JP4161232B2 - Novel protein having sarcosine oxidase activity and method for producing the same - Google Patents

Description

【００４６】
増幅産物（約１．１ｋｂｐ）と制限酵素ＳｍａＩで分解処理したベクターｐＵＣ１９をＴ４ＤＮＡリガーゼでライゲーションして、エシェリヒア・コリーＪＭ１０９のコンピテントセル（東洋紡績製）で形質転換し、形質転換体を得た。該形質転換体の保有する約３．８ｋｂｐのプラスミドには約１．１ｋｂｐの挿入ＤＮＡ断片が存在している。該遺伝子の塩基配列をダイデオキシ法により、配列表の配列番号４のように決定し、また該プラスミドをｐＢＳＯＬＲ２とし、該形質転換体をエシェリヒア・コリーＪＭ１０９（ｐＢＳＯＬＲ２）とした。エシェリヒア・コリーＪＭ１０９（ｐＢＳＯＬＲ２）は、サルコシンオキシダーゼ活性を有する新規なタンパク質をコードする遺伝子を有する形質転換体である。なお、ｐＢＳＯＬＲ２の構造は図１に示す通りである。
【００４７】
実施例３ エシェリヒア・コリーＪＭ１０９（ｐＢＳＯＬＲ２）からのサルコシンオキシダーゼ活性を有する新規なタンパク質の製造
ＴＢ培地５００ｍｌを２Ｌフラスコに分注し、１２１℃、１５分間オートクレーブを行い、放冷後別途無菌ろ過した５０ｍｇ／ｍｌアンピシリン（ナカライテスク製）０．５ｍｌを添加した。この培地にＬＢ培地であらかじめ，３０℃、１６時間振盪培養したエシェリヒア・コリーＪＭ１０９（ｐＢＳＯＬＲ２）の培養液５ｍｌを接種し、３７℃で２４時間通気撹拌培養した。培養終了時のサルコシンオキシダーゼ活性は約０．３５Ｕ／ｍｌであった。
【００４８】
上記菌体を遠心分離にて集菌し、５０ｍＭリン酸緩衝液（ｐＨ８．０）に懸濁した。上記菌体懸濁液を超音波で破砕し、遠心分離を行い上清液を得た。得られた粗酵素液に対し、０．５Ｍ飽和硫安分画処理を行った後、セファデックスＧ−２５（ファルマシアバイオテク製）によるゲルろ過により脱塩し、ＤＥＡＥセファロースＣＬ６−Ｂ（ファルマシアバイオテク製）カラムクロマトグラフィー、セファデックスＧ−２００（ファルマシアバイオテク製）カラムクロマトグラフィーにより分離・精製し、精製タンパク質表品を得た。本方法により得られたサルコシンオキシダーゼ活性を有する新規なタンパク質の標品は、電気泳動（ＳＤＳ−ＰＡＧＥ）的にほぼ単一なバンドを示し、この時の比活性は約０．６Ｕ／ｍｇ−タンパク質であった。
【００４９】
以下に、上記方法により得られたサルコシンオキシダーゼ活性を有する新規なタンパク質の性質を示す。
作用：水、酸素の存在下にサルコシンに作用して、過酸化水素を生成する
Ｋｍ：０．２±０．１ｍＭ
至適温度：４５±５℃
至適ｐＨ：８．０±０．５
熱安定性：４５±５℃以下（ｐＨ８．０、１０分間処理）
ｐＨ安定性：８．０±０．５（２５℃、５時間処理）
分子量：４００００±４０００（ＳＤＳ−ＰＡＧＥ）
１６００００±１６０００（ゲル濾過）
阻害剤：ＰＣＭＢ
基質特異性：サルコシンの他、グリシン、Ｎ−エチルグリシンにも反応性を有する
なお、上記タンパク質のアミノ酸配列は、配列表の配列番号１のように決定された。
【００５０】
【発明の効果】
上述したように、本発明により、Ｋｍが１ｍＭ以下であるサルコシンオキシダーゼ活性を有する新規なタンパク質が単離された。また、該タンパク質を用いることにより、サルコシン、クレアチン、クレアチニンに関する迅速な定量化が可能となった。さらに、タンパク質工学的手法を用いることによる機能改変を行うことにより、理化学的性質の改良を導き出すことができる。
【００５１】
【配列表】

【００５２】

【００５３】

【００５４】

【図面の簡単な説明】
【図１】 ｐＢＳＯＬＲ２の構造を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel protein having sarcosine oxidase activity that can be used for quantification of sarcosine, creatine, and creatinine, and a method for producing the protein.
[0002]
[Prior art]
Conventionally, sarcosine oxidase (EC1.5.3.1) has been used as an enzyme for measuring creatine and creatinine in body fluids, which is clinically an index for diagnosis of muscle diseases and kidney diseases. Used with tininase, creatinase and peroxidase. Sarcosine oxidase acts on the substrate sarcosine in the presence of water and oxygen to produce glycine, formaldehyde and hydrogen peroxide.
[0003]
Such sarcosine oxidases include the genus Bacillus (JP-A-54-52789), the genus Corynebacterium (J. Biochem. 89, 599 (1981)), and the genus Cylindrocarpon. (Japanese Patent Laid-Open No. 56-92790), Pseudomonas genus (Japanese Patent Laid-Open No. 60-43379) and the like have been reported to be produced.
[0004]
[Problems to be solved by the invention]
In Japanese Patent Laid-Open No. 02-265478, sarcosine oxidase produced by Arthrobacter sp. TE1826 (Mikken Kenki No. 10737) is superior in thermal stability to conventional sarcosine oxidase. In addition, it is already known that the Km is about 3 mM, which is smaller than those of other sources, and is a practical enzyme. The value of the Michaelis-Menten constant (hereinafter referred to as Km) of the enzyme is closely related to the time required for measurement using the enzyme, and it can be said that an enzyme having a small Km is more practical. However, the sarcosine oxidase as described above is not sufficient to realize rapid quantification of sarcosine, creatine and creatinine, and development of a sarcosine oxidase having a smaller Km value has been desired.
[0005]
[Means for Solving the Problems]
As a result of various studies to achieve the above object, the present inventors have found a novel protein having sarcosine oxidase activity with a Km of 1 mM or less, and have reached the present invention. And it came to implement | achieve rapid quantification of sarcosine, creatine, and creatinine by using this protein.
[0006]
That is, the present invention is a novel protein having sarcosine oxidase activity characterized in that Km is 1 mM or less. More preferably, the present invention is a novel protein having sarcosine oxidase activity characterized in that Km is 0.2 ± 0.1 mM or less.
[0007]
The present invention is preferably a novel protein having sarcosine oxidase activity having the following physicochemical properties.
Action: acts on sarcosine in the presence of water and oxygen to produce hydrogen peroxide Km: 0.2 ± 0.1 mM
Molecular weight: 40000 ± 4000 (SDS-PAGE)
[0008]
The present invention is more preferably a novel protein having sarcosine oxidase activity having the following physicochemical properties.
Action: acts on sarcosine in the presence of water and oxygen to produce hydrogen peroxide Km: 0.2 ± 0.1 mM
Optimal temperature: 45 ± 5 ° C
Optimum pH: 8.0 ± 0.5
Thermal stability: 45 ± 5 ° C or less (pH 8.0, treatment for 10 minutes)
pH stability: 8.0 ± 0.5 (25 ° C., 5 hours treatment)
Molecular weight: 40000 ± 4000 (SDS-PAGE)
160000 ± 16000 (gel filtration)
Inhibitor: p-chloromercuribenzoic acid (hereinafter referred to as PCMB)
Substrate specificity: reactive to glycine and N-ethylglycine in addition to sarcosine
In addition, the present invention is preferably a novel protein having sarcosine oxidase activity, which is the following protein (a) or (b).
(A) a protein comprising the amino acid sequence described in the sequence listing / SEQ ID NO: 1 (b) comprising the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and sarcosine The protein having oxidase activity or the present invention is more preferably a novel protein having sarcosine oxidase activity, which is a protein consisting of the amino acid sequence described in Sequence Listing / SEQ ID NO: 1.
[0010]
The present invention also relates to a gene encoding a novel protein having sarcosine oxidase activity, which is the following protein (a) or (b).
(A) a protein comprising the amino acid sequence described in the sequence listing / SEQ ID NO: 1 (b) comprising the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and sarcosine The protein having oxidase activity or the present invention is preferably a gene encoding a novel protein having sarcosine oxidase activity, which is a protein consisting of the amino acid sequence described in Sequence Listing / SEQ ID NO: 1.
[0011]
Further, the present invention is preferably a gene encoding a novel protein having sarcosine oxidase activity, which is the following DNA (c) or (d).
(C) DNA consisting of the base sequence described in Sequence Listing / SEQ ID NO: 4
(D) DNA encoding a protein having one or several bases deleted, substituted or added in the sequence (c) and having sarcosine oxidase activity
More preferably, the present invention is a gene encoding a novel protein having sarcosine oxidase activity having a DNA comprising the nucleotide sequence set forth in Sequence Listing / SEQ ID NO: 4.
[0012]
The present invention is also a recombinant vector containing a gene encoding a novel protein having sarcosine oxidase activity as described above. Moreover, this invention is a transformant containing the recombinant vector containing the gene which codes the novel protein which has the above sarcosine oxidase activities.
[0013]
Further, the present invention is characterized by culturing a Bacillus subtilis Marburg 168 strain that produces a protein having sarcosine oxidase activity having a Km of 1 mM or less, and collecting the protein having sarcosine oxidase activity from the culture. And a method for producing a novel protein having sarcosine oxidase activity.
[0014]
In addition, the present invention cultivates a transformant having a gene encoding a novel protein having sarcosine oxidase activity as described above and introduced with a recombinant vector having the productivity of a protein having sarcosine oxidase activity, A method for producing a novel protein having sarcosine oxidase activity, comprising collecting a protein having sarcosine oxidase activity from a culture.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
The novel protein having sarcosine oxidase activity of the present invention is preferably isolated from a sarcosine oxidase-producing microorganism, particularly Bacillus subtilis. Specifically, it can be isolated from, for example, a Bacillus subtilis Marburg 168 strain available from American Type Culture Collection (ATCC), such as Bacillus subtilis MI113, DE100, ASB298 and the like. However, by isolating the gene encoding the protein of the present invention and isolating the expressed protein, the protein can be easily and efficiently obtained.
[0016]
As one embodiment of the present invention, for example, an amino acid sequence as set forth in SEQ ID NO: 1 in the sequence listing, or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence, and Examples include proteins having sarcosine oxidase activity.
[0017]
The degree of amino acid deletion, substitution, and addition includes those in which the basic characteristics are not changed or the characteristics are improved. A method for producing these mutants can be obtained according to a conventionally known method.
[0018]
The gene encoding the novel protein having sarcosine oxidase activity can be extracted from, for example, a Bacillus subtilis Marburg 168 strain, or can be chemically synthesized. Furthermore, a DNA fragment containing a gene encoding a novel protein having sarcosine oxidase activity can be obtained by utilizing the polymerase chain reaction method (PCR method).
[0019]
Examples of the gene include DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence. And a DNA encoding a novel protein having sarcosine oxidase activity. Specifically, for example, DNA consisting of the base sequence described in SEQ ID NO: 4 in the sequence listing, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence can be mentioned.
[0020]
The degree of DNA deletion, substitution, and addition includes those in which the basic characteristics are not changed or the characteristics are improved. A method for producing these mutants can be obtained according to a conventionally known method.
[0021]
As a method for obtaining the above gene, for example, PCR is carried out under known conditions using the chromosomal DNA of Bacillus subtilis Marburg 168 strain as a template and the oligonucleotides of SEQ ID NOs: 2 and 3 in the sequence listing as primers, and has sarcosine oxidase activity. A gene encoding a novel protein may be collected.
[0022]
Specifically, chromosomal DNA of the gene donor Bacillus subtilis Marburg 168 strain is collected by the following steps, for example. That is, a chromosomal DNA-containing lysate can be prepared by collecting a culture obtained by stirring and cultivating a donor microorganism for 1 to 3 days, and then lysing the culture. As a method of lysis, for example, treatment is performed with a lytic enzyme such as lysozyme or β-glucanase, and a protease, other enzyme, or a surfactant such as sodium lauryl sulfate (SDS) is used in combination as necessary, and further freeze-thawing. Or a physical crushing method such as a French press treatment.
[0023]
In order to separate and purify DNA from the lysate obtained as described above, a conventional method is used, for example, by appropriately combining methods such as deproteinization treatment by phenol treatment or protease treatment, ribonuclease treatment, and alcohol precipitation treatment. be able to.
[0024]
As a vector for producing a recombinant, a vector constructed for gene recombination from a phage or plasmid that can autonomously grow in a host microorganism is suitable.
[0025]
Examples of the phage include lambda gt10 and lambda gt11 when Escherichia coli is used as a host microorganism.
[0026]
Examples of the plasmid include pBR322, pUC18, pUC19, and pBluescript when Escherichia coli is used as a host microorganism.
[0027]
On the other hand, any host microorganism may be used as long as the recombinant vector is stable and capable of autonomous propagation and can express a foreign gene. For example, Escherichia coli W3110 and Escherichia coli C600 are generally used. , Escherichia coli HB101, Escherichia coli JM109, and the like.
[0028]
As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method using calcium treatment, an electroporation method, or the like can be used.
[0029]
The microorganism, which is a transformant obtained as described above, can stably produce a large amount of the target protein by being cultured in a nutrient medium. Selection for the presence or absence of transfer of the target recombinant vector to the host microorganism may be performed by searching for a drug resistance marker of the vector holding the target DNA and a microorganism that simultaneously expresses sarcosine oxidase activity, for example, based on the drug resistance marker A microorganism that grows in a selective medium and produces sarcosine oxidase may be selected.
[0030]
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. In many cases, the culture is performed in liquid culture, but industrially, aeration and agitation culture is performed. Is advantageous. As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid and the like are used. As the nitrogen source, any nitrogen compound that can be used may be used. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.
[0031]
The culture temperature can be appropriately changed within the range in which microorganisms grow and produce the target protein. In the case of Escherichia coli, it is preferably about 20 to 42 ° C. Although the culture time varies somewhat depending on conditions, the culture may be terminated at an appropriate time in consideration of the time when the lactate oxidase reaches the maximum yield, and is usually about 6 to 48 hours. The pH of the medium can be changed as appropriate as long as the bacteria grow and produce the target protein, but is preferably about pH 6.0 to 9.0.
[0032]
A culture solution containing bacterial cells producing a novel protein having sarcosine oxidase activity in a culture can be collected and used as it is, but generally, a novel protein having sarcosine oxidase activity is contained in the culture solution according to a conventional method. If the target protein-containing solution is present, the target protein-containing solution and the microbial cells are separated by filtration, centrifugation, or the like. When a novel protein having sarcosine oxidase activity is present in the microbial cells, the microbial cells are collected from the obtained culture by means of filtration or centrifugation, and then the microbial cells are collected by a mechanical method or lysozyme. The target protein is destroyed by an enzymatic method, and if necessary, a chelating agent such as EDTA and / or a surfactant is added to solubilize the target protein, and separated and collected as an aqueous solution.
[0033]
The solution containing the novel protein having sarcosine oxidase activity obtained as described above is subjected to, for example, vacuum concentration, membrane concentration, salting-out treatment such as ammonium sulfate or sodium sulfate, or a hydrophilic organic solvent such as methanol, What is necessary is just to precipitate by the fractional precipitation method by ethanol, acetone, etc. Heat treatment and isoelectric point treatment are also effective purification means. Then, a novel protein having purified sarcosine oxidase activity can be obtained by performing gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, affinity chromatography and the like.
[0034]
For example, separation and purification are performed by gel filtration using Sephadex G-25, G-200 (Pharmacia Biotech), DEAE Sepharose CL6-B (Pharmacia Biotech), octyl Sepharose CL6-B (Pharmacia Biotech) column chromatography. A purified enzyme preparation can be obtained. This purified enzyme preparation is purified to such an extent that it shows an almost single band on electrophoresis (SDS-PAGE).
[0035]
The novel protein having sarcosine oxidase activity of the present invention obtained as described above is characterized in that Km is 1 mM or less. The value of Km is preferably 0.5 mM or less, more preferably 0.2 mM or less, and still more preferably 0.2 ± 0.1 mM. If Km is greater than 1 mM, it is not preferred because it is inferior in terms of rapidity in the determination of sarcosine, creatine and creatinine. That is, conventionally known sarcosine oxidase has a Km of about 3 mM or more, whereas the novel protein having sarcosine oxidase activity of the present invention has a larger Km value than known enzymes that catalyze the same reaction. It is a novel enzyme characterized by being different.
[0036]
Specifically, the novel protein having sarcosine oxidase activity of the present invention preferably has the following physicochemical properties.
Action: acts on sarcosine in the presence of water and oxygen to produce hydrogen peroxide Km: 0.2 ± 0.1 mM
Molecular weight: 40000 ± 4000 (SDS-PAGE)
[0037]
Furthermore, the novel protein having sarcosine oxidase activity of the present invention is more preferably one having the following physicochemical properties.
Action: acts on sarcosine in the presence of water and oxygen to produce hydrogen peroxide Km: 0.2 ± 0.1 mM
Optimal temperature: 45 ± 5 ° C
Optimum pH: 8.0 ± 0.5
Thermal stability: 45 ± 5 ° C or less (pH 8.0, treatment for 10 minutes)
pH stability: 8.0 ± 0.5 (25 ° C., 5 hours treatment)
Molecular weight: 40000 ± 4000 (SDS-PAGE)
160000 ± 16000 (gel filtration)
Inhibitor: PCMB
Substrate specificity: reactive to glycine and N-ethylglycine in addition to sarcosine
As described above, the novel protein having sarcosine oxidase activity of the present invention is useful for quantifying sarcosine, creatine and creatinine, and is suitably used as a reagent for quantifying sarcosine, creatine and creatinine. By using the novel protein having sarcosine oxidase activity in the present invention, the time required for quantification of sarcosine, creatine and creatinine can be shortened several times as compared with the case of using conventional sarcosine oxidase.
[0039]
【Example】
Hereinafter, the present invention will be specifically described by way of examples. In the examples, sarcosine oxidase activity was measured using the following reagents and measurement conditions.
[0040]
<Reagent>
An aqueous solution containing 10 mM sarcosine, 50 mM potassium phosphate (pH 8.0), 0.5 mM 4-aminoantipyrine, 2 mM phenol, 6 U / ml peroxidase was used.
[0041]
<Measurement conditions>
Pre-warm 3 ml of the reagent at 37 ° C for about 2 minutes, add 0.15 ml of sample (enzyme solution), mix gently, and change the absorbance at 500 nm with a spectrophotometer controlled at 37 ° C using water as a control. Was recorded for 2-3 minutes, and the change in absorbance per minute was measured from the initial linear portion. The amount of enzyme that degrades 1 μmol of sarcosine per minute under the above conditions is defined as 1 unit (U).
[0042]
Example 1 Isolation of Chromosomal DNA Chromosomal DNA of Bacillus subtilis MI113 (ATCC 33712), an ATCC-preserving strain and a Marburg 168 strain, was isolated by the following method. The strain was cultured in a brain heart infusion medium (Difco) at 37 ° C. overnight and then collected by centrifugation (8000 rpm, 10 minutes). After washing the cells with a solution of 15 mM sodium citrate (containing 0.15 M sodium chloride), the cells were suspended in 5 ml of a solution containing 20% sucrose, 50 mM Tris-HCl (pH 7.6) and 1 mM EDTA, and 1 ml of lysozyme solution. (100 mg / ml) was added, incubated at 37 ° C. for 30 minutes, and then a solution containing 11 ml of 1% lauroyl sarcosine acid, 0.1 M EDTA (pH 9.6) was added.
[0043]
About 100% of ethidium bromide solution and 0.5% cesium chloride were added to this suspension, mixed with stirring, and DNA was fractionated by ultracentrifugation at 55000 rpm for 20 hours. The collected DNA was dialyzed with 10 mM Tris-HCl buffer (pH 8.0; hereinafter abbreviated as TE) containing 1 mM EDTA to obtain a purified DNA preparation. This was treated with an equal amount of chloroform / phenol solution, and the aqueous layer was separated by centrifugation. Then, twice the amount of ethanol was added, and the DNA was again separated by the above method and dissolved in 2 ml of TE.
[0044]
Example 2 Preparation of a recombinant vector having a gene encoding a novel protein having sarcosine oxidase activity Table 1 shows the DNA obtained in Example 1 as a template and the oligonucleotides of SEQ ID NOs: 2 and 3 in the Sequence Listing as primers. A composition solution as shown was prepared and PCR was performed under known conditions to amplify a gene encoding a novel protein having sarcosine oxidase activity.
[0045]
[Table 1]

[0046]
The vector pUC19 digested with the amplification product (about 1.1 kbp) and the restriction enzyme SmaI was ligated with T4 DNA ligase, and transformed with competent cells of Escherichia coli JM109 (manufactured by Toyobo Co., Ltd.) to obtain transformants. . The inserted DNA fragment of about 1.1 kbp exists in the plasmid of about 3.8 kbp possessed by the transformant. The base sequence of the gene was determined by the dideoxy method as shown in SEQ ID NO: 4 in the sequence listing, the plasmid was designated pBSOLR2, and the transformant was Escherichia coli JM109 (pBSOLR2). Escherichia coli JM109 (pBSOLR2) is a transformant having a gene encoding a novel protein having sarcosine oxidase activity. The structure of pBSOLR2 is as shown in FIG.
[0047]
Example 3 Production of a novel protein having sarcosine oxidase activity from Escherichia coli JM109 (pBSOLR2) 500 ml of TB medium was dispensed into a 2 L flask, autoclaved at 121 ° C. for 15 minutes, allowed to cool and then separately sterile filtered 50 mg 0.5 ml / ml ampicillin (Nacalai Tesque) was added. This medium was inoculated with 5 ml of a culture solution of Escherichia coli JM109 (pBSOLR2) previously cultured in an LB medium with shaking at 30 ° C. for 16 hours, and cultured with aeration and stirring at 37 ° C. for 24 hours. The sarcosine oxidase activity at the end of the culture was about 0.35 U / ml.
[0048]
The cells were collected by centrifugation and suspended in 50 mM phosphate buffer (pH 8.0). The cell suspension was crushed with ultrasonic waves and centrifuged to obtain a supernatant. The obtained crude enzyme solution was subjected to 0.5M saturated ammonium sulfate fractionation treatment, desalted by gel filtration with Sephadex G-25 (Pharmacia Biotech), and DEAE Sepharose CL6-B (Pharmacia Biotech). Separation and purification were performed by column chromatography and Sephadex G-200 (Pharmacia Biotech) column chromatography to obtain a purified protein surface product. The novel protein preparation having sarcosine oxidase activity obtained by this method shows an almost single band on electrophoresis (SDS-PAGE), and the specific activity at this time is about 0.6 U / mg protein. Met.
[0049]
Below, the property of the novel protein which has the sarcosine oxidase activity obtained by the said method is shown.
Action: acts on sarcosine in the presence of water and oxygen to produce hydrogen peroxide Km: 0.2 ± 0.1 mM
Optimal temperature: 45 ± 5 ° C
Optimum pH: 8.0 ± 0.5
Thermal stability: 45 ± 5 ° C or less (pH 8.0, treatment for 10 minutes)
pH stability: 8.0 ± 0.5 (25 ° C., 5 hours treatment)
Molecular weight: 40000 ± 4000 (SDS-PAGE)
160000 ± 16000 (gel filtration)
Inhibitor: PCMB
Substrate specificity: reactive to glycine and N-ethylglycine in addition to sarcosine. The amino acid sequence of the protein was determined as SEQ ID NO: 1 in the sequence listing.
[0050]
【The invention's effect】
As described above, according to the present invention, a novel protein having sarcosine oxidase activity with a Km of 1 mM or less was isolated. Moreover, rapid quantification regarding sarcosine, creatine and creatinine became possible by using the protein. Furthermore, improvement of physicochemical properties can be derived by performing functional modification by using protein engineering techniques.
[0051]
[Sequence Listing]

[0052]

[0053]

[0054]

[Brief description of the drawings]
FIG. 1 shows the structure of pBSOLR2.

Claims (6)

Protein of the following (a) or (b).
(A) a protein comprising the amino acid sequence described in the sequence listing / SEQ ID NO: 1 (b) comprising the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and sarcosine Protein with degradation activity Genes encoding the following proteins (a) or (b).
(A) a protein comprising the amino acid sequence described in the sequence listing / SEQ ID NO: 1 (b) comprising the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and sarcosine Protein with degradation activity Ru DNA der the following (c) or (d) gene.
(C) DNA consisting of the base sequence described in Sequence Listing / SEQ ID NO: 4
(D) DNA encoding a protein having one or several bases deleted, substituted or added in the sequence (c) and having sarcosine degradation activity A recombinant vector containing the gene according to claim 2 or 3 . A transformant comprising the recombinant vector according to claim 4 . According culturing the transformant of claim 5, protein method according to claim 1 you and collecting the protein having a sarcosine decomposition activity from the culture.

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