JPH10248572A - Modified sarcosine oxidase and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent the occurrence of positive errors in measuring creatine and creatinine for diagnoses of muscle diseases and renal diseases by reducing reactivity to proline by modifying a protein having sarcosine oxidase activity using a protein engineering method. SOLUTION: Reactivity of a natural sarcosine oxidase to proline is reduced by a method, etc., in which the 345th phenyl alanine in the amino acid sequence of the formula as a protein having sarcosine oxidase activity, is substituted with alanine, glycine, valine or isoleucine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a protein having sarcosine oxidase activity, which is modified by a protein engineering technique to reduce the reactivity to proline as compared with the wild-type sarcosine oxidase before modification. The present invention relates to a modified sarcosine oxidase, a method for producing the enzyme, and use thereof.

[0002]

2. Description of the Related Art Conventionally, sarcosine oxidase (EC
1.5.3.1) is used together with other enzymes, such as creatinine amidohydrolase, creatine amidinohydrolase, and peroxidase, as enzymes for measuring creatine and creatinine in body fluids, which are clinically indicative of diagnosis of muscle and kidney diseases. Have been. Sarcosine oxidase acts on its substrate, sarcosine, in the presence of water and oxygen to produce glycine, formaldehyde and hydrogen peroxide.

[0003] Such sarcosine oxidases include genus Bacillus (JP-A-54-52789), genus Corynebacterium (J. Biochem. 89, 599 (1981)), and genus Cylindrocarbone (JP-A-56-1981). It is known that bacteria such as Pseudomonas sp. (JP-A-60-43379) produce the same. Above all, Arthrobacter SP T
It has been known that sarcosine oxidase produced by E1826 (FERM P-10637) is a practical enzyme having a higher thermostability and a smaller Km value than conventional sarcosine oxidase (Japanese Patent Laid-Open No. 2-265478 gazette).

[0004] The present inventors have already found that
The sarcosine oxidase gene was successfully isolated from chromosomal DNA extracted from SP TE1826 (FERM P-10637), and the entire structure of the DNA was determined (Journal of Ferme
ntation and BioengineeringVol.75 No.4 pp239-244 (1
993)), the sarcosine oxidase was successfully produced in a transformant by a genetic engineering technique, and high-purity sarcosine oxidase could be supplied in large quantities at low cost (Japanese Patent Laid-Open No. 6-13840). No.).

[0005] However, sarcosine oxidase is known to exhibit reactivity at a low level even with proline, one of the amino acids (see, for example,
5-115281). However, proline, particularly L-proline, is a component of the protein that constitutes a living body and may be present in a body fluid, which may cause a positive error in measuring creatine and creatinine in the body fluid. In fact, Osawa et al. Pointed out that the problem with the reagent for measuring creatinine was the reactivity of sarcosine oxidase contained in the reagent with proline (for example, clinical science,
20, 144-152 (1991), biological sample analysis, 17,332-337 (1994)).

[0006]

Accordingly, it has been desired to reduce the reactivity of wild-type sarcosine oxidase to proline.

[0007]

Means for Solving the Problems As a result of various studies to achieve the above object, the present inventors have found that by modifying a protein having sarcosine oxidase activity by a protein engineering technique, the reactivity to proline can be improved. It has been found that it is possible to construct a modified sarcosine oxidase that is reduced in comparison to wild-type sarcosine oxidase.

[0008] That is, the present invention relates to a protein in which at least one amino acid of an amino acid sequence constituting a protein having sarcosine oxidase activity is mutated by addition, deletion, insertion or substitution, and which has a reactivity to proline. A modified sarcosine oxidase characterized by being reduced as compared to the protein before modification.

[0009] The present invention also provides a method for transforming a host cell with an expression vector incorporating a gene obtained by replacing the gene encoding the amino acid sequence shown in SEQ ID NO: 1 with a gene encoding another amino acid, A method for producing a modified sarcosine oxidase, which comprises culturing the obtained transformant and collecting a modified sarcosine oxidase having reduced reactivity to proline as compared to the protein before modification from the culture. .

Further, the present invention provides a modified sarcosine oxidase, creatine amidinohydrolase, wherein the reactivity to proline is reduced as compared to the protein before modification.
It is a reagent for measuring creatine, including a reagent for detecting peroxidase and hydrogen peroxide.

[0011] The present invention also relates to a reagent for measuring creatinine comprising a modified sarcosine oxidase, creatinine amidohydrolase, creatine amidinohydrolase, peroxidase and a reagent for detecting hydrogen peroxide, the reactivity of which is lower than that of the protein before modification. It is.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The sarcosine oxidase before modification of the present invention is not particularly limited, and examples thereof include sarcosine oxidase derived from Bacillus and sarcosine oxidase derived from Pseudomonas. .

In the present invention, as an example, sarcosine oxidase of Arthrobacter sp. TE1826 (FERMP-10637) (JP-A-2-265478, JP-A-6-13838)
No. 40, Journal of Fermentation and Bioengineeri
ng Vol.75 No.4 pp239-244 (1993)).
The amino acid sequence of sarcosine oxidase derived from Arthrobacter sp. TE1826 is shown in SEQ ID NO: 1 in the sequence listing. In addition, D encoding these amino acid sequences
The NA sequence is shown in SEQ ID NO: 3 in the sequence listing.

The modified sarcosine oxidase of the present invention comprises
A protein in which at least one amino acid of the amino acid sequence constituting a protein having sarcosine oxidase activity is mutated by addition, deletion, insertion or substitution, and the reactivity to proline is lower than that of the protein before modification. It was done. The reactivity with proline is defined as the ratio of the enzymatic activity using proline as a substrate to the enzymatic activity using sarcosine as an original substrate. In the present invention, the reactivity to proline is reduced as compared to the protein before modification, but the degree of the reduction is about 70% or less.

[0015] One embodiment of the present invention is a modified sarcosine oxidase in which at least one amino acid of the amino acid sequence constituting the protein having sarcosine oxidase activity is substituted with another amino acid.

Further, in one embodiment of the present invention, the amino acid sequence of the 345th amino acid sequence represented by SEQ ID NO: 1
The phenylalanine is a protein having an amino acid sequence in which another amino acid has been substituted (sequence list, SEQ ID NO: 2).

One embodiment of the present invention is a modified sarcosine oxidase in which the other amino acid is alanine, glycine, valine or isoleucine.

Further, as a specific embodiment, there is a modified sarcosine oxidase having the following physicochemical properties. Action: acting on sarcosine in the presence of water and oxygen,
Produces glycine, formaldehyde and hydrogen peroxide. Optimum pH: 7.5-8.5 Optimum temperature: 40-50 ° C Stable pH: 6.5-9.0 (25 ° C, 24 hours treatment) Stable temperature: 50 ° C or less (pH 7.5, 10 minutes) Processing) Substrate specificity: Reactivity to proline is 70% or less compared to the protein before modification. Molecular weight: about 43 KDa Amino acid sequence: described in SEQ ID NO: 2 in Sequence Listing.

The modified sarcosine oxidase of the present invention comprises:
It can be manufactured by the following procedure. First, as a method of modifying an amino acid sequence constituting a protein having sarcosine oxidase activity, a method of modifying genetic information which is usually performed is used. That is, a DNA having genetic information of a modified protein is prepared by converting a specific base of a DNA having genetic information of a protein having sarcosine oxidase activity, or by inserting or deleting a specific base. . As a specific method for converting bases in DNA, for example, a commercially available kit (Transformer ™; manufactured by Clonetech, EXOIII / Mung Be
an Deletion Kit; Stratagene) or
Alternatively, use of PCR can be mentioned.

The DNA having the genetic information of the modified protein thus prepared is transferred into a host microorganism in a state of being linked to a plasmid, and becomes a transformant producing the modified protein. As a plasmid at this time, for example, when Escherichia coli is used as a host microorganism, pBluescript,
pUC18 or the like can be used. When another bacterium, for example, a bacterium belonging to the genus Bacillus is used as a host, pUB110,
pHY300PLK or the like can be used. Examples of the host microorganism include Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, and Escherichia coli DH5α. Other host microorganisms include Bacillus and Pseudomonas bacteria. As a method of 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 used. Electroporation may be used.

The microorganism thus obtained as a transformant can stably produce a large amount of the modified protein by culturing it in a nutrient medium. The culture form of the host microorganism which is a transformant may be selected in consideration of the nutritional and physiological properties of the host, and the culture condition is usually selected. In most cases, liquid culture is used, but industrially, aeration-agitation 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, such as glucose, sucrose, lactose, maltose, fructose, molasses,
Pyruvic acid and the like are used. Any nitrogen compound can be used as the nitrogen source, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract. In addition, phosphates, carbonates,
Salts such as sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, and specific vitamins are used as needed.

The cultivation temperature can be appropriately changed within the range in which the bacteria grow and produce the modified protein. In the case of Escherichia coli, the temperature is preferably about 20 to 42 ° C. The cultivation time varies somewhat depending on the conditions, but the cultivation may be terminated at an appropriate time in consideration of the time when the modified protein reaches the maximum yield, and is usually about 6 to 48 hours. The medium pH can be appropriately changed within a range in which the bacteria grow and produce the 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 directly collected and used.
In general, when the modified protein is present in the culture solution, the solution is used after separating the modified protein-containing solution from the microbial cells by filtration, centrifugation or the like according to a conventional method. When the modified protein is present in the cells, the cells are collected from the obtained culture by means such as filtration or centrifugation, and then the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme. The modified protein is solubilized by adding a chelating agent such as EDTA and / or a surfactant, if necessary, and separated and collected as an aqueous solution.

The modified protein-containing solution thus obtained is concentrated, for example, under reduced pressure, membrane concentrated, and further subjected to salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone or the like. What is necessary is just to precipitate by the method. Heat treatment and isoelectric point treatment are also effective purification means. Purified modified sarcosine oxidase can be obtained by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

The reagent for measuring creatine of the present invention includes a modified sarcosine oxidase, a creatine amidinohydrolase, a peroxidase, and a reagent for detecting hydrogen peroxide in which the reactivity to proline is reduced as compared to the protein before modification. Further, the reagent for measuring creatinine of the present invention includes a modified sarcosine oxidase, a creatinine amidohydrolase, a creatine amidinohydrolase, a peroxidase, and a hydrogen peroxide detection reagent in which the reactivity with respect to the proline is reduced as compared with the protein before modification. As creatinine amidohydrolase, creatine amidinohydrolase, and peroxidase, conventionally known ones can be used. Further, the hydrogen peroxide detection reagent is a reagent for measuring hydrogen peroxide generated by the modified sarcosine oxidase together with peroxidase, and includes, for example, 4-aminoantipyrine and a phenol derivative or an aniline derivative. The present invention is not limited to these hydrogen peroxide measurement reagents.

A method for measuring creatine or creatinine using these reagents is a conventionally known method. In the measurement reagent of the present invention, the modified sarcosine oxidase has reduced reactivity to proline as compared to the protein before modification, without being affected by proline in body fluids, accurately, and simply. These components can be measured.

[0027]

The present invention will be described below in more detail with reference to examples. In Examples, sarcosine oxidase activity was measured as follows. That is, 48 mM Tris-HC
1 buffer (pH 8.0), 95 mM sarcosine, 0.47 mM
4-aminoantipyrine, 2.0 mM phenol, 0.
In 45% Triton X-100, 4.5 U / ml peroxidase, the enzyme was reacted at 37 ° C. for 10 minutes, and 500 nm
The absorbance at is measured. 1 unit of enzyme activity (U)
Was the amount of enzyme that produced 1 micromol of hydrogen peroxide per minute under these conditions. The reactivity to L-proline was measured as a relative ratio of the activity when sarcosine in the above composition was replaced with L-proline.

Example 1 Modification of sarcosine oxidase pSAOEP3, a recombinant plasmid having the genetic information of sarcosine oxidase, was obtained from Journal of Fermentation and Bioengineering.
f Fermentation and Bioengineering) Vol.75 No.4 pp2
According to the method described in 39-244 (1993), it was prepared as follows. First, Arthrobacter SP TE18
Chromosomal DNA of No. 26 (FERM P-10637) was isolated by the following method. The strain was diluted with 100 ml of 2 × YT medium (1.6% polypeptone, 1% yeast extract, 0.5% sodium chloride (pH 7.2)).
After shaking culture at 7 ° C. overnight, the cells were 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 EDTA, 50 mM Tris-HCl (pH
Was suspended in 5 ml of a solution containing 7.6), 0.5 ml of a lysozyme solution (100 mg / ml) was added, and the mixture was incubated at 37 ° C. for 30 minutes. Then, 11 ml of a solution containing 1% lauroyl sarcosine acid, 0.1 M EDTA (pH 9.6) was added.

To this suspension was added 0.5 ml of an ethidium bromide solution.
About 100% cesium chloride, and stir and mix.
The DNA was collected by ultracentrifugation at rpm for 20 hours. The collected DNA was 10 mM Tris-HCl (pH 8.0), 1 mM ED
It was dialyzed against a solution containing TA (TE) to obtain a purified DNA sample.

[0030] 1 µg of the purified DNA preparation was subjected to restriction enzyme Sau3AI.
(Toyobo Co., Ltd.) to perform a partial decomposition reaction to decompose into fragments of 2 kbp or more, and then cut with SalIII Toyobo Co., Ltd., pUC1
Using 80.5 μg of T4-D according to the BACKFILLING method of MGLoftus et al. (Biotechniques Vol 12, No. 2 (1992)).
One unit of NA ligase (manufactured by Toyobo) was reacted at 16 ° C. for 12 hours to ligate DNA. The ligated DNA is Ha
Escherichia coli JM10 created by nahan's method
Transformation was performed using 9 competent cells. About 1 × 10 ⁶ transformant colonies were obtained per μg of DNA used. The obtained colonies were grown at 37 ° C. on L medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride) containing 50 μg / ml ampicillin, 0.5% sarcosine, 0.005% pararose aniline and 0.025% sodium hydrogen sulphite. For 18 hours, and the recombinant DNA containing the sarcosine oxidase gene was screened using the red colony as an indicator.

As a result, one out of about 1,000 colonies
A red colony was obtained at the ratio of the strain. One of the strains contained an inserted DNA fragment of about 8.7 kbp in the plasmid, and this plasmid was designated as pSAO1. Next, the inserted DNA fragment was cut from pSAO1 with various restriction enzymes and subcloned into pUC18 to obtain a DNA fragment of about 1.7 k.
pSAOEP3 having a bp inserted DNA fragment was obtained.

Insertion of pSAOEP3 into SEQ ID NO: 3 in the Sequence Listing
The DNA sequence of the A fragment was added to pSAOEP3 in SEQ ID NO: 1 in the sequence listing.
The amino acid sequences of sarcosine oxidase encoded in the inserted DNA fragment are described. Based on the recombinant plasmid pSAOEP3, TransformerTM (manufactured by Clonetech), which is a kit for converting bases in DNA and oligonucleotides of SEQ ID NO: 5 in the sequence listing,
Mutation treatment was performed according to the protocol of merTM.
As a result, the modified protein F34 in which phenylalanine at position 345 of SEQ ID NO: 1 in the sequence listing was substituted with alanine was used.
DN having genetic information of 5A (described in SEQ ID NO: 2 in the sequence listing)
A plasmid carrying A was made.

A DNA having the genetic information of the modified sarcosine oxidase is digested with various restriction enzymes to prepare subclones.
The nucleotide sequence was determined using QUENING PRO 7-deaza-dGTP kit (manufactured by Toyobo), and it was confirmed that the DNA sequence had been modified. Escherichia coli JM10 is a recombinant plasmid carrying DNA having the genetic information of the modified sarcosine oxidase.
Nine competent cells were transformed to obtain transformants.

Example 2 Culture of transformant 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, allowed to cool, and then 0.1% of 50 mg / ml ampicillin (manufactured by Nacalai Tesque), which was separately sterile-filtered, was added. This culture medium was inoculated with 1 ml of a culture solution of a transformant previously cultured with shaking at 37 ° C. for 18 hours in a medium having the same composition as described above, and cultured at 37 ° C. with aeration and stirring.

[0035] The modified protein is obtained from the culture solution by using the Journal of Fermentation and Bioengineering.
According to the purification method of sarcosine oxidase described in Vol.75 No.4 pp239-244 (1993), the steps of sonication, removal of nucleic acid, ammonium sulfate salting out, DEAE-Sephadex column chromatography, and gel filtration column chromatography are sequentially performed. And purified by SDS-polyacrylamide gel electrophoresis until a single band was formed.

Example 3 Evaluation of Modified Protein Purified modified sarcosine oxidase and wild-type sarcosine oxidase were compared in terms of reactivity with L-proline and sarcosine. Table 1 shows the results.

[0037]

[Table 1]

As is clear from Table 1, the reactivity of the modified sarcosine oxidase F345A with L-proline was as follows.
This shows that the reactivity of wild-type sarcosine oxidase to L-proline is lower. The specific activity indicating the absolute reactivity to sarcosine is about 20 U / mg for wild-type sarcosine oxidase, while about 18 U / g for modified sarcosine oxidase F345A.
It was almost comparable to mg. That is, it became clear that the modified sarcosine oxidase had a lower reactivity to L-proline than the wild-type sarcosine oxidase without substantially impairing the absolute enzyme performance. Other properties were almost the same as those of wild-type sarcosine oxidase.

[0039]

Industrial Applicability According to the present invention, it has become possible to modify a protein having sarcosine oxidase activity using a protein engineering technique to supply a modified sarcosine oxidase having reduced reactivity to proline. The modified sarcosine oxidase of the present invention can be mass-produced by a genetic engineering technique in a bacterial system. Also,
The modified sarcosine oxidase of the present invention is clinically used as an enzyme for measuring creatine and creatinine in body fluids, which is an indicator of diagnosis of muscular disease and renal disease, without being affected by proline, creatine and creatinine in a sample. Can be used to accurately and quickly measure the amount of

[0040]

[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 290 295 300 300 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 385 389

SEQ ID NO: 2 Sequence length: 389 Sequence type: amino acid Topology: linear Sequence type: protein Other information: Xaa represents an amino acid other than phenylalanine. 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 290 295 300 300 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 Xaa 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 385 389

SEQ ID NO: 3 Sequence length: 1670 Sequence type: nucleic acid (DNA) Number of strands: double-stranded Topology: linear Sequence type: genomic cDNA Origin Organism: Arthrobacter sp.
P.) Ltd. Name: TE1826 sequence 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 GA A TTA GAA AAG 537 Val Pro Phe Ala 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 633 100 105 110 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 Asp Phe Glu Ile Ala Glu Asp Phe V al 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 260 265 270 270 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 ACA 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 1564G ATTGAAGCA CAAACTGTAC TTGAACGGCT TTTTTATTAA CTTGTAA CGA TAACAGGAAC 1624GC TAAAATAA GAAGACCGCT GCATAAGAAT AGTACGGGAG GAATTC 1670

SEQ ID NO: 4 Sequence length: 1670 Sequence type: nucleic acid (DNA) Number of strands: double-stranded Topology: linear Sequence type: genomic cDNA Origin Organism: Arthrobacter sp.
P.) Stock name: TE1826 Other information: N indicates A or C or G or T or U. Xaa represents an amino acid other than phenylalanine. 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 NNN TCC GGA CAT GGG TTT AAA TTC 1353 Ser Asn Val Ala Ile Ala Ala Gly Xaa 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

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 15/09 ZNA C12R 1:06) (C12N 1/21 C12R 1:19) (C12N 9/02 C12R 1:19 )

Claims (10)

[Claims] 1. A protein in which at least one amino acid of an amino acid sequence constituting a protein having sarcosine oxidase activity has been mutated by addition, deletion, insertion or substitution, wherein the protein has no reactivity to proline before modification. A modified sarcosine oxidase, characterized in that it is reduced as compared to 2. The modified sarcosine oxidase according to claim 1, wherein at least one amino acid of the amino acid sequence constituting the protein having sarcosine oxidase activity is substituted with another amino acid. 3. An amino acid sequence in which phenylalanine at position 345 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing has been substituted with another amino acid (SEQ ID NO: 2 in the sequence listing).
The modified sarcosine oxidase according to claim 2, which is a protein having: 4. The modified sarcosine oxidase according to claim 3, wherein the other amino acid is alanine, glycine, valine or isoleucine. 5. A modified sarcosine oxidase having the following physicochemical properties. Action: acting on sarcosine in the presence of water and oxygen,
Produces glycine, formaldehyde and hydrogen peroxide. Optimum pH: 7.5-8.5 Optimum temperature: 40-50 ° C Stable pH: 6.5-9.0 (25 ° C, 24 hours treatment) Stable temperature: 50 ° C or less (pH 7.5, 10 minutes) Processing) Substrate specificity: Reactivity to proline is 70% or less compared to the protein before modification. Molecular weight: about 43 KDa Amino acid sequence: described in SEQ ID NO: 2 in Sequence Listing. 6. A host cell is transformed with an expression vector incorporating a gene in which the gene encoding the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing has been substituted with a gene encoding another amino acid. Culturing the transformant,
A method for producing a modified sarcosine oxidase, comprising collecting from the culture a modified sarcosine oxidase having reduced reactivity to proline as compared to the protein before modification. 7. The method for producing a modified sarcosine oxidase according to claim 6, wherein the gene encoding phenylalanine at position 345 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is replaced with a gene encoding another amino acid. . 8. A gene obtained by replacing the gene encoding the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing with a gene encoding another amino acid is the DNA sequence shown in SEQ ID NO: 4 in the sequence listing. A method for producing the modified sarcosine oxidase according to claim 6. 9. A reagent for detecting modified sarcosine oxidase, creatine amidinohydrolase, peroxidase and hydrogen peroxide, which has reduced reactivity to proline according to any one of claims 1 to 5 as compared to the protein before modification. And a reagent for measuring creatine. 10. A modified sarcosine oxidase, creatinine amidohydrolase, creatine amidinohydrolase, peroxidase and a peroxidase having reduced reactivity to the proline according to any one of claims 1 to 5 as compared to the protein before modification. A reagent for measuring creatinine, including a reagent for detecting hydrogen oxide.