JP2015023859A - Measurement of l-tryptophan - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an efficient method for production of soluble L-tryptophan oxidase and a specific and accurate method for measurement of L-tryptophan in a short time using the obtained enzyme.SOLUTION: The invention uses L-tryptophan oxidase comprising (i), (ii), and (iii): (i) a protein having a specific amino acid sequence, (ii) a protein having an amino acid sequence with 90% or more similarity to a specific amino acid sequence, and having L-tryptophan oxidase activity, (iii) a protein having L-tryptophan oxidase activity comprising deleting, substituting, and/or adding preferably 55 or less of amino acids in a specific amino acid sequence. The invention provides L-tryptophan oxidase in which an action against L-phenylalanine and L-tyrosine is 1% or less if an action against L-tryptophan is 100%, and a method for measurement of L-tryptophan using the L-tryptophan oxidase.

Description

  The present invention relates to a polynucleotide encoding L-tryptophan oxidase, a recombinant vector containing the polynucleotide, a transformed cell into which the polynucleotide is inserted, a method for producing L-tryptophan oxidase, L-tryptophan oxidase, and the The present invention relates to a method for measuring L-tryptophan using L-tryptophan oxidase, a measuring reagent composition, and a measuring kit.

  L-tryptophan is a typical amino acid and one of the essential amino acids. It is also contained in the human brain, intestines, mast cells, etc., and is converted into serotonin as a neurotransmitter and melatonin as a neurohormone in the brain, and converted into NAD which is a coenzyme of biological oxidoreductase in the living body. ing.

  Thus, since L-tryptophan is an important amino acid, a simple and accurate quantitative measurement method for L-tryptophan has been sought.

  As an enzyme that acts on L-tryptophan, a basidiomycete, Coprinus sp. Derived amino acid oxidase for L-tryptophan is known (Patent Document 1 and Non-Patent Document 1). However, it is described that the enzyme is also reactive with L-phenylalanine and L-tyrosine. Furthermore, in order to obtain cultured cells containing the enzyme, it takes a long culture period of about one month. In addition, the enzyme is a membrane-bound enzyme and needs to be solubilized in the purification process. The described and cumbersome manufacturing method was a problem.

  As an L-tryptophan analysis method, an analysis method using VioA which is an oxidase derived from Chromobacterium violaceum and StaO which is an oxidase derived from Streptomyces sp. TA-A0724 is known (Patent Document 2). . However, in the L-tryptophan analysis method using these enzymes, linearity is not obtained between the L-tryptophan concentration and the absorbance in the measurement for about 10 minutes, and in order to obtain linearity, it takes 20 minutes or more. It took a long time. Furthermore, both the StaO enzyme solution and the VioA enzyme solution containing glycerol, which is a stabilizer, show remarkable heat inactivation after heat treatment at 40 ° C. for 1 hour, resulting in poor heat stability. was there.

  On the other hand, as an oxidase that acts on L-tryptophan, various L-amino acid oxidases derived from basidiomycete Hebeloma cylindrosporum and ascomycete Neurospora crassa are known (Non-Patent Documents 2 to 4). However, since L-amino acid oxidase acts on various L-amino acids, it could not be used for specific measurement of L-tryptophan in samples contaminated with various amino acids.

JP 2001-69974 A JP 2012-196207 A

Biosci. Biotechnol. Biochem. , 64 (7), 1486-1493, 2000 Journal of Biological Chemistry, 192 (2), 755-767, 1951 Journal of Biological Chemistry, 265 (28), 17246-17251, 1990 Microbiology, 158, 272-283, 2012

  The present invention provides an efficient method for producing soluble L-tryptophan oxidase, and an L-tryptophan assay that enables accurate L-tryptophan measurement in a short time using the enzyme having high thermal stability and substrate specificity. A measuring method, a measuring reagent composition, and a measuring kit are provided.

  Thus, by using a polynucleotide isolated by screening, a method for producing soluble L-tryptophan oxidase that can be efficiently produced in a short period of time is found, and the thermal stability and substrate specificity of L-tryptophan oxidase are high. From the knowledge, the inventors have found that accurate L-tryptophan measurement can be performed in a short time, and thus the present invention has been achieved.

That is, the present invention relates to the following aspects [1] to [8].
[1] A polynucleotide comprising the following (a), (b) or (c):
(A) a polynucleotide having the base sequence represented by SEQ ID NO: 1,
(B) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 2 or 6;
(C) A polynucleotide encoding a protein having an amino acid sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO: 2 or 6 and having L-tryptophan oxidase activity.
[2] A recombinant vector having the polynucleotide according to [1].
[3] A transformed cell having the polynucleotide according to [1].
[4] A method for producing L-tryptophan oxidase, comprising culturing the transformed cell according to [3] and collecting L-tryptophan oxidase from the obtained culture.
[5] Soluble L-tryptophan oxidase comprising the following (i), (ii) or (iii):
(I) a protein having the amino acid sequence of SEQ ID NO: 2 or 6,
(Ii) a protein having an amino acid sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO: 2 or 6, and having L-tryptophan oxidase activity;
(Iii) A protein having L-tryptophan oxidase activity having a deletion, substitution and / or addition of at most 55 amino acids in the amino acid sequence of SEQ ID NO: 2 or 6.
[6] The L-tryptophan oxidase according to [5], wherein the activity on L-phenylalanine and L-tyrosine is 1% or less when the activity on L-tryptophan is 100%.
[7] A method for measuring L-tryptophan using the tryptophan oxidase according to [5] or [6].
[8] The method for measuring L-tryptophan according to [7], wherein the activity on L-phenylalanine and L-tyrosine is 1% or less when the activity on L-tryptophan is 100%.
[9] The method for measuring blood L-tryptophan according to [7] or [8].
[10] A reagent composition for L-tryptophan measurement comprising the tryptophan oxidase according to [5] or [6].
[11] The reagent composition for L-tryptophan measurement according to [10], wherein the activity on L-phenylalanine and L-tyrosine is 1% or less when the activity on L-tryptophan is 100%.
[12] The reagent composition for measuring blood L-tryptophan according to [10] or [11].
[13] A kit for measuring L-tryptophan comprising the tryptophan oxidase according to [5] or [6].
[14] The kit for measuring L-tryptophan according to [13], wherein the activity on L-phenylalanine and L-tyrosine is 1% or less when the activity on L-tryptophan is 100%.
[15] The blood L-tryptophan measurement kit according to [13] or [14].

  According to the present invention, soluble L-tryptophan oxidase with high thermal stability and substrate specificity can be easily produced, and the production cost of the enzyme and the reagent composition and measurement kit containing the enzyme can be reduced. It became possible. In addition, it was possible to measure L-tryptophan accurately and specifically in a short time even in samples contaminated with various amino acids.

It is a figure which shows the effect | action to each substrate. It is a figure which shows the L-tryptophan measurement result in presence / absence of L-glutamine. It is a figure which shows the first half part of alignment of the base sequences which encode the amino acid sequence of sequence number 2-11. It is a figure which shows the latter half part of the alignment of the base sequences which encode the amino acid sequence of sequence number 2-11.

The polynucleotide of the present invention is a polynucleotide comprising (a), (b), (c), (d), (e), (f) or (g).
(A) a polynucleotide having the base sequence represented by SEQ ID NO: 1,
(B) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 2 or 6;
(C) having an amino acid sequence of at least 80%, preferably at least 85%, 90%, 92%, 95% or 98%, similar to the amino acid sequence set forth in SEQ ID NO: 2 or 6, and having L-tryptophan oxidase activity A polynucleotide encoding a protein having,
(D) having at least 60%, preferably at least 65%, 70%, 75%, 80%, 85%, 90% or 95% of the base sequence shown in SEQ ID NO: 1 and A polynucleotide encoding a protein having L-tryptophan oxidase activity;
(E) a polynucleotide that hybridizes with the polynucleotide of SEQ ID NO: 1 under a stringent condition and encodes a protein having L-tryptophan oxidase activity;
(F) deletion of at most 60, more preferably at most 55, 50, 40, 30, 20, 10 or 5 amino acids in the amino acid sequence set forth in SEQ ID NO: 2 or 6 A polynucleotide encoding a protein having L-tryptophan oxidase activity having substitutions and / or additions,
(G) having an amino acid sequence of at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90% or 95% with the amino acid sequence set forth in SEQ ID NO: 2 or 6; A polynucleotide encoding a protein having L-tryptophan oxidase activity.
For example, the polynucleotide which codes the protein which has the amino acid sequence of sequence number 3-5, 7-11, 16-18 can be illustrated, and this base sequence is shown in FIG.3 and FIG.4. Having an amino acid sequence of at least 80%, preferably at least 85%, 90%, 92%, 95% or 98% similarity to the sequences set forth in SEQ ID NOs: 3-5, 7-11, 16-18; It may be a polynucleotide encoding a protein having tryptophan oxidase activity.

  Furthermore, the polynucleotide of the present invention is preferably a polynucleotide encoding a protein having L-tryptophan oxidase activity having a sequence length of at most 650 amino acids, at most 630 amino acids or at most 600 amino acids. Further, in the amino acid sequence of the active enzyme which is a mature protein, the number of amino acids from the N-terminus to the first Gly of Gly-Xaa-Gly-Xaa-Xaa-Gly which is a FAD binding motif sequence is preferably at most 100 , 80, 70, 60 or 50, a polynucleotide encoding a protein having L-tryptophan oxidase activity.

  The identity is based on the value of identity calculated by homology analysis between the base sequences of GENETYX (manufactured by Genetics) or between amino acid sequences.

  Similarity is based on the value of Similarity calculated by homology analysis between amino acid sequences of GENETYX (Genetics).

  In the present invention, specific conditions for “hybridization under stringent conditions” for hybridization include, for example, 50% formamide, 5 × SSC (150 mM sodium chloride, 15 mM trisodium citrate, 10 mM sodium phosphate). After incubation at 42 ° C. with 1 mM ethylenediaminetetraacetic acid, pH 7.2), 5 × Denhardt's solution, 0.1% SDS, 10% dextran sulfate and 100 μg / mL denatured salmon sperm DNA, the filter was 0.2 X Washing at 42 ° C. in SSC can be exemplified.

  In the present invention, “polynucleotide” includes synthetic DNA encoding L-tryptophan oxidase, chromosomal DNA, cDNA synthesized from RNA, or polynucleotide obtained by PCR amplification using them as a template.

  Although the origin of chromosomal DNA or RNA is not particularly limited, it is preferably a fungus, more preferably a basidiomycete or ascomycete, more preferably a fungus belonging to the class Agaricidae, and particularly preferably the genus Coprinopsis or the genus Volvariella , Trichoroma genus, Bjerkandara genus, Cortinarius genus or Phanerochaete genus, and can be Colinopsin cinerea, Volvariella volvacea, Tricholoma matsutake, Bjerkandera, Bjerk.

  Chromosomal DNA or RNA can be extracted from the fungus to prepare a DNA or cDNA library. Subsequently, a primer for obtaining a polynucleotide encoding the enzyme is prepared based on analysis data of the N-terminal amino acid sequence and / or internal amino acid sequence of the purified L-tryptophan oxidase. Alternatively, a plurality of oligonucleotide probes or degenerate primers are prepared based on the alignment shown in FIGS. The polynucleotide of the present invention can be obtained from the library by a conventional method such as hybridization, PCR, or RT-PCR using the probe or primer.

  Specifically, the acquisition method based on the alignment comparing the base sequence homology was designed by designing a forward primer and a reverse primer for decoding the internal sequence from a site with high identity in the alignment, and using the library as a template. PCR is performed as follows. The primer design site is preferably a site with a sequence length of about 15 to 40 bases and a high identity, and the forward primer is preferably a site where the amplification side (downstream side) matches at least 2 bases, and a reverse primer Is preferably designed by selecting a site where the amplification side (upstream side) matches at least 2 bases and using mixed bases as appropriate. When PCR is performed using a primer designed from this site, the annealing temperature is set low, preferably 40-50 ° C, more preferably 40-45 ° C. The second stage PCR may be performed using the primer set inside the primer set used in the first stage. The size of the PCR product is predicted from the position of the base used for the primer, and the corresponding PCR product is decoded. If the decoded PCR product has at least 50% identity, preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identity with SEQ ID NO: 1 The internal sequence of the polynucleotide of the invention has been obtained. Next, the full length of the polynucleotide of the present invention can be obtained from the decoded internal sequence by a known method. That is, a primer is designed to elucidate the vicinity of the start codon and the stop codon of the ORF of the polynucleotide encoding the L-tryptophan oxidase of the present invention, and 5′-RACE is used using the primer as a template. And 3′-RACE method. As a result, the vicinity of the start codon and the stop codon of the ORF can be clarified. Subsequently, based on the alignment comparing the clarified sequence and the homology between SEQ ID NO: 2 and determining the N terminus of the clarified sequence corresponding to the N terminus (first amino acid) of SEQ ID NO: 2, Primers capable of amplifying a polynucleotide from the base encoding to the stop codon are designed. Preferably, using the primer, the polynucleotide of the present invention can be obtained using the library as a template.

  The polynucleotide of the present invention is subjected to homology search using, for example, BLAST (blastp or tblastn) with respect to a public sequence whose function is unknown using the amino acid sequence set forth in SEQ ID NO: 2, and the similarity is at least 80%, preferably at least It can be obtained from the base sequence encoding the amino acid sequence hit at 82%, 85%, 88%, 90%, 92%, 95%, 98%. Based on the alignment comparing the homology between the hit public sequence and SEQ ID NO: 2, the N-terminus of the hit sequence corresponding to the N-terminus (first amino acid) of SEQ ID NO: 2 is determined, and the N-terminus is encoded Primers that can amplify a polynucleotide from the base to the stop codon are designed from the published sequence. Subsequently, it can be obtained by amplification by PCR or RT-PCR using the DNA or RNA of the strain derived from the base sequence as a template. Further, a polynucleotide obtained by amplifying a recombinant protein by using a polynucleotide obtained by amplification and confirming the L-tryptophan oxidase activity by a conventional method is a polynucleotide encoding a protein having L-tryptophan oxidase activity. It only needs to be confirmed. DNA or RNA can also be obtained from strains of the same or same genus as the strain from which the public sequence is derived.

  The polynucleotide of the present invention can be prepared by modification by a known mutation introduction method, mutation introduction PCR method or the like. In addition, the polynucleotide of the present invention is synthesized by a well-known chemical synthesis technique based on the base sequence encoding the amino acid sequence shown in SEQ ID NOs: 2 to 11, for example, the base sequence information shown in FIGS. Can do.

  The recombinant vector of the present invention is a cloning vector or an expression vector, and the vector is appropriately selected and contains the polynucleotide of the present invention as an insert. As the insert, a polynucleotide having an optimized codon usage frequency may be introduced corresponding to the host cell. Further, the insert may contain an intron when the host is a eukaryotic cell. When the gene does not include a start codon, a vector that is expressed as a fusion protein may be selected by adding the start codon or using the start codon on the vector side. The expression vector may be either a prokaryotic expression vector or a eukaryotic expression vector, and examples thereof include a pUC system, pBluescript II, pET expression system, pGEX expression system, and pCold expression system. If necessary, genes such as chaperones and lysozymes can be introduced into the same and / or different vectors as the polynucleotide of the present invention.

  Examples of the transformed cells of the present invention include prokaryotic cells such as Escherichia coli and Bacillus subtilis, eukaryotic cells such as fungi (yeast, Aspergillus or other ascomycetes, basidiomycetes, etc.), insect cells, mammalian cells, and the like. Can be used.

  Soluble L-tryptophan oxidase can be produced by collecting L-tryptophan oxidase from the culture obtained by culturing the transformed cells of the present invention.

  For culturing L-tryptophan oxidase producing bacteria used in the present invention, a normal culture medium for microorganisms can be used. Carbon sources, nitrogen sources, vitamins, inorganic substances, and other micronutrients required by microorganisms to be used are moderately used. As long as it contains, any of a synthetic medium and a natural medium can be used. As the carbon source, glucose, sucrose, lactose, dextrin, starch, glycerin, molasses and the like can be used. Nitrogen sources include inorganic salts such as ammonium chloride, ammonium nitrate, ammonium sulfate and ammonium phosphate, amino acids such as DL-alanine and L-glutamic acid, nitrogen content such as peptone, meat extract, yeast extract, malt extract and corn steep liquor Natural products can be used. As vitamins, riboflavin, pyridoxine, niacin (nicotinic acid), thiamine and the like can be used. As the inorganic substance, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, ferric chloride and the like can be used. Furthermore, compounds necessary for adjusting protein expression may be added as appropriate.

  The culture for obtaining the L-tryptophan oxidase of the present invention is not particularly limited as long as the culture conditions are suitable for the production of L-tryptophan oxidase, but the culture temperature is in the range of 10 ° C to 45 ° C and pH 4 to pH10. It is preferred to do so. If the culture period is batch culture, a range of 1 to 8 days is preferable. However, if the production amount is increased by fed-batch culture or continuous culture, the period is extended within a range where productivity is determined to be optimal. be able to.

  As a method for obtaining L-tryptophan oxidase from the culture, an ordinary protein purification method can be used. In this method, for example, after culturing L-tryptophan oxidase producing bacteria, the culture solution is centrifuged to obtain a culture supernatant, or a cultured microorganism is obtained, and the cultured microorganism is disrupted and / or lysed by an appropriate method. In this method, a supernatant is obtained from the treatment solution by centrifugation or the like. L-tryptophan oxidase contained in these supernatants is appropriately purified by ultrafiltration, salting out, solvent precipitation, heat treatment, dialysis, ion exchange chromatography, hydrophobic adsorption chromatography, gel filtration, affinity chromatography, etc. It can be purified by a combination of operations.

  The soluble L-tryptophan oxidase of the present invention is preferably a recombinant L-tryptophan oxidase obtained from the transformed cell transformed with the polynucleotide of the present invention.

The L-tryptophan oxidase of the present invention is the following (1), (2), (3) and (4), preferably (5), (6) or (7).
(1) An enzyme that catalyzes a reaction that oxidizes L-tryptophan in the presence of an electron acceptor to produce indolepyruvic acid, hydrogen peroxide, and ammonia, and can exchange electrons with oxygen.
(2) Flavin is bound as a coenzyme. Examples of the flavin include flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN).
(3) A protein comprising the following (i), (ii), (iii) or (iv).
(I) A protein having the amino acid sequence of SEQ ID NO: 2 or 6.
(Ii) L-tryptophan having an amino acid sequence that is at least 80%, preferably at least 85%, 88%, 90%, 92%, 95% or 98% similar to the amino acid sequence set forth in SEQ ID NO: 2 or 6; It is a protein having oxidase activity.
(Iii) Deletion of at most 60, more preferably at most 55, 50, 40, 30, 20, 10, or 5 amino acids in the amino acid sequence set forth in SEQ ID NO: 2 or 6 A protein having L-tryptophan oxidase activity having substitutions and / or additions.
(Iv) having at least 60%, preferably at least 65%, 70%, 75%, 80%, 85%, 90% or 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 or 6; It is a protein having L-tryptophan oxidase activity.
(4) It is a non-membrane bound enzyme and is soluble.

(5) High thermal stability, assuming that the activity before heat treatment is 100%, the activity is preferably 80% or more, more preferably 90% or more after treatment at 40 ° C. for 1 hour.
(6) A protein having L-tryptophan oxidase activity having a sequence length of at most 650 amino acids, at most 630 amino acids, and at most 600 amino acids. More preferably, it is a modified enzyme in which 1 to several amino acids starting from methionine are added to the tip of the amino acid sequence of the mature protein.
(7) In the amino acid sequence of the active enzyme that is a mature protein, the number of amino acids from the N-terminus to the first Gly of Gly-Xaa-Gly-Xaa-Xaa-Gly that is the FAD-binding motif sequence is preferably at most 100 It is a protein that is 80, 70, 60 or 50.
For example, a protein having the amino acid sequence set forth in SEQ ID NOs: 3-5, 7-11, 16-18, and similarity to the sequence is at least 80%, preferably at least 85%, 90%, 92%, 95% or 98 % Of the amino acid sequence and a protein having L-tryptophan oxidase activity.

  The L-tryptophan oxidase of the present invention has a low activity on L-phenylalanine and L-tyrosine when the activity on L-tryptophan is taken as 100%, preferably 3% or less, more preferably 2% or less. More preferably, it is 1% or less. Since the L-tryptophan oxidase of the present invention has a high substrate specificity for L-tryptophan, a specific L-tryptophan measurement is possible.

The enzyme activity of L-tryptophan oxidase of the present invention can be measured by the following method.
(Enzyme activity measurement method)
Potassium phosphate buffer (pH 7.0) is final concentration 33.3 mM, 4-aminoantipyrine is final concentration 0.833 mM, phenol solution is final concentration 14 mM, horseradish peroxidase is final concentration 3.33 units / ml, L-tryptophan Is placed in a quartz cell so as to have a final concentration of 33.3 mM, set in a spectrophotometer equipped with a thermostatic cell holder and incubated at 37 ° C. for 10 minutes, and then added with 0.1 ml of enzyme solution and stirred (total liquid volume 3. 0 ml), the absorbance change (ΔABS / min) for 5 minutes at 500 nm is measured. Then, the enzyme activity is calculated by the following formula. In addition, n in a formula represents the dilution rate of an enzyme. The enzyme activity that oxidizes 1 μmol of L-tryptophan per minute is defined as 1 unit (U).

  L-tryptophan can be efficiently measured using the L-tryptophan oxidase of the present invention. The method includes a step of bringing a test sample containing L-tryptophan into contact with the L-tryptophan oxidase of the present invention. By producing hydrogen peroxide, L-tryptophan in the test sample can be quantified. Although the measuring object of this invention is not specifically limited, For example, a biological sample can be illustrated and it is suitable for the measurement of a blood sample.

  Moreover, the reagent composition for L-tryptophan measurement can be manufactured using the L-tryptophan oxidase of this invention. The reagent composition of this invention should just be a reagent composition which contains L-tryptophan oxidase of this invention as an enzyme. The amount of enzyme in the composition is not particularly limited as long as L-tryptophan in the sample can be measured, but is preferably about 0.001 to 200 U, more preferably about 0.05 to 50 U per sample. The reagent composition can be produced by mixing the enzyme and other components. For example, a stability selected from the group consisting of bovine serum albumin (BSA) or ovalbumin, saccharides (eg trehalose, etc.) or sugar alcohols, carboxyl group-containing compounds, alkaline earth metal compounds, ammonium salts, sulfates or proteins. Stabilizing components known to those skilled in the art, such as agents, antioxidants, or buffers, can be added as appropriate to enhance the thermal stability and storage stability of the enzyme and reagent components. Moreover, the said stabilizer and another substance can also be used for the said reagent composition for the purpose of a reactivity improvement or a specific improvement.

  An L-tryptophan measurement kit can be produced using the L-tryptophan oxidase of the present invention or the reagent composition.

  The measuring method, measuring reagent composition and measuring kit of the present invention have low activity on L-phenylalanine and L-tyrosine, and are preferably 200 μM or less, more preferably 150 μM or less, even more preferably 100 μM or less, particularly preferably in the sample. Preferably, the activity on L-phenylalanine and L-tyrosine of 80 μM or less is low. In the measurement using the measurement method of the present invention and the measurement reagent composition or measurement kit, when the activity on L-tryptophan is 100%, the activity on L-phenylalanine and L-tyrosine in the sample is: In any case, it is preferably 3% or less, more preferably 2% or less, and still more preferably 1% or less. In addition, the measuring method, measuring reagent composition and measuring kit of the present invention are hardly affected by L-glutamine, preferably 1000 μM or less, more preferably 800 μM or less, and even more preferably 600 μM or less. Hardly receive. The difference in the measured L-tryptophan value in the presence and absence of L-glutamine is preferably within 15%, more preferably within 10%, and even more preferably within 5%. Therefore, if the measurement method, measurement reagent composition, and measurement kit of the present invention are used, L-tryptophan in a sample containing impurities can be specifically measured. In particular, the measuring method, measuring reagent composition and measuring kit of the present invention can be used for measuring L-tryptophan in the presence of L-glutamine, and L-glutamine is present in the highest concentration of amino acids in blood. Therefore, it is suitable for use in measuring L-tryptophan in blood.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by a following example.

[Example 1]
(Method for producing L-tryptophan oxidase (TRO))
(1) Acquisition of polynucleotide encoding L-tryptophan oxidase A strain producing L-tryptophan oxidase isolated from soil was cultured, and L-tryptophan oxidase was purified from the obtained culture.
As a result of analyzing the N-terminus and the internal amino acid sequence of the purified enzyme, the N-terminus was TDPHQ.
Primers for 5′-RACE method and 3′-RACE method are prepared from the internal amino acid sequence, and 5′-RACE method and 3′-RACE method are performed using DNA extracted from the enzyme production strain as a template, The base sequence of a part of the polynucleotide coding for was elucidated.
From the elucidated base sequence, an ORF containing a polynucleotide encoding the enzyme was predicted, and a primer for obtaining a polynucleotide from the initiation codon to the termination codon was prepared. Using this primer, RT-PCR was performed using RNA extracted from the enzyme production strain as a template to elucidate the base sequence of ORF.
From the N-terminal sequence of the enzyme, a base sequence encoding an enzyme that is a mature protein was predicted and shown in SEQ ID NO: 1. The amino acid sequence of the enzyme is shown in SEQ ID NO: 2.

(2-1) Production of L-tryptophan oxidase by recombinant microorganisms Inserted by RT-PCR using the following primers 1 and 2 (SEQ ID NOs: 12 and 13) using RNA extracted from an L-tryptophan oxidase producing strain as a template Was prepared. The insert is DNA having atg added to the upstream of the base sequence shown in SEQ ID NO: 1, having a NheI restriction enzyme site, and having a XhoI restriction enzyme site downstream. The insert was inserted into pET-21 (a) + vector to obtain vector pET21-T into which a polynucleotide encoding L-tryptophan oxidase was inserted. The vector was inserted into E. coli JM109, amplified and isolated, and then transformed into E. coli BL21 (DE3). As a result, transformed cell 1 (recombinant E. coli) having DNA containing SEQ ID NO: 1 was obtained.
Primer 1: 5'-CTAGCTAGCATG ACCGATCCTCACCAGCCC-3 '
Primer 2: 5'-CCGCTCGAGTTA TTAAGGGGTTTGCAAGTCCAACTT-3 '

The recombinant Escherichia coli was collected by culturing for 2 days, and the collected cells were disrupted in 50 mM Tris buffer (pH 8.0) and centrifuged to recover the centrifugal supernatant (soluble fraction). . The culture was started at 20 ° C., changed to 15 ° C. after 24 hours, and then IPTG was added.
Since L-tryptophan oxidase activity was confirmed in the centrifugal supernatant, it was found that soluble L-tryptophan oxidase was obtained, and L-tryptophan oxidase was obtained by short-term culture.
L-tryptophan oxidase obtained by this production method has the amino acid sequence set forth in SEQ ID NO: 16.
Next, the supernatant was heat-treated at 40 ° C. for 1 hour, and then the supernatant obtained by centrifugation was collected and used as an L-tryptophan oxidase sample.

(2-2) Production of L-tryptophan oxidase by recombinant microorganism Using the vector pET21-T obtained in 2-1 as a template, the insert was obtained by PCR using the following primers 3 and 4 (SEQ ID NOs: 14 and 15). Prepared. The insert is DNA having atg added to the upstream of the base sequence shown in SEQ ID NO: 1, having a HindIII restriction enzyme site, and having an XbaI restriction enzyme site downstream. The insert was inserted into a pColdIII vector to obtain a vector pCold-T into which a polynucleotide encoding tryptophan oxidase was inserted. The vector was inserted into E. coli JM109, amplified and isolated, and then transformed into E. coli BL21 (DE3). As a result, transformed cell 2 (recombinant E. coli) carrying DNA containing SEQ ID NO: 1 was obtained.
Primer 3: 5'-CCCAAGCTT ATGACCGATCCTCACCAGCCC-3 '
Primer 4: 5'-CTATCTAGATTA TTAAGGGGTTTGCAAGTCCAACTT-3 '

The recombinant Escherichia coli was collected by culturing for 1 day, and the collected cells were disrupted in 50 mM Tris buffer (pH 8.0) and centrifuged to collect the centrifugal supernatant (soluble fraction). . In addition, culture | cultivation started culture | cultivation at 37 degreeC, and after changing to 15 degreeC after 5.5 hours, IPTG was added.
Since L-tryptophan oxidase activity was confirmed in the centrifugal supernatant, it was found that soluble L-tryptophan oxidase was obtained, and L-tryptophan oxidase was obtained by short-term culture.
L-tryptophan oxidase obtained by this production method has the amino acid sequence set forth in SEQ ID NO: 17.

[Example 2]
(Characteristics of L-tryptophan oxidase)
(1) Thermal stability The enzyme activity before and after the heat treatment in Example 1 (2-1) was measured. As a result, assuming that the L-tryptophan oxidase activity before the heat treatment was 100%, the activity after the heat treatment was 90%. The inventive L-tryptophan oxidase has been found to have high thermal stability.

(2) Activity Using L-tryptophan oxidase of Example 1 (2-1), the activity on L-tryptophan, L-phenylalanine or L-tyrosine was confirmed. In the activity measurement method, 0, 20, 40, 60 and 80 μM L-tryptophan, L-phenylalanine or L-tyrosine was used as a substrate. Note that 0.37 U of any L-tryptophan oxidase was used.
The measurement results (calibration curve) for each substrate are shown in FIG. The activity against 80 μM L-tryptophan was taken as 100% and expressed as the relative activity at each substrate concentration.
The slope of the calibration curve for each substrate was calculated, and the activity for L-phenylalanine and L-tyrosine was calculated with the value for L-tryptophan as 100%. The activity on each substrate was in the range of 0 to 80 μM. 0.59% and 0.86%.
As described above, the L-tryptophan oxidase of the present invention is an enzyme having a low substrate activity of 1% or less and a high substrate specificity, and can be used for specific measurement of L-tryptophan. It turns out that it is available.

[Example 3]
(Measurement of L-tryptophan)
Using L-tryptophan oxidase of Example 1 (2-1), L-tryptophan was measured in the presence or absence of L-glutamine. In the activity measurement method, 0, 20, 40, 60, and 80 μM L-tryptophan was used as a substrate, and in the presence of L-glutamine, 600 μM L-glutamine was further added and measured.
It is known that L-glutamine is present at the highest concentration of about 600 μM among the amino acids present in the blood.

The measurement results are shown in FIG. Each activity was shown as a relative activity, assuming that the activity against 80 μM L-tryptophan in the absence of L-glutamine was 100%.
In the measurement of L-tryptophan using the L-tryptophan oxidase of the present invention, there was almost no difference in the relative activity of L-tryptophan in the presence and absence of L-glutamine.
Therefore, it was found that the measurement using the L-tryptophan oxidase of the present invention enables accurate L-tryptophan measurement even in the presence of 600 μM L-glutamine. Furthermore, good linearity was observed between the L-tryptophan concentration and the relative activity even when the measurement time was as short as 5 minutes.
As described above, the measurement method of the present invention is hardly affected by L-glutamine. For example, even a sample in which other amino acids are contaminated, such as a blood sample having a high L-glutamine concentration, can be rapidly and accurately detected. It seems that the measurement of is possible.

[Example 4]
(Method 2 for producing L-tryptophan oxidase)
(1) Search for L-tryptophan oxidase Using the amino acid sequence described in SEQ ID NO: 2, a homology search was performed on the public sequence by BLAST (blastp) to obtain a sequence having high similarity. By comparing each acquired sequence with SEQ ID NO: 2, the amino acid sequence of the active enzyme which is a mature protein was determined, and shown in SEQ ID NOs: 3 to 11. As a result of homology analysis of the amino acid sequence described in SEQ ID NO: 2 and each sequence using GENETYX, the similarity between the amino acid sequence described in SEQ ID NO: 2 and SEQ ID NO: 3 to 11 is 95% of SEQ ID NO: 3 and 4 SEQ ID NO: 5 was 93%, SEQ ID NO: 6 was 92%, SEQ ID NOs: 7 to 10 were 88%, and SEQ ID NO: 11 was 87%. It was speculated that the proteins having these sequences have L-tryptophan oxidase activity.
The results of alignment of the base sequences encoding the amino acid sequences described in SEQ ID NOs: 2 to 11 using GeneDoc are shown in FIGS. 3 and 4.

(2) Production of L-tryptophan oxidase by recombinant microorganisms 2
Based on the result of (1), recombinant production of L-tryptophan oxidase was performed using a polynucleotide encoding the amino acid sequence described in SEQ ID NO: 6.
An insert DNA was synthesized in which NdeI containing the start codon atg was added upstream of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6 and the codon usage frequency was optimized, and an XhoI restriction enzyme site was added downstream. The insert was inserted into the pET-21 (a) + vector to obtain a vector into which the polynucleotide encoding the amino acid sequence described in SEQ ID NO: 6 was inserted. The vector was inserted into E. coli JM109, amplified and isolated, and then transformed into E. coli BL21 (DE3). As a result, a transformed cell (recombinant Escherichia coli) having a DNA containing a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 6 was obtained.

The recombinant Escherichia coli was collected by culturing for 3 days, and the collected cells were disrupted in 50 mM Tris buffer (pH 8.0) and centrifuged to collect the centrifugal supernatant (soluble fraction). . The culture was started at 37 ° C., changed to 15 ° C. after 3.5 hours, and then IPTG was added.
Since L-tryptophan oxidase activity was confirmed in the centrifugal supernatant, it was found that soluble L-tryptophan oxidase was obtained, and that L-tryptophan oxidase was obtained by short-term culture.
L-tryptophan oxidase obtained by this production method has the amino acid sequence set forth in SEQ ID NO: 18.

From the above, it was found that L-tryptophan oxidase can be produced from the polynucleotide encoding the amino acid sequence described in SEQ ID NO: 6.
Therefore, a protein having the amino acid sequence described in SEQ ID NOs: 3 to 5 and 7 to 11 also has L-tryptophan oxidase activity, and also from a polynucleotide encoding the amino acid sequence described in SEQ ID NOs: 3 to 5 and 7 to 11, Similarly, it is speculated that L-tryptophan oxidase can be produced.

Claims (15)

A polynucleotide comprising the following (a), (b) or (c):
(A) a polynucleotide having the base sequence represented by SEQ ID NO: 1,
(B) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 2 or 6;
(C) A polynucleotide encoding a protein having an amino acid sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO: 2 or 6 and having L-tryptophan oxidase activity.   A recombinant vector carrying the polynucleotide according to claim 1.   A transformed cell having the polynucleotide according to claim 1.   A method for producing L-tryptophan oxidase, comprising culturing the transformed cell according to claim 3 and collecting L-tryptophan oxidase from the obtained culture. Soluble L-tryptophan oxidase consisting of the following (i), (ii) or (iii):
(I) a protein having the amino acid sequence of SEQ ID NO: 2 or 6,
(Ii) a protein having an amino acid sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO: 2 or 6, and having L-tryptophan oxidase activity;
(Iii) A protein having L-tryptophan oxidase activity having a deletion, substitution and / or addition of at most 55 amino acids in the amino acid sequence of SEQ ID NO: 2 or 6.   6. The L-tryptophan oxidase according to claim 5, wherein the activity on L-phenylalanine and L-tyrosine is 1% or less when the activity on L-tryptophan is 100%. A method for measuring L-tryptophan using the tryptophan oxidase according to claim 5 or 6.   The method for measuring L-tryptophan according to claim 7, wherein the activity on L-phenylalanine and L-tyrosine is 1% or less when the activity on L-tryptophan is 100%.   The method for measuring L-tryptophan in blood according to claim 7 or 8.   A reagent composition for measuring L-tryptophan comprising the tryptophan oxidase according to claim 5 or 6.   The reagent composition for measuring L-tryptophan according to claim 10, wherein the activity for L-phenylalanine and L-tyrosine is 1% or less when the activity for L-tryptophan is 100%.   The reagent composition for measuring blood L-tryptophan according to claim 10 or 11.   A kit for measuring L-tryptophan comprising the tryptophan oxidase according to claim 5 or 6.   14. The kit for measuring L-tryptophan according to claim 13, wherein the activity on L-phenylalanine and L-tyrosine is 1% or less when the activity on L-tryptophan is 100%.   The kit for measuring blood L-tryptophan according to claim 13 or 14.

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