JPH07170976A - Novel enzyme having n-methylvaline oxidase - Google Patents

Abstract

PURPOSE:To obtain a novel N-methylvaline oxidase which is useful in determination of N-methylvaline. CONSTITUTION:This oxidase acts on N-methylvaline in the presence of water and oxygen to form valine, formaldehyde and hydrogen peroxide where the substrate specificity is 100% for N-methylvaline and less than 1% for sarcosine, the optimal pH is 7-9, the optimal temperature is 40 to 50 deg.C, and the molecular weight is 43kd. The enzyme is obtained by substituting another amino acid (preferably valine) for phenylalanine at No.103 in the amino acid sequence of a gene given in the formula of the protein having the sarcosine oxidase activity.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel enzyme having N-methylvaline oxidase activity, a method for producing the same and its use, and in particular, it is obtained by modifying an amino acid sequence constituting a protein having sarcosine oxidase activity. The present invention relates to a novel enzyme having N-methylvaline oxidase activity, a method for producing the same, and use thereof.

[0002]

2. Description of the Related Art An enzyme that acts on N-methylvaline to produce hydrogen peroxide has not yet been found in nature.
Nor is it artificially produced.

[0003]

DISCLOSURE OF THE INVENTION The present invention has been earnestly studied in order to find N-methylvaline oxidase which is useful for the measurement of N-methylvaline. As a result, a protein having sarcosine oxidase activity was obtained by a protein engineering technique. It was found to be modified to create a new enzyme with N-methylvaline oxidase activity.

[0004]

That is, the present invention provides an N-methylvaline oxidase activity characterized by acting on N-methylvaline in the presence of water and oxygen to produce valine, formaldehyde and hydrogen peroxide. It is a novel enzyme having.

Further, the present invention is characterized in that phenylalanine in the amino acid sequence constituting a protein having sarcosine oxidase activity is replaced with another amino acid.
-A method for producing a novel enzyme having methylvaline oxidase activity. Specifically, the gene encoding phenylalanine at the 103rd position in the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing is site-specifically mutated to be replaced with another amino acid to obtain N-methylvaline oxidase activity. It is characterized by producing an enzyme having
Valine is preferred as the other amino acid.

The present invention further relates to N-methylvaline in a sample, which is a novel enzyme having N-methylvaline oxidase activity which acts on N-methylvaline in the presence of water and oxygen to produce valine, formaldehyde and hydrogen peroxide. Is used to measure oxygen consumed and valine, formaldehyde or hydrogen peroxide produced, and is a method for quantifying N-methylvaline.

Specific examples of the enzyme of the present invention having an N-methylvaline oxidase activity which acts on N-methylvaline in the presence of water and oxygen to produce valine, formaldehyde and hydrogen peroxide are as follows: And an enzyme having Action: acts on N-methylbatine in the presence of water and oxygen to produce valine, formaldehyde and hydrogen peroxide. Substrate specificity: N-methylvaline 100% Sarcosine ≤1% Optimum pH: 7-9 Optimum temperature: 40-50 ° C Molecular weight: 43Kd

[0008] As a more specific example, the amino acid sequence is the first of the amino acid sequences listed in SEQ ID NO: 1 of the Sequence Listing.
N- in which the 03rd phenylalanine is replaced with valine
A novel protein having methylvaline oxidase activity can be mentioned.

The novel enzyme of the present invention is produced by substituting phenylalanine with another amino acid in the amino acid sequence constituting the protein having sarcosine oxidase activity.

The sarcosine oxidase used in the present invention includes, for example, sarcosine oxidase belonging to the genus Bacillus, Corynebacterium, and Arthrobacter, and is not particularly limited. Lorobacter sp. TE1826 sarcosine oxidase (Jounal of Fermentation and Bioen
gineering Vol.75 No.4 pp239-244 (1993)) was used.

As a method for modifying an amino acid sequence constituting a protein having sarcosine oxidase activity, a commonly used method for modifying genetic information is used. That is, by converting a specific base of DNA having genetic information of a protein having sarcosine oxidase activity, or by inserting or deleting a specific base, D having the genetic information of a modified protein can be obtained.
NA is created.

A specific method for converting the bases in DNA is, for example, a commercially available kit (Transformer ™; Clonet).
ech, T7-GEN in vitro mutagenesis kit ； Stratage
ne)) or the use of PCR method.

Specifically, first, chromosomal DNA is isolated from cells that produce the parent protein. Obtained chromosomal DNA
Is partially digested with a restriction enzyme, for example, Sau3aI, separated into fragments, and the DNA is ligated with a plasmid and a DNA ligase cleaved with the same restriction enzyme. The ligated DNA is transformed with Escherichia coli competent cells. The obtained colonies are cultured in a medium and the recombinant DNA having the gene inserted therein is screened. Then, the inserted DNA fragment is cleaved with various restriction enzymes and subcloned into another plasmid to obtain a plasmid having the inserted DNA fragment. Various subclones are
QUENASE VERSION2.0 7-deaza-d GTP kit (manufactured by Toyobo)
Is used for sequencing.

Next, using an oligonucleotide in which the base encoding phenylalanine is replaced with a base encoding another amino acid and TransformerTM (manufactured by Clonetech),
According to the Transformer ™ protocol, a DNA having the genetic information of a modified protein in which a phenylalanine residue is replaced with another amino acid, preferably valine, is prepared.

The prepared DNA containing the genetic information of the modified protein is transferred into the host microorganism in a state of being linked with the plasmid, and becomes a transformant producing the modified protein. The plasmid used in this case is, for example, pBluescr when Escherichia coli is used as the host microorganism.
You can use ipt, pUC18, etc.

As the host microorganism, for example, Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, Escherichia coli DH5α and the like can be used. As a method for transferring the recombinant vector to the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, a method of transferring the recombinant DNA in the presence of calcium ions can be adopted. An electroporation method may be used.

The thus obtained transformant microorganism can stably produce a large amount of modified protein by culturing in a nutrient medium. Regarding the culture form of the host microorganism that is a transformant, the culture conditions may be selected in consideration of the nutritional physiological properties of the host. Usually, in most cases, liquid culture is performed, but industrially, aeration stirring culture is performed. Is advantageous.
As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, and for example, glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid, etc. are used. Any available nitrogen compound may be used as the nitrogen source, for example, peptone, meat extract,
Yeast extract, casein hydrolyzate, soybean meal alkaline extract and the like are used. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids, specific vitamins and the like are used as necessary.

The culture temperature can be appropriately changed within the range in which the bacterium grows and produces the modified protein, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C. Although the culturing time varies depending on the conditions, it may be completed at a suitable time in consideration of the time when the maximum yield of the modified protein is reached, and it is usually about 6 to 48 hours. The medium pH can be appropriately changed within a range in which the bacterium grows and produces a modified protein,
Particularly preferably, the pH is about 6.0 to 9.0.

The culture solution containing the cells producing the modified protein in the culture can be collected and used as it is. Generally, when the modified protein is present in the culture solution, filtration, centrifugation or the like is carried out. Is used after separating the modified protein-containing solution from the microbial cells. When the modified protein is present in the microbial cells, the obtained culture is harvested by means such as filtration or centrifugation, and then the microbial cells are disrupted by a mechanical method or an enzymatic method such as lysozyme. If necessary, a chelating agent such as EDTA and / or a surfactant is added to solubilize the modified protein, and the solution is separated and collected as an aqueous solution.

The modified protein-containing solution thus obtained is subjected to, for example, vacuum concentration, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol or acetone. It can be made to precipitate by. Further, heat treatment and isoelectric point treatment are also effective refining means. A purified modified protein having N-methylvaline oxidase activity can be obtained by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, or affinity chromatography.

The novel enzyme having N-methylvaline oxidase activity of the present invention acts on N-methylvaline oxidase in a sample in the presence of water and oxygen to produce valine, formaldehyde and hydrogen peroxide.
Then, by measuring the amount of oxygen consumed or the amount of produced alline, formaldehyde or hydrogen peroxide, N-
Methylvaline can be quantified. As a sample,
Examples include amino acid derivative synthetic intermediates, pharmaceutical synthetic intermediates, and N-methyl amino acid research samples.

As a measuring method of oxygen consumed, there is a method of utilizing an oxygen electrode. Further, as a method for measuring the produced valine, there is a method using an amino acid analyzer. Further, as a method of measuring the formaldehyde produced, there is a method of utilizing formaldehyde dehydrogenase. As a method for measuring the produced hydrogen peroxide, a conventionally known method may be used, for example, a method using peroxidase, 4-aminoantipyrine and a phenol derivative or an aniline derivative.
Examples of the phenol derivative include phenol, 2,4-dichlorophenol, p-chlorophenol and the like. Examples of the aniline derivative include dimethylaniline, diethylaniline, N-ethyl-N- (2-hydroxy-3
-Sulfopropyl) -m-toluidine and the like.

[0023]

EXAMPLES The present invention will be specifically described below with reference to examples. In the examples, Km and kca, which are indices showing enzyme characteristics,
Measurement of N-methylvaline oxidase activity or sarcosine oxidase activity when determining t was performed as follows. That is, 48 mM Tris buffer (pH 8.0), substrate at any concentration (N-methylvaline or sarcosine), 0.
47 mM 4-aminoantipyrine, 2.0 mM phenol, 0.04
The enzyme is reacted in 5% Triton X-100, 4.5 U / ml peroxidase at 37 ° C. for 10 minutes, and the absorbance at 500 nm is measured.

Example 1 Preparation of DNA carrying genetic information for N-methylvaline oxidase A recombinant plasmid, pSAOEP3 carrying genetic information for sarcosine oxidase, was prepared by the following method. EARTHROBACTER SP TE1826
No. 0637) was isolated by the following method. The strain was cultured in 100 ml of 2 × YT medium (1.6% polypeptone, 1% yeast extract, 0.5% sodium chloride (pH7.2) at 37 ° C. overnight with shaking, and then collected by centrifugation (8000 rpm, 10 minutes). After washing the cells with a solution containing 15 mM sodium citrate and 0.15 M sodium chloride, 20% sucrose, 1 mM EDT
A, suspend in 5 ml of a solution containing 50 mM Tris-HCl (pH 7.6) and add 0.5 ml of lysozyme solution (100 mg / ml).
The temperature was kept at 30 ° C for 30 minutes. Then 11 ml of a solution containing 1% lauroyl sarcosinic acid, 0.1 M EDTA (pH 9.6) was added.
To this suspension, 0.5% of ethidium bromide solution and about 100% of cesium chloride were added, stirred and mixed, and DNA was fractionated by ultracentrifugation at 55.000 rpm for 20 hours. The separated DNA was dialyzed against a solution (TE) containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA to obtain a purified DNA preparation. Escherichia Collie
Competent cells of JM109 were prepared by the method of Hanahan and used as a host for library preparation.

1 μg of chromosomal DNA was digested with the restriction enzyme Sau3AI
PUC18 0.5 cleaved with SalI (manufactured by Toyobo) after partial decomposition reaction (manufactured by Toyobo) to decompose into fragments of 2 kbp or more.
μg and MG Loftus et al.'s BACKFILLING method (Biotechniques
Vol12, No. 2 (1992)), T4-DNA ligase (manufactured by Toyobo) was reacted at 16 ° C. for 12 hours to ligate the DNA. The ligated DNA is Escherichia coli JM109
Transformation was performed using the competent cells of. Used D
About 1 × 10 ⁶ transformant colonies were obtained per 1 μg of NA. The resulting colonies were 50 μg / ml ampicillin, 0.5% sarcosine, 0.005% pararose aniline and 0.
The recombinant DNA containing the sarcosine oxidase gene was cultured in an L medium containing 025% sodium hydrogen sulfite (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride) at 37 ° C. for 18 hours, and the red colony was used as an index. Screened. As a result, a colony showing a red color was obtained at a rate of 1 strain out of about 1,000 colonies. An inserted DNA fragment of about 8.7 kbp was present in the plasmid possessed by one of these strains, and this plasmid was designated as pSAO1. Then, the inserted DNA fragment from pSAO1 was cleaved with various restriction enzymes and subcloned into pUC18 to obtain about 1.7 k.
pSAOEP3 having a bp inserted DNA fragment was obtained.

Subclones were prepared by digesting the approximately 1.7 kbp insert DNA fragment of pSAOEP3 with various restriction enzymes. Various subclones can be
Using VERSION2.0 7-deaza-dGTP kit (manufactured by Toyobo),
The DNA sequence was determined (Sequence Listing, SEQ ID NO: 2). The amino acid sequence determined from the DNA sequence is shown in SEQ ID NO: 1 in the sequence listing. Then, using the oligonucleotide of SEQ ID NO: 3 in the sequence listing and TransformerTM (manufactured by Clonetech),
According to the ansformer ™ protocol, a DNA having the genetic information of a modified protein (F103V) in which phenylalanine at the 103rd position in SEQ ID NO: 1 was replaced with valine was prepared. A recombinant plasmid carrying a DNA having the genetic information of F103V was designated as pSAOEP3-F103.
I named it V. (See Figure 1)

Example 2 Preparation of transformant Escherichia coli JM with plasmid pSAOEP3-F103V
After contacting 109 competent cells in ice for 30 minutes, 42 ℃
Transformation was performed by heat shock for 45 seconds to obtain a transformant JM109 (pSAOEP3-F103V).

Example 3 Cultivation of transformants and purification of modified protein 2 × YT medium (1.6% polypeptone, 1% yeast extract, 0.5%
50 ml of sodium chloride (pH 7.2) was dispensed into a 500 ml flask, autoclaved at 121 ° C. for 15 minutes and allowed to cool, and then 0.1% of 50 mg / ml ampicillin (manufactured by Nacalai Tesque) that had been separately sterile filtered was added. A transformant (JM109 (pSA
1 ml of a culture solution of OEP3-F103V)) was inoculated and cultured at 37 ° C. with aeration and stirring. Modified protein (F103V) from the culture solution,
After cell disruption, nucleic acid removal, and salting out, ion exchange column chromatography was performed (Journal of Fermenta
tion and Bioengineering Vol.75 No.4 pp239-244 (199
See 3)), and purified by SDS-polyacrylamide gel electrophoresis until a single band was formed. The molecular weight of the purified protein was 43 Kd, the optimum pH was 7 to 9, and the optimum temperature was 40 to 50 ° C.

Purified modified protein (F103V)
Table 1 shows the measured Km and kcat values of sarcosine oxidase (produced by Toyobo Co., Ltd.) of Arthrobacter SP TE1826.

[0030]

[Table 1]

The characteristics of the modified protein (F103V) are as follows:
It has N-methylvaline oxidase activity. Looking at kcat / Km, which is an index indicating the reactivity of the enzyme, the reactivity of sarcosine oxidase to sarcosine is about 54 times that of N-methylvaline, whereas the modified protein (F103V) is N-methylvaline. About 10 times the reactivity to sarcosine
It was 5 times. That is, the modified protein (F103
It was revealed that V) is a novel enzyme having N-methylvaline oxidase activity.

Example 4 Measurement of N-methylvaline using N-methylvaline oxidase The concentration of N-methylvaline in a sample was measured by the following measuring method using the following reagents. Reagents Tris buffer (pH 8.0) 50 mM F103V or sarcosine oxidase 0.1 mg / ml 4-aminoantipyrine 0.42 mM phenol 1.8 mM peroxidase 4.7 U / ml

Measuring method: N-methylvaline aqueous solution 2 mM (dissolved in 100 mM Tris buffer (pH 8.0)) 10-step dilution was used as a sample, 100 μl of each was sampled, 3 ml of the above reagent was added thereto, and the mixture was added at 30 ° C. After reacting for 3 minutes, the absorbance at 500 nm was determined. As a blank, distilled water was used instead of the test liquid containing N-methylvaline. FIG. 1 shows the dilution linearity of the sample. As is clear from FIG. 1, N- of the present invention
By using the novel enzyme having methylvaline oxidase activity, N-methylvaline can be measured accurately and easily in a short time. With sarcosine oxidase, N-methylvaline could not be measured due to lack of sensitivity.

[0034]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to modify a protein having sarcosine oxidase activity using a protein engineering technique to supply a novel enzyme having N-methylvaline oxidase activity. The novel enzyme having N-methylvaline oxidase activity of the present invention can be used for measuring the amount of N-methylvaline in a sample.

[0035]

[Sequence list]

SEQ ID NO: 1 Sequence length: 389 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Arthrobacter S
P.) Strain name: TE1826 sequence Met Ser Ile Lys Lys Asp Tyr Asp Val Ile Val Val Gly Ala Gly Ser 1 5 10 15 Met Gly Met Ala Ala Gly Tyr Tyr Leu Ser Lys Gln Gly Val Lys Thr 20 25 30 Leu Leu Val Asp Ser Phe His Pro Pro His Thr Asn Gly Ser His His 35 40 45 Gly Asp Thr Arg Ile Ile Arg His Ala Tyr Gly Glu Gly Arg Glu Tyr 50 55 60 Val Pro Phe Ala Leu Arg Ala Gln Glu Leu Trp Tyr Glu Leu Glu Lys 65 70 75 80 Glu Thr His His Lys Ile Phe Thr Lys Thr Gly Val Leu Val Phe Gly 85 90 95 Pro Lys Gly Glu Ala Pro Phe Val Ala Glu Thr Met Glu Ala Ala Lys 100 105 110 Glu His Ser Leu Asp Val Asp Leu Leu Glu Gly Ser Glu Ile Asn Lys 115 120 125 Arg Trp Pro Gly Val Thr Val Pro Glu Asn Tyr Asn Ala Ile Phe Glu 130 135 140 Lys Asn Ser Gly Val Leu Phe Ser Glu Asn Cys Ile Arg Ala Tyr Arg 145 150 155 160 Glu Leu Ala Glu Ala Asn Gly Ala Lys Val Leu Thr Tyr Thr Pro Val 165 170 175 Glu Asp Phe Glu Ile Ala Glu Asp Phe Val Lys Ile Gln Thr Ala Tyr 180 185 190 Gly Ser Phe Thr Ala Ser Lys Leu Ile Val Ser Met Gly Ala Trp Asn 195 200 205 Ser Lys Leu Leu Ser Lys Leu Asn Ile Glu Ile Pro Leu Gln Pro Tyr 210 215 220 Arg Gln Val Val Gly Phe Phe Glu Cys Asp Glu Lys Lys Tyr Ser Asn 225 230 235 240 Thr His Gly Tyr Pro Ala Phe Met Val Glu Val Pro Thr Gly Ile Tyr 245 250 255 Tyr Gly Phe Pro Ser Phe Gly Gly Cys Gly Leu Lys Ile Gly Tyr His 260 265 270 Thr Tyr Gly Gln Lys Ile Asp Pro Asp Thr Ile Asn Arg Glu Phe Gly 275 280 285 Ile Tyr Pro Glu Asp Glu Gly Asn Ile Arg Lys Phe Leu Glu Thr Tyr 290 295 295 Met Pro Gly Ala Thr Gly Glu Leu Lys Ser Gly Ala Val Cys Met Tyr 305 310 315 320 Thr Lys Thr Pro Asp Glu His Phe Val Ile Asp Leu His Pro Gln Phe 325 330 335 Ser Asn Val Ala Ile Ala Ala Gly Phe Ser Gly His Gly Phe Lys Phe 340 345 350 Ser Ser Val Val Gly Glu Thr Leu Ser Gln Leu Ala Val Thr Gly Lys 355 360 365 Thr Glu His Asp Ile Ser Ile Phe Ser Ile Asn Arg Pro Ala Leu Lys 370 375 380 Gln Lys Glu Thr Ile 385

SEQ ID NO: 2 Sequence length: 1670 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomicDNA Origin: Organism name: Arthrobacter sp
p.) Strain name: TE1826 Sequence features Characteristic symbol: -35signal Location: 114. ． 119 Method for determining feature: S Characteristic symbol: -10signal Location: 237. ． 242 Method of determining the feature: S Feature symbol: CDS Location: 298. ． 1464 Method of determining feature: P Symbol representing feature: mat peptide Location of existence: 301. ． 1464 Method of characterizing: E Other information: Gene encoding a protein with sarcosine oxidase activity. SEQ CTGCAGTTCT TCCTCCAGCT TTTGAATCCT CACGGTAACA TAAGATTGAA CATAATTTAA 60 ACTTTTGGCC GCCTTTGAAA CGCTGCCATA TTCAACTACC TTTTGAAAAA TCTGCAAATC 120 TTTAATTTCC AAGTATAATC ACTCCCAAAA CGTTCTTTTA CTACTAGCAC TAGAATATTT 180 CTAAAAGTGA TAGCTGCTAT CACTTTTAAG CATTTTACAT GATGGCCAAT AGGCCGTATG 240 ATGTAAATAG ATAATTAAGA AAATTCAAAT TACCTGTTTG AAAAAGGAGA GGAAACA 297 ATG AGT ATT AAA AAA GAT TAT GAT GTA ATT GTG GTT GGC GCT GGT TCC 345 Met Ser Ile Lys Lys Asp Tyr Asp Val Ile Val Val Gly Ala Gly Ser 1 5 10 15 ATG GGA ATG GCA GCT GGG TAC TAT CTG TCT AAA CAA GGT GTT AAA ACA 393 Met Gly Met Ala Ala Gly Tyr Tyr Leu Ser Lys Gln Gly Val Lys Thr 20 25 30 CTA TTG GTA GAT TCA TTT CAT CCT CCC CAT ACA AAT GGC AGC CAT CAT 441 Leu Leu Val Asp Ser Phe His Pro Pro His Thr Asn Gly Ser His His 35 40 45 GGC GAT ACA CGG ATC ATT CGT CAC GCA TAT GGC GAA GGA AGA GAG TAT 489 Gly Asp Thr Arg Ile Ile Arg His Ala Tyr Gly Glu Gly Arg Glu Tyr 50 55 60 GTA CCG TTT GCC TTG AGA GCA CAA GAG TTA TGG TAT GAA TTA GAA AAG 537 Val Pro Phe Al a Leu Arg Ala Gln Glu Leu Trp Tyr Glu Leu Glu Lys 65 70 75 80 GAG ACT CAT CAT AAA ATA TTT ACA AAA ACA GGT GTA CTC GTT TTT GGT 585 Glu Thr His His Lys Ile Phe Thr Lys Thr Gly Val Leu Val Phe Gly 85 90 95 CCT AAA GGA GAA GCT CCT TTC GTT GCC GAA ACA ATG GAA GCC GCA AAG 633 Pro Lys Gly Glu Ala Pro Phe Val Ala Glu Thr Met Glu Ala Ala Lys 100 105 110 GAA CAT TCA TTA GAT GTT GAT TTA CTA GAA GGA AGT GAA ATA AAT AAG 681 Glu His Ser Leu Asp Val Asp Leu Leu Glu Gly Ser Glu Ile Asn Lys 115 120 125 CGT TGG CCA GGT GTA ACG GTT CCT GAG AAT TAT AAT GCT ATT TTT GAA 729 Arg Trp Pro Gly Val Thr Val Pro Glu Asn Tyr Asn Ala Ile Phe Glu 130 135 140 AAA AAT TCT GGT GTC TTA TTT AGT GAA AAT TGT ATT CGC GCT TAC CGT 777 Lys Asn Ser Gly Val Leu Phe Ser Glu Asn Cys Ile Arg Ala Tyr Arg 145 150 155 160 GAA TTG GCG GAA GCA AAT GGT GCG AAA GTT CTA ACG TAC ACA CCC GTT 825 Glu Leu Ala Glu Ala Asn Gly Ala Lys Val Leu Thr Tyr Thr Pro Val 165 170 175 GAA GAT TTC GAG ATT GCC GAG GAC TTC GTC AAA ATC CAA ACC GCC TAT 873 Glu A sp Phe Glu Ile Ala Glu Asp Phe Val Lys Ile Gln Thr Ala Tyr 180 185 190 GGC TCC TTT ACA GCC AGT AAA TTA ATT GTT AGC ATG GGC GCT TGG AAT 921 Gly Ser Phe Thr Ala Ser Lys Leu Ile Val Ser Met Gly Ala Trp Asn 195 200 205 AGC AAA CTG CTA TCA AAA TTA AAT ATT GAA ATC CCA TTG CAG CCA TAC 969 Ser Lys Leu Leu Ser Lys Leu Asn Ile Glu Ile Pro Leu Gln Pro Tyr 210 215 220 CGT CAA GTT GTC GGA TTC TTC GAA TGT GAT GAA AAA AAA TAT AGC AAT 1017 Arg Gln Val Val Gly Phe Phe Glu Cys Asp Glu Lys Lys Tyr Ser Asn 225 230 235 240 ACA CAT GGT TAT CCG GCG TTC ATG GTC GAA GTC CCA ACT GGC ATC TAT 1065 Thr His Gly Tyr Pro Ala Phe Met Val Glu Val Pro Thr Gly Ile Tyr 245 250 255 TAC GGA TTT CCA AGC TTC GGC GGC TGC GGC TTG AAA ATA GGC TAT CAT 1113 Tyr Gly Phe Pro Ser Phe Gly Gly Cys Gly Leu Lys Ile Gly Tyr His 260 265 270 ACG TAT GGT CAA AAA ATC GAT CCA GAT ACG ATT AAT CGT GAA TTT GGT 1161 Thr Tyr Gly Gln Lys Ile Asp Pro Asp Thr Ile Asn Arg Glu Phe Gly 275 280 285 ATT TAC CCG GAG GAT GAA GGG AAT ATT CGC AAA TTC CTG GAA A CA TAT 1209 Ile Tyr Pro Glu Asp Glu Gly Asn Ile Arg Lys Phe Leu Glu Thr Tyr 290 295 300 ATG CCG GGA GCA ACC GGC GAA TTA AAA AGT GGG GCA GTT TGC ATG TAC 1257 Met Pro Gly Ala Thr Gly Glu Leu Lys Ser Gly Ala Val Cys Met Tyr 305 310 315 320 ACA AAA ACA CCT GAT GAG CAT TTC GTG ATT GAT TTA CAT CCT CAA TTC 1305 Thr Lys Thr Pro Asp Glu His Phe Val Ile Asp Leu His Pro Gln Phe 325 330 335 TCG AAT GTC GCG ATT GCA GCC GGA TTC TCC GGA CAT GGG TTT AAA TTC 1353 Ser Asn Val Ala Ile Ala Ala Gly Phe Ser Gly His Gly Phe Lys Phe 340 345 350 TCA AGC GTA GTT GGT GAA ACA TTA AGT CAA TTA GCT GTA ACC GGT AAA 1401 Ser Ser Val Val Gly Glu Thr Leu Ser Gln Leu Ala Val Thr Gly Lys 355 360 365 ACA GAA CAC GAT ATT TCC ATC TTT TCA ATC AAT CGC CCT GCT TTA AAA 1449 Thr Glu His Asp Ile Ser Ile Phe Ser Ile Asn Arg Pro Ala Leu Lys 370 375 380 CAA AAA GAA ACG ATT TAAAAACGCA AGCAAGCCGT ACATAAATTT CGATAGATAT 1504 Gln Lys Glu Thr Ile 385 TATGTACGGC TTACTTTATT TACAACTTAA AAATCTGCAT ATCAATCCTG TCCCTCTACT 1564 GATTGAAGCA CAAACTGT AC TTGAACGGCT TTTTTATTAA CTTGTAACGA TAACAGGAAC 1624 GCTAAAATAA GAAGACCGCT GCATAAGAAT AGTACGGGAG GAATTC 1670

SEQ ID NO: 3 Sequence length: 39 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GGTCC TAAAG GAGAA GCTCC TGTCG TTGCC GAAAC AATG 39

[Brief description of drawings]

FIG. 1 shows the structure of the plasmid, pSAOEP3-F103V.

FIG. 2 shows the dilution linearity of N-methylvaline measurement.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1:19) (C12N 1/21 C12R 1:19)

Claims (7)

[Claims] 1. A novel enzyme having N-methylvaline oxidase activity, which acts on N-methylvaline in the presence of water and oxygen to produce valine, formaldehyde and hydrogen peroxide. 2. The novel enzyme having N-methylvaline oxidase activity according to claim 1, which has the following physicochemical properties. Action: acts on N-methylvaline in the presence of water and oxygen to produce valine, formaldehyde and hydrogen peroxide. Substrate specificity: N-methylvaline 100% Sarcosine ≤1% Optimum pH: 7-9 Optimum temperature: 40-50 ° C Molecular weight: 43Kd 3. A novel enzyme having N-methylvaline oxidase activity according to claim 1, wherein the 103rd phenylalanine in the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing is replaced with valine. . 4. A method for producing a novel enzyme having N-methylvaline oxidase activity, which comprises substituting another amino acid for phenylalanine in the amino acid sequence constituting a protein having sarcosine oxidase activity. 5. N-methylvaline oxidase activity by substituting another amino acid by site-specific mutation of the gene encoding phenylalanine at the 103rd position of the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing. The method for producing a novel enzyme having N-methylvaline oxidase activity according to claim 4, which comprises producing an enzyme having 6. The method for producing a novel enzyme having N-methylvaline oxidase activity according to claim 4 or 5, wherein the other amino acid is valine. 7. N-methylvaline in a sample is reacted with N-methylvaline in the presence of water and oxygen to give valine,
A novel enzyme having an N-methylvaline oxidase activity that produces formaldehyde and hydrogen peroxide is caused to act, and oxygen consumed or valine, formaldehyde or hydrogen peroxide produced is measured.
-A method for the determination of methylvaline.