisting amino acids such as valine. In particular, the concentration of L-methionine contained in a test sample was determined by enzyme fluorescence coloring using resazurin, diaphorase, and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ). It can be detected with high sensitivity.

  In addition, L-methionine analysis using phenylalanine dehydrogenase functionally modified to have substrate specificity for L-methionine can be performed with high sensitivity by enzyme fluorescence coloring method, so that other than homocystinuria Homocystinuria can be diagnosed using the same measurement procedure, reagent composition, and fluorescence wavelength of detection as important inborn errors of metabolic disorders. By using the same specifications as diagnostic kits for other inborn errors of metabolic disorders It can provide a significant improvement in screening work efficiency.

  The present invention relates to a method for analyzing L-methionine contained in a test sample using a modified phenylalanine dehydrogenase (hereinafter also referred to as a modified enzyme) having improved substrate specificity for methionine.

[Preprocessing]
The L-methionine analysis method of the present invention includes a step (pretreatment step) in which a test sample is subjected to a treatment capable of converting at least a part of a branched chain amino acid into an oxo acid. This pretreatment step is performed by incubating the test sample with a reaction solution containing branched-chain amino acid transaminase (EC 2.6.1.42), 2-oxoglutarate and pyridoxal phosphate. Branched chain amino acid transaminase (EC 2.6.1.42) is derived, for example, from archaea, microorganisms or eukaryotes having substrate specificity for branched chain amino acids, specifically from Escherichia coli, lactic acid bacteria, hyperthermophilic bacteria, etc. It can be an enzyme. Here, the branched chain amino acid is, for example, at least one selected from the group consisting of L-leucine, L-isoleucine, and L-valine.

  The present invention includes a method for reducing the amount of a branched chain amino acid in a test sample, which is an inhibitor of L-methionine analysis. A method for reducing the amount of branched-chain amino acids in a test sample that becomes an inhibitor of L-methionine analysis is at least a part of the branched-chain amino acids that may be included in the test sample for analyzing L-methionine. Conversion to an oxoacid. The method for reducing the amount of branched chain amino acids in the test sample in this method is the same as the pretreatment.

  The test sample can be, for example, a blood sample, and the blood sample can be dry filter paper blood. The dry filter paper blood in the present invention is obtained by, for example, making a special blood collection filter paper soak in a sufficient amount of blood collected from a newborn's sputum. The time for blood collection is 5 to 7 days after birth, and about 2 hours after suckling. If it comes out, blood collection is good after bathing. It is desirable that the blood soaked in the filter paper sufficiently permeates the back side of the filter paper. Further, the test object is not limited to the blood of a newborn, and dry filter paper blood other than the newborn can be used as a test sample.

  For dried filter paper blood, for example, as described in the examples, an extract obtained through immobilization and elution steps can be used. The steps of immobilization and elution are, for example, punched into a 3 mm disk shape with a filter paper puncher, and placed in a well of a 96-well microplate, 1 to 10, preferably 2 to 5, respectively, and an immobilization solution (for example, acetone: Ethanol: purified water = 7: 7: 2) is added to the well, soaked in the filter paper, allowed to stand in the incubator, and the eluate (for example, 0.1 M glycine-KCl-KOH buffer, pH 9.6) is added to the well. It is done by extracting. The extract is dispensed, for example, into each well of a black 96-well plate and subjected to the pretreatment step. Deionized water can also be used as the eluate. The objective is to extract water-soluble low-molecular components after immobilizing hemoglobin or the like in the sample with an organic solvent, so that any aqueous solvent and deionized water can be used as the eluent. The reason why the buffer solution is used above is to adjust the pH to be suitable for the subsequent enzyme reaction and the like.

  It is preferable from the viewpoint that a large number of samples can be processed at once in the well of the microplate, the pretreatment step and the color development step capable of converting at least a part of the branched chain amino acid into oxo acid.

Specifically, the pretreatment step is a transaminase (EC 2.6.1.42) having a substrate specificity for a branched chain amino acid in a test sample in the presence of 2-oxoglutarate and pyridoxal phosphate. The reaction is carried out by transferring the amino group of the branched chain amino acid to produce glutamic acid, thereby converting the branched chain amino acid in the test sample into an oxo acid. The reaction temperature is appropriately determined in consideration of the optimum temperature of transaminase and the like, and can be performed at 37 ° C., for example. The reaction time can be appropriately determined in consideration of the degree of conversion of the branched chain amino acid contained in the test sample to oxo acid. The amount of transaminase, 2-oxoglutarate and pyridoxal phosphate used can be, for example, in the following range.
Transaminase usage: 0.1 to 10 U / ml
Amount of 2-oxoglutaric acid used: 1 to 100 mM
Amount of pyridoxal phosphate used: 2 to 100 μM

  Branched-chain amino acid transaminase (EC 2.6.1.42) is an enzyme that catalyzes the reversible transamination reaction between branched-chain amino acids (L-leucine, L-isoleucine, L-valine) and 2-oxoglutarate. Pyridoxal phosphate (PLP) is required as a coenzyme. This enzyme has been found in a variety of microorganisms and animals and is encoded by the ilvE gene. Examples of the branched chain amino acid transaminase include, for example, branched chain amino acid transaminases derived from archaea, microorganisms and eukaryotes, specifically, E. coli, lactic acid bacteria, hyperthermophilic bacteria and the like.

In particular, the branched chain amino acid transaminase preferably has high substrate specificity for branched chain amino acids such as L-leucine, L-isoleucine and L-valine, and the K _m value for these substrates is L-methionine or L More preferably lower than -phenylalanine.

[Color development process]
In the first aspect of the present invention, the test sample after the pretreatment is incubated with a reaction solution containing resazurin, diaphorase, and nicotinamide adenine dinucleotide (NAD ⁺ ) using a modified phenylalanine dehydrogenase. , And the step of detecting color development of the reaction solution after the incubation, L-methionine contained in the test sample is analyzed. The color development can be a color development of a fluorescent dye derived from resazurin. Alternatively, the absorbance of a resazurin-derived dye can also be measured. Mixing of the test sample with resazurin, diaphorase and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) can be performed, for example, in the well of a microplate as described above. Instead of oxidized nicotinamide adenine dinucleotide (NAD ⁺ ), oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) can also be used. The reaction temperature is appropriately determined in consideration of the optimum temperature of the modified phenylalanine dehydrogenase and the like, and can be performed at 37 ° C., for example. The reaction time can be appropriately determined in consideration of the degree of fluorescence development. The detection and measurement of fluorescent color development can be performed using, for example, a microplate reader.

The reaction scheme of the first aspect of the present invention is shown below.

The amount of the modified phenylalanine dehydrogenase, resazurin, diaphorase, and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) can be set, for example, in the following range.
Amount of modified phenylalanine dehydrogenase used: 0.001 to 1 U / ml
Resazurin usage: 10 to 100 μM
Amount of diaphorase used: 10 mU / ml to 0.5 U / ml
Amount of oxidized nicotinamide adenine dinucleotide (NAD ⁺ ): 0.1 to 10 mM

In the second aspect of the present invention, the step of mixing the test sample after pretreatment with modified phenylalanine dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ), and the step of measuring the absorption of the produced NADH To analyze L-methionine contained in the test sample. In place of oxidized nicotinamide adenine dinucleotide (NAD ⁺ ), oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) can also be used. When oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) is used, the absorption of the produced NADPH is measured. The reaction temperature is appropriately determined in consideration of the optimum temperature of the modified phenylalanine dehydrogenase and the like, and can be performed at 37 ° C., for example. The reaction time can be appropriately determined in consideration of the degree of NADH production. Oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) is reduced by the modified phenylalanine dehydrogenase, and the produced NADH has an absorption at 340 nm, and the increase in the absorbance value is measured, so that the test sample L-methionine contained in can be analyzed. The absorption value at 340 nm can be measured using, for example, a microplate reader.

The reaction scheme of the second aspect of the present invention is shown below.

The usage amount of the modified phenylalanine dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) can be, for example, in the following range.
Amount of modified phenylalanine dehydrogenase used: 0.001 to 1 U / ml
Amount of oxidized nicotinamide adenine dinucleotide (NAD ⁺ ): 0.1 to 10 mM

In the third aspect of the present invention, the test sample after the pretreatment is treated with modified phenylalanine dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), L-methionine contained in the test sample is analyzed by a step of incubation with a reaction solution containing an electron carrier and a reducing coloring reagent, and a step of detecting formazan dye coloration in the reaction solution after the incubation. The reaction temperature is appropriately determined in consideration of the optimum temperature of the modified phenylalanine dehydrogenase and the like, and can be performed at 37 ° C., for example. The reaction time can be appropriately determined in consideration of the degree of formazan coloring. Detection and measurement of formazan coloring can be performed using, for example, a microplate reader.

  The electron carrier is not particularly limited as long as it is a substance that is electrochemically reduced by NADH or NADPH generated as a result of the enzyme reaction and reduces the reducing coloring reagent. For example, quinones, cytochromes, viologens, phenazines, phenoxazines, phenothiazines, ferricyanides, ferredoxins, ferrocene and derivatives thereof can be used. Among them, phenazines such as electron carriers (donors) such as phenazine methosulfate (PMS) and 1-methoxy-phenazine methosulfate (1-Methoxy-PMS) can be mentioned. Examples of the reducing coloring reagent include int (INT), nitro blue tetrazolium (NBT), and dimethyl monotetrazolium bromide (MTT). By measuring the color development of the formazan dye, L-methionine contained in the test sample can be analyzed.

The reaction scheme of the third aspect of the present invention is shown below.

The amounts of the modified phenylalanine dehydrogenase, oxidized nicotinamide adenine dinucleotide (NAD ⁺ ), electron carrier, and reducing-type coloring reagent can be as follows.
Amount of modified phenylalanine dehydrogenase used: 0.001 to 1 U / ml
Amount of oxidized nicotinamide adenine dinucleotide (NAD ⁺ ): 0.1 to 10 mM
Amount of electron carrier used: 0.1 to 10 mM
Amount of reducing coloring reagent used: 0.1 to 10 mM

In the fourth aspect of the present invention, the test sample after the pretreatment is treated with modified phenylalanine dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), L-methionine contained in the test sample is analyzed by a step of incubation with a reaction solution containing an electron carrier, a metal ion and a chelate indicator, and a step of detecting color development of the reaction solution after the incubation. The reaction temperature is appropriately determined in consideration of the optimum temperature of the modified phenylalanine dehydrogenase and the like, and can be performed at 37 ° C., for example. The reaction time can be appropriately determined in consideration of the degree of coloring of the dye. The detection and measurement of color development can be performed using, for example, a microplate reader.

As the electron carrier, those mentioned in the second embodiment of the present invention can be used. For example, an electron carrier (donor may be a donor) such as 1-methoxy-phenazine methosulfate (1-Methoxy-PMS). As the metal ion, for example, Co ³⁺ can be used, and as the chelate indicator, for example, 5-Br-PAPS, etc. can be used, and the absorbance value of the color developed by the reaction is measured and detected. Thus, L-methionine contained in the test sample can be analyzed.

The reaction scheme of the fourth aspect of the present invention is shown below.

The amounts of the modified phenylalanine dehydrogenase, oxidized nicotinamide adenine dinucleotide (NAD ⁺ ), electron carrier, metal ion and chelate indicator can be as follows.
Amount of modified phenylalanine dehydrogenase used: 0.001 to 1 U / ml
Amount of oxidized nicotinamide adenine dinucleotide (NAD ⁺ ): 0.1 to 10 mM
Amount of electron carrier used: 0.1 to 10 mM
Metal ion usage: 0.01 to 1 mM
Amount of chelating indicator used: 0.1 to 10 mM

[Modified Phenylalanine Dehydrogenase]
The modified phenylalanine dehydrogenase used in the present invention is described below.

Modified phenylalanine dehydrogenase (part 1)
The modified phenylalanine dehydrogenase (part 1) has, for example, at least one, preferably two amino acids modified so as to improve the substrate specificity of phenylalanine dehydrogenase (EC 1.4.1.20) to methionine It is a modified enzyme. The modified enzyme used in the present invention is a protein in which the 124th amino acid of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a is substituted with serine (Ser) and the 310th amino acid is substituted with serine (Ser). By substituting the two amino acids with serine (Ser), the substrate specificity of phenylalanine dehydrogenase is modified to have specificity for methionine and branched chain amino acids. The modified enzyme catalyzes the oxidative deamination reaction of amino acids using oxidized NAD ⁺ as a coenzyme, and reductive amination reaction using reduced NADH as a coenzyme in a neutral pH region containing ammonia. To catalyze.

The modified phenylalanine dehydrogenase (part 1) will be described in detail below.
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 Yamada A, Dairi T, Ohno Y, Huang XL and Asano Y. (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 high substrate specificity for L-phenylalanine. FIG. 1 shows the substrate specificities of phenylalanine dehydrogenases derived from Bacillus badius IAM11059, Bacillus sphaericus R79a and Sporosarcina ureae R04 to L-trypsin, L-leucine, L-isoleucine, L-phenylalanine, L-methionine and L-valine. Shown by relative activity. When the relative activity with respect to L-phenylalanine is set to 100, the relative activity with respect to amino acids other than L-phenylalanine is low, and especially the relative activity with respect to L-methionine is 10 or less.

  In the present invention, as described above, a modified form in which phenylalanine dehydrogenase having a substrate specificity for L-methionine is much lower than that for L-phenylalanine is modified to improve the substrate specificity for methionine. Enzyme (part 1) is used. Specifically, it is a modified enzyme obtained by modifying at least one amino acid of a wild-type enzyme so as to improve the substrate specificity for methionine. For example, when the relative activity to phenylalanine is 100, the relative activity to methionine is preferably 50 or more, more preferably 100 or more, and further preferably 500 or more.

  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.

* Position of glycine (Gly) in a continuous sequence of threonine (Thr) glycine (Gly)
** Sequence of wild-type enzyme

  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 serine (Ser), threonine (Thr) or alanine (Ala) in 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.

* Position of valine (Val) in contiguous sequence of glutamine (Gln) valine (Val)
** Sequence of wild-type enzyme

Specific examples of the modified enzyme (part 1) are as follows:
(1a) a modified enzyme in which the 123rd amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus badius IAM11059 is substituted with serine (Ser),
(1b) The 123rd amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus badius IAM11059 is serine (Ser), and the 309th amino acid is either serine (Ser), threonine (Thr) or alanine (Ala). A modified enzyme,
(2a) a modified enzyme in which the 124th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a is substituted with serine (Ser),
(2b) The amino acid sequence of the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a is serine (Ser), and the 310th amino acid is serine (Ser), threonine (Thr) or alanine (Ala). A modified enzyme substituted with either
(3a) a modified enzyme in which the 125th amino acid residue of the amino acid sequence of the phenylalanine dehydrogenase derived from Sporosarcina ureae R04 is substituted with serine (Ser),
(3b) The 125th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Sporosarcina ureae R04 is serine (Ser), and the 308th amino acid is either serine (Ser), threonine (Thr) or alanine (Ala). A modified enzyme,
(4a) a modified enzyme in which the 122th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus halodurans is substituted with serine (Ser),
(4b) The amino acid sequence of the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus halodurans is serine (Ser) and the 308th amino acid is either serine (Ser), threonine (Thr) or alanine (Ala) A modified enzyme substituted with
(5a) a modified enzyme in which the 123rd amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Geobacillus kaustophilus is substituted with serine (Ser),
(5b) The 123rd amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Geobacillus kaustophilus is serine (Ser), and the 309th amino acid is either serine (Ser), threonine (Thr) or alanine (Ala) A modified enzyme substituted with
(6a) a modified enzyme in which the 122nd amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Oceanobacillus iheyensis is substituted with serine (Ser),
(6b) The amino acid sequence of the amino acid sequence of phenylalanine dehydrogenase derived from Oceanobacillus iheyensis is serine (Ser) and the 308th amino acid is either serine (Ser), threonine (Thr) or alanine (Ala) A modified enzyme substituted with
(7) a modified enzyme in which the 117th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Rhodococcus sp. M-4 is replaced with serine (Ser), and (8a) a phenylalanine dehydrogenase derived from Thermoactinomyces intermedius A modified enzyme in which the 114th amino acid residue of the amino acid sequence is replaced with serine (Ser),
(8b) The 114th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Thermoactinomyces intermedius is serine (Ser), and the 297th amino acid is either serine (Ser), threonine (Thr) or alanine (Ala) Is a modified enzyme substituted with

  Furthermore, the modified enzyme (part 1) uses two phenylalanine dehydrogenases of different origins as a boundary between glycine (Gly) in the continuous sequence of threonine (Thr) glycine (Gly) in the 114th to 125th range. Thus, it can be a recombinant chimeric modified enzyme. Specifically, the amino acid sequence immediately before glycine (Gly) in the above range of one phenylalanine dehydrogenase and the glycine in the above range of another phenylalanine dehydrogenase having a different origin from this phenylalanine dehydrogenase ( Gly) is a chimeric modified enzyme in which the amino acid sequence from immediately after to the last is linked in this order via serine (Ser).

  The chimera-modified enzyme is a protein in which any one amino acid sequence in the following group (A) and any one amino acid in the group (B) are consecutive in this order via serine (Ser). However, (B) is selected from an amino acid sequence of an enzyme of a different origin from (A). That is, the numbers (A) and (B) are different combinations.

Group (A)
(A-1) amino acid sequence 1-122 of phenylalanine dehydrogenase derived from Bacillus badius IAM11059,
(A-2) amino acid sequence 1-123 of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a,
(A-3) 1 to 124 of the amino acid sequence of phenylalanine dehydrogenase derived from Sporosarcina ureae R04,
(A-4) 1-121th amino acid sequence of phenylalanine dehydrogenase derived from Bacillus halodurans,
(A-5) amino acid sequence 1-122 of phenylalanine dehydrogenase derived from Geobacillus kaustophilus,
(A-6) amino acid sequence 1-121 of phenylalanine dehydrogenase derived from Oceanobacillus iheyensis,
(A-7) amino acid sequence 1-161 of phenylalanine dehydrogenase derived from Rhodococcus sp. M-4, and
(A-8) 1st to 113th amino acid sequence of phenylalanine dehydrogenase from Thermoactinomyces intermedius

Group (B)
(B-1) amino acid sequence 124-380 of phenylalanine dehydrogenase derived from Bacillus badius IAM11059,
(B-2) Phenylalanine dehydrogenase amino acid sequence 125-381 from Bacillus sphaericus R79a,
(B-3) 126-379th amino acid sequence of phenylalanine dehydrogenase derived from Sporosarcina ureae R04
(B-4) 123rd to 379th amino acid sequence of phenylalanine dehydrogenase from Bacillus halodurans
(B-5) 124th to 380th amino acid sequence of phenylalanine dehydrogenase from Geobacillus kaustophilus
(B-6) 123rd to 379th amino acid sequence of phenylalanine dehydrogenase from Oceanobacillus iheyensis
(B-7) 118-356th amino acid sequence of phenylalanine dehydrogenase derived from Rhodococcus sp. M-4
(B-8) 115th to 366th amino acid sequence of phenylalanine dehydrogenase from Thermoactinomyces intermedius

The amino acids in the group (B) may have the following amino acid sequences.
(B-1) is an amino acid sequence in which the 309th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus badius IAM11059 is substituted with serine (Ser), threonine (Thr) or alanine (Ala),
(B-2) is an amino acid sequence in which the 310th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a is substituted with serine (Ser), threonine (Thr) or alanine (Ala),
(B-3) is an amino acid sequence in which the 308th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Sporosarcina ureae R04 is substituted with serine (Ser), threonine (Thr) or alanine (Ala),
(B-4) is an amino acid sequence in which the 308th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus halodurans is substituted with serine (Ser), threonine (Thr) or alanine (Ala),
(B-5) is an amino acid sequence obtained by substituting serine (Ser), threonine (Thr) or alanine (Ala) for the 309th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Geobacillus kaustophilus,
(B-6) is an amino acid sequence in which the 308th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Oceanobacillus iheyensis is substituted with serine (Ser), threonine (Thr) or alanine (Ala),
(B-8) is an amino acid sequence in which the 297th amino acid residue of the amino acid sequence of phenylalanine dehydrogenase derived from Thermoactinomyces intermedius is substituted with serine (Ser), threonine (Thr) or alanine (Ala).

Specific examples of the chimera modified enzyme include the following.
(1) Modification in which amino acid sequence 125 to 381 of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a is linked in this order via serine (Ser) to amino acid sequence 1 to 122 of phenylalanine dehydrogenase derived from Bacillus badius IAM11059 Type enzyme,
(2) Modification in which amino acid sequence 126 to 379 of Sporosarcina ureae R04-derived phenylalanine dehydrogenase is linked in this order via serine (Ser) to amino acid sequence 1-122 of phenylalanine dehydrogenase derived from Bacillus badius IAM11059 Type enzyme,
(3) Modification in which the amino acid sequence 124-380 of the phenylalanine dehydrogenase derived from Bacillus badius IAM11059 is linked in this order via serine (Ser) to the amino acid sequence 1-123 of the phenylalanine dehydrogenase derived from Bacillus sphaericus R79a Type enzyme,
(4) Modification in which the amino acid sequence of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a is linked to amino acid sequence 126 to 379 of the amino acid sequence of Sporosarcina ureae R04 derived from the amino acid sequence 1 to 123 of serine (Ser) in this order. Type enzyme,
(5) Modification in which amino acid sequence 124-380 of Bacillus badius IAM11059-derived phenylalanine dehydrogenase is linked in this order via serine (Ser) to amino acid sequence 1-144 of Sporosarcina ureae R04-derived phenylalanine dehydrogenase Type enzyme,
(6) Modification in which amino acid sequence 125-379 of phenylalanine dehydrogenase derived from Bacillus sphaericus R79a is linked in this order via serine (Ser) to amino acid sequence 1-124 of phenylalanine dehydrogenase derived from Sporosarcina ureae R04 Type enzyme.

  In addition to the above, a chimeric modified enzyme can be prepared by combining two of any of the eight types of phenylalanine dehydrogenases shown in Table 1 or 2 above.

  Furthermore, in the amino acid sequence of the modified enzyme (part 1), phenylalanine dehydrogenation having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids and improved substrate specificity for methionine It may be a modified enzyme having an amino acid sequence having enzyme 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. Alternatively, the chimera-modified enzyme does not include serine (Ser) that links (A) and (B) to the deleted and / or substituted amino acid. The modified enzyme further having substitutions, insertions or deletions has improved substrate specificity for methionine compared to the wild-type enzyme. Improvement of substrate specificity can be achieved, for example, when the relative activity for phenylalanine is 100. The relative activity with respect to methionine is preferably 50 or more, more preferably 100 or more.

  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.

  The following is a list of specific examples of the modified enzyme (part 1) and the relative activities with respect to methionine when the relative activity with respect to phenylalanine is defined as 100.

Modified phenylalanine dehydrogenase (Part 2)
The modified phenylalanine dehydrogenase (part 2) is a modified enzyme in which amino acids in at least 3 sites, preferably 3 to 5 sites, are modified so as to improve the substrate specificity of phenylalanine dehydrogenase for methionine. The modified enzyme used in the present invention, the phenylalanine dehydrogenase derived from Bacillus sphaericus R79a, the 124th amino acid serine (Ser), the 145th amino acid methionine (Met), the 66th amino acid cysteine (Cys), A protein in which the 295th amino acid is replaced by asparagine (Asn) and the 310th amino acid is replaced by proline (Pro). By the amino acid substitution, the substrate specificity of phenylalanine dehydrogenase has a particularly high specificity for methionine, and the substrate specificity for natural branched chain amino acids such as phenylalanine and leucine, isoleucine and valine is higher than that of methionine. The enzyme has been modified to be low. The modified enzyme is also an enzyme that reversibly catalyzes oxidative deamination and reductive amination of amino acids using oxidized NAD ⁺ as a coenzyme.

Hereinafter, the modified phenylalanine dehydrogenase (part 2) will be described in detail.
As described above, wild-type phenylalanine dehydrogenase has a very high substrate specificity for L-phenylalanine, and has a relative activity to L-phenylalanine in enzymes derived from Bacillus badius IAM 11059, Bacillus sphaericus R79a or Sporosarcina ureae R04. Then, the relative activity of a natural amino acid such as L-methionine, L-leucine, L-isoleucine or L-valine with respect to the substrate is 10 or less.

  The modified phenylalanine dehydrogenase (Part 2) is a mutant enzyme in which the substrate specificity for L-methionine is remarkably improved by modifying the phenylalanine dehydrogenase having a very low substrate specificity for L-methionine as described above. It is. 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.

* Position of glycine (Gly) in a continuous sequence of threonine (Thr) glycine (Gly)
** Sequence of wild-type enzyme

  Furthermore, the modification that improves the substrate specificity for methionine specifically includes a continuous sequence of glutamine (Gln) valine (Val) in the range 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 to 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.

* Position of valine (Val) in contiguous sequence of glutamine (Gln) valine (Val)
** Sequence of wild-type enzyme

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

* Position of valine (Val) / asparagine (Asn) / alanine (Ala) in consecutive sequences of isoleucine (Ile), valine (Val) / asparagine (Asn) / alanine (Ala) and glycine (Gly)
** Sequence of wild-type enzyme

  The modified enzyme (part 2) 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 acids shown in Tables 1 to 3 are preferred. Tables 7 and 8 below further show amino acids of wild-type phenylalanine dehydrogenase to be modified. These amino acids may not need 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 7 and 8.

  Modifications that further improve the substrate specificity for methionine are specifically glycine (Gly), isoleucine (Ile), leucine (Leu) in the 281 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 281 to 295th range) Asn). Consecutive 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 281 to 295th range depends on the origin of the enzyme and is in the position shown in the following table.

* Position of leucine (Leu) in the contiguous sequence of glycine (Gly), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), alanine (Ala), proline (Peo) and aspartic acid (Asp)
** Sequence of wild-type enzyme

  Modifications that improve the substrate specificity for methionine specifically include a sequence of alanine (Ala) and leucine (Leu) in the 54th to 67th amino acid sequence of wild type phenylalanine dehydrogenase (however, This is the substitution of leucine (Leu) with cysteine (Cys) in leucine (Leu) in the 54th 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 54th to 67th range) is derived from the enzyme. Depending on the location, it is in the position shown in the table below.

* Position of leucine (Leu) in a continuous sequence of alanine (Ala) and leucine (Leu)
** Sequence of wild-type enzyme

  Furthermore, in the amino acid sequence of the modified enzyme (part 2), phenylalanine dehydrogenase having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids 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 a substitution, insertion or deletion has an improved substrate specificity for methionine as compared to the wild-type enzyme, and the improvement of the 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.

  As specific examples of the modified enzyme (No. 2), 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 taken as 100 is shown in a list.

  The method for obtaining the modified phenylalanine dehydrogenase (Part 1 and Part 2) used in 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 preparing 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, a modified enzyme can be produced. The expression of the protein in the expression system will be described later in this specification.

The modified enzyme preferably contains 1 to 12, preferably 6 to 9 consecutive histidine residues located on the N-terminal side of the methionine residue (first amino acid) of the enzyme protein. The histidine tag linked to the N-terminal side of the modified enzyme (protein) facilitates the purification of the enzyme from the recombinant culture and is a protein via a divalent metal ion such as nickel (Ni ²⁺ ). It is effective for immobilizing

  The method for obtaining DNA encoding the amino acid sequence of the modified enzyme 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 a gene encoding the amino acid sequence of the modified enzyme.

  A gene encoding the amino acid sequence of the modified enzyme 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.

  The vector on which the DNA encoding the amino acid sequence of the modified enzyme is placed is not particularly limited, but may be, for example, a self-replicating vector (such as a plasmid) or the host when introduced into the host cell. It may be integrated into the cell's genome and replicated along 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 encoding the amino acid sequence of the modified enzyme may be operably linked 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 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 linking a gene encoding the amino acid sequence of the modified enzyme, a promoter, and optionally a terminator and / or secretory signal sequence, respectively, and inserting them into an appropriate vector are well known to those skilled in the art.

  A transformant can be prepared by introducing a DNA (gene) encoding a modified enzyme amino acid sequence or a recombinant vector into an appropriate host. The host cell into which the gene encoding the amino acid sequence of the modified enzyme or the recombinant vector is introduced may be any cell as long as it can express the gene encoding the amino acid sequence of the modified enzyme. Bacteria, yeast, fungi and higher eukaryotic cells Etc.

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, such as 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-specific mutagenesis gene amplified by a PCR reaction using a site-specific mutation 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 a modified enzyme.

  The transformant is cultured, and the modified enzyme (part 1) modified with at least one amino acid so as to improve the substrate specificity of phenylalanine dehydrogenase (EC 1.4.1.20) to methionine from the culture, or at least A modified enzyme can be prepared by collecting a modified enzyme (part 2) in which one amino acid is modified.

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 from the culture of the transformant, a usual method for isolating and purifying the modified enzyme may be used.
For example, if the modified enzyme is expressed in a dissolved state in the cell, the cell is collected by centrifugation after culturing, suspended in an aqueous buffer, disrupted by an ultrasonic crusher, etc. A cell 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.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1
[Preparation of branched chain amino acid transaminase]
Add 10 ml of 0.1 M phosphate buffer (pH 7.3) to 10 mg of branched-chain amino acid transaminase (EC 2.6.1.42) (B2549-10MG, Branched-chain amino acid transaminase, form bacterial source, Sigma). Was used (10 U / ml). Here, the branched chain amino acid transaminase has high substrate specificity for branched chain amino acids such as L-leucine, L-isoleucine and L-valine, and the substrate specificity for L-methionine is much higher than that of branched chain amino acids. As long as the transaminase is extremely low, it may be any enzyme.

[Fluorescence determination of methionine in standard filter paper blood]
The procedure for quantifying methionine fluorescence in dry filter paper blood using mutant phenylalanine dehydrogenase and branched chain amino acid transaminase is described below. A standard dry filter paper blood (Lot No. and various added concentrations are shown in Table 10), which is a mixture of the collected blood with a known concentration of amino acid and galactose, is punched into a 3 mm disk shape with a filter paper puncher and placed in a 96-well microplate well. I put three each. Immobilization solution (acetone: ethanol: purified water = 7: 7: 2) 20 μL / well was added, soaked in the filter paper, and allowed to stand in an incubator at 37 ° C. for 1 hour. After adding 150 μL / well of eluate (0.1 M glycine-KCl-KOH buffer, pH 9.6) and stirring, the mixture was extracted at room temperature for 1 hour.

  Dispense 30 μL / well of the extract into a black 96-well plate, transaminase solution (10 U / ml branched chain amino acid transaminase: 0.2 ml, 10 mM PLP: 0.04 ml, 1 M potassium phosphate buffer (pH 7.8): 0.4 ml, 0.1 M 2-oxoglutarate: 0.4 ml and purified water: 2.96 ml) After adding 30 μL / well and stirring, the mixture was reacted in an incubator at 37 ° C. for 1 hour.

100 μL / well of resazurin buffer (50 mM Tris-HCl buffer (pH 8.9) containing 40 μM resazurin) was added to the microplate, and mutant phenylalanine dehydrogenase solution (80 mU mutant phenylalanine dehydrogenase) was added. Enzyme (pBS124S310S), 2 mM NAD ⁺ and 0.02 mg / ml diaphorase (produced by Oriental Yeast Co., Ltd.) were dispensed at 40 μL / well in an enzyme solution prepared with 20 mM Tris-HCl buffer (pH 8.0). After the microplate was stirred, it was allowed to stand in an incubator at 37 ° C. for 1 hour to carry out an enzyme reaction to develop a color of the fluorescent dye. The fluorescence obtained by the above reaction was measured with a microplate reader (GENios, manufactured by Tecan) using a filter having an excitation wavelength of 545 nm and a fluorescence wavelength of 590 nm.

Since the standard dry filter paper blood of Sample 1 is prepared by adding the above amino acid to unwashed blood, other amino acids are present in a state close to that of a general specimen.
Since the standard dry filter paper blood of Sample 2 is prepared by adding the above amino acid to the washed blood, the influence of other amino acids is extremely low.

Preparation of standard dry filter paper blood of sample 1: Filter human erythrocyte pack with gauze and absorbent cotton to remove aggregates 2: Prepare hematocrit to 55% 3: Mix above blood: added standard solution = 9: 1 4 : Drop on filter paper and dry to make filter paper blood.

  FIG. 1 shows the results of a condition study on the amount of branched chain amino acid transaminase contained in the pretreated transaminase solution. As a result, it was found that it is appropriate to add 0.2 ml (0.5 U / ml) of an enzyme solution of 10 U / ml for the addition amount of branched chain amino acid transaminase.

  Further, even when the addition amount of the branched chain amino acid transaminase was increased, the effect of addition of 0.2 ml or more was not observed. The slope of fluorescence intensity of the leucine-added standard dry filter paper blood sample at this time was about 18% of the slope of the L-methionine-added standard dry filter paper blood sample. In particular, the effect at a high concentration (leucine addition concentration of 10 mg / dl or more) was remarkable.

  From the results shown in FIG. 1, the effect of 1 to 2.7 mg / dl was confirmed when the L-leucine concentration was 10 mg / dl or more. In the concentration range of 10 mg / dl or less, no influence by amino acids other than L-methionine such as L-leucine and L-phenylalanine was observed. From these results, it was confirmed that the enzyme fluorescence assay according to the present invention is effective when the concentration of L-leucine and L-phenylalanine contained in the blood sample is 10 mg / dl or less.

Example 2
Fluorescence quantification of L-methionine was performed in the same manner as in Example 1 except that the mutant phenylalanine dehydrogenase (pBS124S310S) was replaced with the mutant phenylalanine dehydrogenase (BS124S145M66C295N310P). The result is shown in figure 2.

  From the results shown in FIG. 2, the effect of 1 to 2.7 mg / dl was confirmed when the L-leucine concentration was 10 mg / dl or more. In the concentration range of 10 mg / dl or less, no influence by amino acids other than L-methionine such as L-leucine and L-phenylalanine was observed. From these results, it was confirmed that the enzyme fluorescence assay according to the present invention is effective when the concentration of L-leucine and L-phenylalanine contained in the blood sample is 10 mg / dl or less.

Example 3
[experimental method]
material
Lactobacillus delbrueckii subsp. Bulgaricus ATCC 11842 strain and the hyperthermophilic bacterium Thermus thermophilus HB27 (ATCC BAA-163) chromosomal DNA were obtained from ATCC. In production of the recombinant, Escherichia coli JM109 strain (manufactured by Invitrogen) was used as the host E. coli, and pUC19 was used as the vector DNA. All primers used in the PCR method were requested to synthesize by Hokkaido System Science Co., Ltd. All other reagents were analytical grade.

The base sequence and amino acid sequence of branched-chain amino acid transaminase derived from Lactobacillus delbrueckii subsp. Bulgaricus ATCC 11842 are described in Genbank Database (Accession No. CR954253), and are described in SEQ ID NOs: 31 and 32 in the sequence listing.
Themus thermophilus is a highly thermophilic bacterium that can grow at 65-85 ° C. The base sequence and amino acid sequence of branched-chain amino acid transaminase derived from Themus thermophilus HB27 are described in Genbank Database (Accession No. AE017221), and are described in SEQ ID NOs: 33 and 34 in the sequence listing.

Culture and purification of chromosomal DNA
The lyophilized pellet of Lactobacillus delbrueckii subsp. Bulgaricus ATCC11842 obtained from ATCC was suspended in MRS broth (manufactured by DIFCO) based on the ATCC protocol and inoculated into 5 ml of MRS broth at 37 ° C. Static culture was performed for 30 hours. Chromosomal DNA was purified from 5 ml of this cell culture solution and subjected to the following experiment.

Preparation of plasmid
The pUC19 plasmid was purified from an Escherichia coli JM109 culture solution into which the pUC19 vector had been incorporated, using an Invisorb Spin Plasmid Mini Kit (manufactured by Invitek). As a vector plasmid for L. delbrueckii ilvE gene recombinant production, add 7 μl of purified plasmid pUC19 vector DNA, 2.5 μl of 10 × H buffer (Takara Bio Inc.), 0.8 μl of PstI and EcoRI respectively. Then, the total volume of the reaction solution was adjusted to 25 μl with sterilized ultrapure water and subjected to restriction enzyme treatment at 37 ° C. for 3 hours. As a vector plasmid for T. thermophilus ilvE gene recombinant production, add 7 μl of purified plasmid pUC19 vector DNA, 2.5 μl of 10 × K buffer (Takara Bio Inc.), 0.8 μl each of PstI and BamHI, and sterilize The total volume of the reaction solution was 25 μl with ultrapure water, and restriction enzyme treatment was performed at 37 ° C. for 3 hours.

  2 μl of shrimp-derived alkaline phosphatase (Roche) and 5 μl of x10 alkaline phosphatase buffer are added to 25 μl of the above vector plasmid reaction solution, respectively, and the total amount of the reaction solution is 50 μl with sterile ultrapure water, at 37 ° C. for 1 hour. Reacted. The reaction solution was purified by phenol / chloroform extraction and ethanol precipitation, and dissolved in TE solution to obtain 20 μl of dephosphorylated pUC19 vector DNA.

ilvE gene amplification
In the amplification of L. delbrueckii chromosomal DNA, synthetic oligonucleotide primer containing PstI recognition site sequence (underlined) using L. delbrueckii chromosomal DNA (base sequence 2) as a template 1: 5'- ctagtga ctgcag tttaaggaaatagcatggcaaaaaaagatctc-3 ' (Hokkaido System Science Co., Ltd., SEQ ID NO: 35) and synthetic oligonucleotide primer containing EcoRI recognition site sequence (underlined) 2: 5'- cttcttctc gaattc gatttttacacgtggtgg-3 '(Hokkaido System Science Co., Ltd. ), SEQ ID NO: 36). For amplification of the T. thermophilus ilvE gene, a synthetic oligonucleotide primer containing a PstI recognition site sequence (underlined): 5'-aaagcgct ctgcag gaaggaggaataggccatgaccaaggctgaggcc-3 '(Hokkaido System Science Co., Ltd., SEQ ID NO: 37) And a synthetic oligonucleotide primer containing a BamHI recognition site sequence (underlined): 5′-cctggtgctcgc ggatcc gcgcctggtagc-3 ′ (Hokkaido System Science Co., Ltd., SEQ ID NO: 38) was used. PCR reaction composition: 50 ng chromosomal DNA, 1 μl each of 100 pmol / μl synthetic nucleotide primer, 5 μl ExTaq × 10 buffer (Takara Bio Inc.), 2.5 mM dNTP mixture (Takara Bio Inc.) 5 μl and TaKaRa ExTaq DNA Polymerase (Takara Bio Inc.) 1 μl were added, and the total amount of the reaction solution was adjusted to 50 μl with sterile ultrapure water. For PCR, a PTC-200 Peltier Thermal Cycler (manufactured by MJ Research Japan Co., Ltd.) was used, and the reaction conditions were a reaction cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 2 minutes 30 times. The PCR product was electrophoresed and the target amplification product (about 1 kb) excised under UV irradiation was extracted and purified with Gel-M ^TM Gel Extraction Kit (VIOGENE), 50 μl of purified product Got. Add 6 μl of 10 × H buffer (Takara Bio Inc.), 1 μl each of PstI and EcoRI to 41 μl of the purified product, make the total reaction volume 60 μl with sterile ultrapure water, and react at 37 ° C. for 3 hours Then, restriction enzyme treatment was performed on both ends of the purified PCR product. The reaction solution was incubated at 60 ° C. for 15 minutes to inactivate the restriction enzyme, followed by ethanol precipitation to obtain a purified insert.

Ligation and transformation Mix 1 μl of the dephosphorylated plasmid obtained above, 5 μl of the purified insert, and 6 μl of Ligation mix (Takara Bio Inc.), making the total reaction volume 12 μl overnight at 16 ° C. After the ligation reaction, 6 μl of the reaction solution was transformed into E. coli JM109, applied to an LB agar medium containing 50 μg / ml ampicillin, and cultured at 37 ° C. for 10 hours. The obtained colonies were inoculated on a master plate, and the colonies formed were inoculated into 5 mL of LB medium containing 50 μg / ml ampicillin and cultured overnight. Plasmid DNA was purified from the culture and analyzed by ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The clones in which the correct sequences of the L. delbrueckii and T. thermophilus ilvE genes were confirmed were E E. coli JM109 / pUCLDB and E. coli JM109 / pUCTTH were used in the following experiments.

Measurement of branched-chain amino acid transaminase activity The activity of branched-chain amino acid transaminase during enzyme purification was measured according to the method of Collier et al. (J. Bacteriol, 112, 365 (1972)). ) And 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 Tris-HCl buffer, pH 8.0, 0.1 ml of 0.5 M NH ₄ Cl solution, 0.1 ml of 2.5 mM pyridoxal 5 'phosphate (PLP) solution, 0.2 M L-glutamic acid (manufactured by Nihon Riken) ) 0.2 ml of the solution and 0.1 ml of 1 mM NADH (Oriental Yeast Co., Ltd.) were added to 0.1 ml of 0.1 M -keto isocaproic acid and an appropriate amount of the enzyme solution to make the total reaction volume 1.0 ml. The molecular extinction coefficient (ε) of NADH is 6,220 l · mol ⁻¹ · cm ⁻¹ and the rate of change in absorbance per unit time (min) ((340 nm / min) is determined from the decrease in absorbance at 340 nm. One unit (U) of enzyme activity during enzyme purification was defined as the amount of enzyme that oxidizes 1 μmol of NADH per minute, and the specific activity (U / mg) is the enzyme activity per 1 mg of protein (U / mg). U) defined.

Purification of branched chain amino acid transaminase from L. delbrueckii E. coli recombinant E. coli JM109 / pUCLDB was cultured at 37 ° C. for 12 hours in 5 ml of LB medium containing 50 μg / ml ampicillin, and then 50 μg / ml ampicillin. And 500 ml of LB medium containing 0.1 mM IPTG, and cultured at 37 ° C. for 12 hours. Bacteria are collected by centrifugation (6,000xg, 10 minutes, 4 ° C), washed with 0.85% physiological saline, and then 5 times the volume of the wet cell weight 1 mM EDTA, 0.1 mM PLP And suspended in 0.1 M KPB, pH 7.4 containing 0.1 mM dithiothreitol (DTT), 1 mM 2-oxoglutarate and 10% glycerol. Cell suspension is crushed with an ultrasonic crusher (KUBOTA INSONATOR model210, 19 kHz, 10 minutes, 4 ° C), and the residue is removed by centrifugation (28,000 xg, 20 minutes, 4 ° C), and cell-free extraction is performed. A liquid was prepared. This cell-free extract was dialyzed overnight in 50 mM KPB, pH 7.4 (Buffer A) containing 1 mM EDTA, 0.1 mM PLP, 0.1 mM DTT, 1 mM 2-oxoglutarate and 10% glycerol, Add to a DEAE Toyopearl column (φ5.5 cm x 11 cm) equilibrated with Buffer A and elute the adsorbed enzyme protein by increasing the linear concentration gradient in Buffer A containing 0.5 M sodium chloride. Minutes were collected. This crude enzyme solution is dialyzed overnight against Buffer A, then added to a Butyl Toyopearl column equilibrated with buffer A that has been saturated with 20% ammonium sulfate in advance, and the ammonium sulfate saturation in Buffer A is determined by a linear concentration gradient. The adsorbed enzyme protein was eluted by decreasing. The purity of the active fraction was confirmed by SDS polyacrylamide gel electrophoresis to prepare an enzyme solution. The enzyme solution was dialyzed against Buffer A and stored in a 50% glycerol solution at 30 ° C. In the methionine quantitative experiment, these branched chain amino acid transaminases were dialyzed in 50 mM KPB, pH 7.4 containing 1 mM EDTA and 0.1 mM PLP.

Purification of branched-chain amino acid transaminase from T. thermophilus
E. coli JM109 / pUCTTH contains 1 mM EDTA and 0.1 mM PLP in a volume 5 times the wet cell weight after culturing, collecting and washing in the same manner as E. coli JM109 / pUCLDB It was suspended in 0.1M KPB, pH 7.4. A cell-free extract prepared by crushing the cell suspension with an ultrasonic crusher (19 kHz, 10 minutes, 4 ° C) and removing the residue by centrifugation (28,000 xg, 20 minutes, 4 ° C) After incubating at 70 ° C. for 10 minutes, the denatured protein was removed by centrifugation (6,000 × g, 10 minutes, 4 ° C.). The supernatant was dialyzed overnight in 50 mM KPB, pH 7.4 (Buffer B) containing 1 mM EDTA and 0.1 mM PLP, and then DEAE Toyopearl column (φ2.5 cm x, previously equilibrated with Buffer B). 30 cm), and the adsorbed enzyme protein was eluted by increasing with a linear concentration gradient in Buffer B containing 0.5 M sodium chloride, and the active fraction was collected. The purity was confirmed by SDS polyacrylamide gel electrophoresis to obtain an enzyme solution. The enzyme solution was dialyzed against Buffer B and stored in a 50% glycerol solution at −30 ° C.

Preparation of branched chain amino acid transaminase supplied by SIGMA 10 ml of branched chain amino acid transaminase (B2549-10MG, from bacterial source, manufactured by SIGMA) 3.6 ml of 0.1 M phosphate buffer (pH 7.3) Was added and dissolved (labeled 10 U / ml).

Substrate specificity of branched chain amino acid transaminase The substrate specificity of branched chain amino acid transaminase was measured according to the method of Yvon et al. (Appl. Environ. Microbiol, 33, 414 (1997)). One unit (U) of this enzyme activity was defined as the amount of enzyme that catalyzes the production of 1 μmol of L-glutamic acid per minute. The reaction solution was 17.5 μl of 1 M Tris-HCl buffer (pH 8.0), 25 μl of 100 mM 2-oxoglutarate, 25 μl of 0.5 mM pyridoxal phosphate, 25 μl of 30 mM amino acids, and appropriate amounts of enzyme solution. The total reaction volume was 250 μl. This was subjected to an enzyme reaction at 37 ° C for 5 minutes, and then the reaction was stopped by adding the same amount of ethanol as the reaction solution, and the glutamic acid concentration produced in the reaction system was determined by HPLC (SUMICHIRAI OA5000 column; Sumitomo Chemical Co., Ltd.). Product). Table 11 shows the relative activity with respect to each substrate when the relative activity with respect to L-leucine is defined as 100.

Purification of methionine dehydrogenase E. coli JM109 / pUCHis BS124S145M66C295N310P modified with modified phenylalanine dehydrogenase gene (BS124S145M66C295N310P) was cultured in LB medium containing 50 μg / ml ampicillin at 37 ° C. for 12 hours at a final concentration of 0.1 mM. Then IPTG was added and cultured at 30 ° C. for 4 hours. Bacteria are collected by centrifugation (6,000xg, 10 minutes, 4 ° C), washed with 0.85% physiological saline, then 5 times the volume of the wet cell weight 300 mM NaCl, 20 mM imidazole And suspended in 20 mM Tris-HCl buffer, pH 8.0 containing 5 mM 2-mercaptoethanol. The suspension is crushed with an ultrasonic crusher (KUBOTA INSONATOR model210, 19 kHz, 20 minutes, 4 ° C), and the residue is removed by centrifugation (28,000 xg, 20 minutes, 4 ° C). Prepared.

  Add the cell-free extract to a Ni-Chelating Sepharose FF column (resin amount: approx. 10 ml) previously equilibrated with the same buffer, and use the same buffer containing 75 mM imidazole in a volume 10 times the column volume. After washing, the adsorbed enzyme protein was eluted with the same buffer containing 500 mM imidazole. Fractionation with solid ammonium sulfate was performed in ice-cold water, and the active fraction was collected and dialyzed against 20 mM Tris-HCl buffer, pH 8.0 containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol. Purity was confirmed by SDS polyacrylamide gel electrophoresis, and a 50% glycerol solution was stored at -30 ° C.

The standard activity of the mutant enzyme (BS124S145M66C295N310P) was measured by measuring the reduction of NAD ⁺ at 340 nm at 25 ° C using a disposable cuvette made of PMMA with an optical path length of 1 cm. The enzyme reaction solution was a 1.0 mL reaction solution containing 100 μmol glycine-KCl-KOH buffer (pH 10.4), 2.5 μmol β-NAD ⁺ , 10 μmol of various L-amino acids and an appropriate amount of enzyme solution. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the production of 1 μmol NADH per minute in the oxidative deamination reaction. Specific activity (U / mg) was defined as the amount of enzyme per mg protein.
[Test Example 1]
Methionine fluorescence determination in standard filter paper blood using branched chain amino acid transaminase derived from L. delbrueckii Methionine fluorescence in dry filter paper using mutant phenylalanine dehydrogenase (BS124S145M66C295N310P) and branched chain amino acid transaminase derived from L. delbrueckii subsp. Bulgaricus The procedure for quantification was performed as follows. A standard dry filter paper blood (sample No. and various addition concentrations are shown in Table 12), in which the collected blood is mixed with amino acids and galactose at known concentrations, is punched into a 3 mm disk with a filter puncher and placed in a 96-well microplate well. I put three each. Immobilization solution (acetone: ethanol: purified water = 7: 7: 2) was added at 20 μL / well, soaked in filter paper, and dried in an incubator at 37 ° C. for 1 hour. 150 μL / well of eluate (0.1 M glycine-KCl-KOH buffer, pH 9.6) was added to the dried filter paper, and the mixture was stirred and extracted at 25 ° C. for 1 hour.

  The extract was dispensed at 30 μL / well into a black 96-well plate, 10 mU / ml L. delbrueckii-derived branched chain amino acid transaminase enzyme solution (4.0 U / ml branched chain amino acid transaminase: 0.01 ml, 10 mM PLP: 0.04 ml, 1 M potassium phosphate buffer (pH 7.8): 0.4 ml, 0.1 M 2-oxoglutarate: 0.4 ml and purified water: 3.15 ml) or SIGMA branched chain amino acid transaminase enzyme solution (labeled 10 U / ml branched chain) Amino acid transaminase: 0.2 ml, 10 mM PLP: 0.04 ml, 1 M potassium phosphate buffer (pH 7.8): 0.4 ml, 0.1 M 2-oxoglutarate: 0.4 ml and purified water: 2.96 ml), 30 μL / well each After adding and stirring each, it was made to react at 37 degreeC for 1 hour.

100 μL / well of resazurin buffer (50 mM Tris-HCl buffer (pH 8.9) containing 40 μM resazurin) was added to the microplate, and mutant phenylalanine dehydrogenase solution (9.5 U / ml mutant phenylalanine dehydrogenation was added. Enzyme: 0.1 ml, 25 mM NAD ⁺ : 0.4 ml, 0.2 mg / ml Diaphorase (from Clostridium, manufactured by Oriental Yeast): 0.5 ml, 20 mM Tris-HCl buffer (pH 8.0): 4.0 ml) 40 μL / well After the microplate was stirred, it was allowed to stand at 37 ° C. for 1 hour to carry out an enzyme reaction to develop a color of the fluorescent dye.The fluorescence obtained in the above reaction was analyzed with a microplate reader (Infinite M200, Tecan). Fluorescence intensity was measured at an excitation wavelength of 545 nm and a fluorescence wavelength of 590 nm.

  FIG. 3 and FIG. 4 show the fluorescence intensities for the methionine-added standard filter paper blood sample and the L-leucine and L-phenylalanine-added standard sample using SIGMA supplied branched chain amino acid transaminase and L. delbrueckii derived branched chain amino acid transaminase, respectively. When using a branched chain amino acid transaminase supplied by SIGMA, it is necessary to measure the fluorescence intensity while suppressing the decrease in L-methionine by the branched chain amino acid transaminase, so the effects of L-phenylalanine and L-leucine in the sample are completely eliminated. It cannot be removed (FIG. 3). However, when BCAT derived from L. delbrueckii was used, the effect of amino acids such as L-leucine and L-phenylalanine was 1.2 mg / dl or less in any concentration range, and it was effective as a methionine quantification method in filter paper blood samples. It was shown that there is.

  In addition, when the branched chain amino acid transaminase enzyme solution was 5 mU / ml or less, L-leucine in the filter paper blood could not be completely removed in the measurement of the filter paper blood sample 1, and the fluorescence intensity increased, allowing quantification. Although no further effect was observed even at 10 mU / ml or more, it was found that it was appropriate to add a 10 mU / ml branched-chain amino acid transaminase enzyme solution.

[Test Example 2]
Determination of methionine fluorescence in standard filter paper blood using branched chain amino acid transaminase derived from T. thermophilus Procedure for fluorescence determination of methionine in dry filter paper blood using mutant phenylalanine dehydrogenase (BS124S145M66C295N310P) and branched chain amino acid transaminase derived from T. thermophilus Was carried out in the same manner as in Example 1. However, the branched chain amino acid transaminase enzyme solution derived from T. thermophilus is 8.0 U / ml branched chain amino acid transaminase: 0.005 ml, 10 mM PLP: 0.04 ml, 1 M potassium phosphate buffer (pH 7.8): 0.4 ml, 0.1 M A branched amino acid removal reaction was performed by adding 30 μL / well of a 10 mU / ml branched-chain amino acid transaminase enzyme solution containing 2-oxoglutarate: 0.4 ml and purified water: 3.155 ml.

  FIG. 5 shows the fluorescence intensities for the methionine-added standard filter paper blood sample, L-leucine and L-phenylalanine-added standard sample. In the filter paper blood sample 2, even when L-leucine and L-phenylalanine were at high concentrations, the effects were hardly confirmed. In sample 1, the effect of amino acids such as L-leucine and L-phenylalanine was 0.8 mg / ml or less in any concentration range, and it was confirmed that the method was effective as a method for quantifying methionine in filter paper blood samples.

Reference example 1
Preparation method of mutant phenylalanine dehydrogenase pBS124S310S and BS124S145M66C295N310P (2; vector preparation pUC18His)
In the vector used in the present invention, EcoRI restriction enzyme recognition sequence was added upstream of the ribosome binding sequence site of pRSET-B vector DNA (Invitrogen) with a synthetic primer (Hokkaido System Science), and BamHI restriction enzyme was added. The sequence up to the recognition sequence was amplified by PCR and inserted into the restriction enzyme recognition sites of the multicloning sites EcoRI and BamHI of pUC18 vector DNA (manufactured by Takara Bio Inc.). This vector induces the target gene with the lac promoter of the pUC18 vector DNA, and expresses the protein in a state where 6 consecutive histidine sequences derived from pRSET-B are fused to the N-terminal side of the expressed protein. The recombination and expression of the function-modified amino acid dehydrogenase gene prepared in the present invention can be performed without being limited to the prepared vector DNA. For example, a commercially available vector DNA such as pRSET-B vector DNA, pUC18 vector DNA, and pET21 (+) vector DNA may be used.

The procedure for preparing the vector DNA used is shown below.
Prepare pRSET-B and pUC18 vector DNAs from recombinant E. coli (Escherichia coli JM109) culture solution containing pRSET-B and pUC18 vector DNAs, and purify the vector DNA plasmid by phenol / chloroform extraction and PEG precipitation. went.

(2-1; pRSET-B PCR)
Synthetic oligonucleotide primer 1: 5′-g ccgaat tcttaagaaggagatatacata-3 ′ (Hokkaido System Science Co., Ltd.) (SEQ ID NO: 21) containing EcoRI recognition site (single underline) using the pRSET-B vector DNA obtained above as a template DNA ) And BamHI recognition site (single underlined) synthetic oligonucleotide primer 2: 5'-gc tcggat ccttatcgtcatcgtc-3 '(Hokkaido System Science Co., Ltd.) (SEQ ID NO: 22) is used for PCR (Polymerase Chain Reaction) An approximately 0.1 kb fragment was obtained. The PCR was performed with the following reaction solution composition. 10 ng template DNA plasmid (purified pRSET-B vector DNA), 100 pmol / μl of the synthetic oligonucleotide primer 0.5 μl each, 10 × KOD PCR buffer (TOYOBO) 2.5 μl, 2.5 mM dNTP mixture (Takara Bio) 2.5 μl) and KOD-Plus-DNA polymerase (TOYOBO) 0.5 μl were added to make the total reaction volume 25 μl. As the PCR reaction conditions, a reaction cycle of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 20 seconds was repeated 35 times. PCR was performed using a PTC-200 Peltier thermal cycler (manufactured by MJ Research Japan Co., Ltd.). Run the PCR reaction solution, excise the resulting amplification target product (approximately 0.1 kb) under UV irradiation, extract and purify with Gel Extraction Kit Gel-M ^TM Gel Extraction Kit (VIOGENE), and purify 30 μl The product was obtained. Add 29 μl of 10 × K buffer (Takara Bio), 0.8 μl each of EcoRI and BamHI, and 0.9 μl of sterilized ultrapure water to 29 μl of the purified product to give a total volume of 35 μl and react at 37 ° C. for 3 hours. Restriction enzyme treatment was performed on both ends of the purified PCR product. The reaction mixture was electrophoresed, and the band of the target fragment (about 0.1 kb) was extracted and purified using Gel-M ^™ Gel Extraction Kit (manufactured by VIOGENE), and purified insert fragment containing 30 μl of His-tag sequence. Got.

(2-2; Preparation of pUC18 vector DNA)
10 μg of the purified pUC18 vector DNA obtained in the above plasmid extraction, 2 μl of 10 × K buffer (Takara Bio), 1 μl each of EcoRI and BamHI, and sterilized ultrapure water to make a total volume of 20 μl for 3 hours at 37 ° C. Reacted. Add 20 μl of 10 × SAP buffer (Boehringer Mannheim), 2 μl of shrimp-derived alkaline phosphatase (Boehringer Mannheim) and 23 μl of sterilized ultrapure water to 20 μl of the reaction solution to make a total volume of 50 μl and react for 1 hour at 50. 1 μl of phosphatase was added and reacted at 50 ° C. for 30 minutes. The reaction solution was purified by conventional methods such as phenol / chloroform extraction and ethanol precipitation, and 30 μl of dephosphorylated pUC18 vector DNA dissolved in TE solution was prepared.

(2-3; ligation reaction)
The purified insert fragment and vector DNA obtained in (2-1 and 2-2) above were ligated according to the following procedure. 16 μl of dephosphorylated pUC18 vector DNA, 5 μl of the purified insert fragment, 1 μl of 10 × Reaction buffer (New England Biolabs) and 0.5 μl of T4 DNA ligase (New England Biolabs) were mixed to make a total volume of 10 μl. The ligation reaction was performed at 0 ° C. for 10 hours.

(2-4; Transformation of Escherichia coli JM109)
A part of the reaction solution was transformed into Escherichia coli JM109. The grown colonies of Escherichia coli were inoculated into an LB liquid medium containing ampicillin salt and cultured with shaking at 37 ° C. for 12 hours. Plasmid DNA was purified from the culture solution according to a conventional method. The base sequence analysis of the purified plasmid DNA was performed using LI-COR dNA Sequencer model 4000L (Aloka). The analysis sample was prepared by PCR using a Thermo Sequenase Cycle Sequencing Kit (manufactured by usb) according to the protocol of the company. A clone in which the correct sequence of the inserted fragment was confirmed was used as Escherichia coli JM109 / pUC18His in the experiment of the present invention.

(Production of site-directed mutagenesis enzyme of pdh gene derived from Bacillus sphaericus R79a)
[Production of site-specific mutation enzyme pBS124S310S]
The pPDH-DBL plasmid containing the pdh gene of Bacillus sphaericus R79a was used as the template DNA. Based on the template DNA, amplification of the pdh gene was performed by a conventional PCR method using a sense primer 5'-aaggatccgatggcaaaacagcttgaaaag-3 '(SEQ ID NO: 23) and an antisense primer 5'-ccgctgcagttactcttttatg-3' (SEQ ID NO: 24). It was. The pdh gene obtained above was ligated to the BamHI-PstI site of the pUC18His vector and used as template plasmid DNA. The plasmid DNA as a template, was subjected to introduction of site-directed mutagenesis according to the manufacturer's protocol by PCR reaction using ^{QuikChange (R) Multi Site-Directed} Mutagenesis Kit (STRATAGENE (R) , Inc.). The synthetic oligonucleotide primer used for mutagenesis uses 5'-cgattttacacaagtactgacatgggg-3 '(SEQ ID NO: 25) at the 124th amino acid substitution, and 5'-ggcttgatccagwsngctgacgaactttatggg-3' (sequence) for the 310th amino acid substitution. Number 26) (w is adenine (a) or thymine (t), s is cytosine (c) or guanine (g), n is adenine (a), cytosine (c), guanine (g) or thymine (t) Any of the bases) was used. A clone in which the correct sequence of the inserted fragment was confirmed was used in the experiment of the present invention as Escherichia coli JM109 / pBS124S310S.

[Preparation of site-specific mutation enzyme BS124S145M66C295N310P]
The Bacillus sphaericus R79a-derived phenylalanine dehydrogenase gene pdh incorporated into the pUC18His vector was used as the template plasmid DNA in the same manner as the preparation of pBS124S310S described above. Site-specific mutation was introduced by PCR reaction using QuikChange ^(R) Multi Site-Directed Mutagenesis Kit (manufactured by STRATAGENE ^(R)) according to the protocol of the company. The synthetic oligonucleotide primer used for mutagenesis was 5'-cgattttacacaagtactgacatgggg-3 '(SEQ ID NO: 25) for the 124th amino acid substitution, and 5'-gagacgaatttcattatgggaattcctgag-3' (SEQ ID NO: 25) for the 145th amino acid substitution. 27), 5'-gtggatgaagcttgygaagatgtgcttcgc-3 '(SEQ ID NO: 28) (y is a base of either cytosine (c) or thymine (t)) and 295th amino acid substitution for the 66th amino acid substitution 5'-gaaaagggaattaaytatgcacccgattatatc-3 '(SEQ ID NO: 29) (y is a base of either cytosine (c) or thymine (t)), and 5'-ggcttgatccagccngctgacgaactttatggg for the 310th amino acid substitution -3 ′ (SEQ ID NO: 30) (n is a base of either adenine (a) or cytosine (c) or thymine (t) or guanine (g)), respectively. A clone in which the correct sequence of the inserted fragment was confirmed was used in the experiment of the present invention as Escherichia coli JM109 / pBS124S145M66C295N310P.

(3: Mutant expression and enzyme protein purification)
The expression of the site-specific mutant gene obtained in the present invention and the expressed enzyme protein were performed according to the method described above. The purity of the purified enzyme was confirmed by sodium dodecyl sulfate / polyacrylamide / gel electrophoresis. The obtained purified enzyme was used in Examples 1 and 2 described above.

  By using the branched-chain amino acid removal step in the test sample in the present invention, highly sensitive and rapid enzyme fluorescence determination of L-methionine contained in the biological sample becomes possible. This creates unprecedented sensitivity, accuracy and convenience in newborn mass screening leading to early detection of homocystinuria, a congenital amino acid metabolism disorder, and physical and mental health for children and parents. It is possible to dramatically reduce the burden.

  According to the present invention, particularly in diseases such as homocystinuria, phenylketonuria and maple syrup urine disease, which are congenital amino acid metabolism disorders, neonatal mass screening for early detection, or regular health of the patient An available examination method can be provided for diagnosis. Furthermore, the analysis method of the present invention can also be used for detection and quantification of L-methionine contained in food or the environment.

Effects of branched chain amino acid transaminase addition concentration (A) L-methionine added filter paper blood, (B) L-methionine-free filter paper blood Effect of pretreatment with branched-chain amino acid transaminase (fluorimetric determination of methionine using 5-point mutant enzyme) (A) L-methionine added filter paper blood, (B) L-methionine-free filter paper blood function modified phenylalanine dehydrogenase For analysis of L-methionine in biological samples Fluorescence intensity is shown for a methionine-added standard filter paper blood sample using a branched chain amino acid transaminase supplied by SIGMA, and a L-leucine and L-phenylalanine added standard sample. The fluorescence intensity with respect to the methionine addition standard filter paper blood sample using L. delbrueckii derived branched-chain amino acid transaminase, and the L-leucine and L-phenylalanine addition standard samples is shown. The fluorescence intensities of a methionine-added standard filter paper blood sample using a branched-chain amino acid transaminase derived from T. thermophilus, and a L-leucine and L-phenylalanine added standard sample are shown.

Claims (18)

Subjecting the test sample to a treatment capable of converting at least a part of the branched chain amino acid into an oxo acid;
The test sample after the treatment is a reaction solution containing modified phenylalanine dehydrogenase and resazurin, diaphorase, and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ). A method for analyzing L-methionine contained in a test sample, comprising a step of incubation together with the step of detecting color development of a reaction solution after the incubation. The method according to claim 1, wherein the color development is a color development of a fluorescent dye derived from resazurin. Subjecting the test sample to a treatment capable of converting at least a part of the branched chain amino acid into an oxo acid;
A step of mixing a test sample after treatment with modified phenylalanine dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), and generated NADH Or the analysis method of L-methionine contained in a test sample including the process of measuring the absorption of NADPH. Subjecting the test sample to a treatment capable of converting at least a part of the branched chain amino acid into an oxo acid;
After the treatment, the test sample is treated with modified phenylalanine dehydrogenase, oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), electron carrier and reducing chromogenic reagent. Incubating with a reaction solution containing,
A method for analyzing L-methionine contained in a test sample, comprising a step of detecting formazan dye color development in a reaction solution after incubation. Subjecting the test sample to a treatment capable of converting at least a part of the branched chain amino acid into an oxo acid;
The test sample after the treatment is modified with phenylalanine dehydrogenase, oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), electron carrier, metal ion and chelate indicator Incubating with a reaction solution comprising:
A method for analyzing L-methionine contained in a test sample, comprising a step of detecting color development of a reaction solution after incubation. The treatment that can convert at least a part of the branched chain amino acid into oxo acid is performed by incubating the test sample with a reaction solution containing branched chain amino acid transaminase (EC 2.6.1.42), 2-oxoglutarate and pyridoxal phosphate. The method according to claim 1, wherein the method is performed. The method according to claim 6, wherein the branched chain amino acid transaminase (EC 2.6.1.42) is an enzyme derived from archaebacteria, microorganisms or eukaryotes having substrate specificity for branched chain amino acids. The method according to claim 7, wherein the branched chain amino acid is at least one selected from the group consisting of L-leucine, L-isoleucine and L-valine. The method according to any one of claims 1 to 8, wherein the modified phenylalanine dehydrogenase is an enzyme having substrate specificity for L-methionine. The method according to any one of claims 1 to 9, which is a method for quantifying L-methionine in a test sample. The method according to any one of claims 1 to 10, wherein the treatment step and the color development step capable of converting at least a part of the branched chain amino acid into an oxo acid are carried out in a well of a microplate. The method according to any one of claims 1 to 11, wherein the test sample is a blood sample. Testing for branched-chain amino acids that inhibit L-methionine analysis, including converting at least a portion of the branched-chain amino acids that may be included in the test sample for L-methionine analysis to oxoacids A method for reducing the amount in a sample. The method according to claim 13, wherein the conversion of at least a part of the branched chain amino acid into an oxo acid is performed using a branched chain amino acid transaminase (EC 2.6.1.42) in the presence of 2-oxoglutarate and pyridoxal phosphate. The method according to claim 14, wherein the branched-chain amino acid transaminase (EC 2.6.1.42) is an enzyme derived from archaea, microorganisms or eukaryotes having substrate specificity for branched-chain amino acids. The method according to claim 15, wherein the branched chain amino acid is at least one selected from the group consisting of L-leucine, L-isoleucine and L-valine. The method according to any one of claims 13 to 16, wherein the analysis of L-methionine is performed using a modified phenylalanine dehydrogenase. The method according to any one of claims 12 to 16, wherein the test sample is a blood sample.