JP2546273B2 - Pyruvate oxidase composition - Google Patents
Pyruvate oxidase compositionInfo
- Publication number
- JP2546273B2 JP2546273B2 JP62154175A JP15417587A JP2546273B2 JP 2546273 B2 JP2546273 B2 JP 2546273B2 JP 62154175 A JP62154175 A JP 62154175A JP 15417587 A JP15417587 A JP 15417587A JP 2546273 B2 JP2546273 B2 JP 2546273B2
- Authority
- JP
- Japan
- Prior art keywords
- pyruvate oxidase
- pyruvate
- activity
- oxidase
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明はピルビン酸オキシダーゼの活性化に関するも
のである。TECHNICAL FIELD The present invention relates to activation of pyruvate oxidase.
ピルビン酸オキシダーゼは血清や尿中のピルビン酸の
比色定量に用いられる酵素である。本発明によれば、ピ
ルビン酸オキシダーゼを活性化することにより、従来の
ピルビン酸オキシダーゼの使用量を効果的に減少させる
ことができる。Pyruvate oxidase is an enzyme used for colorimetric determination of pyruvate in serum and urine. According to the present invention, by activating pyruvate oxidase, the amount of conventional pyruvate oxidase used can be effectively reduced.
（従来の技術） 従来よりアルコール類、有機溶媒及びポリオール類が
酵素の安定化に一般的に効果があることは知られていた
（別冊蛋白質核酸酵素植物酵素蛋白質研究法187〜195頁
（1976）発行所：共立出版株式会社）。しかしながらこ
れら物質の添加効果は酵素の精製時や保存時の失活防止
が主な効果であり、酵素活性化が必ずしも生じ得なかっ
た。(Prior Art) It has been conventionally known that alcohols, organic solvents and polyols are generally effective in stabilizing enzymes (Separate volume Protein Nucleic Acid Enzyme Plant Enzyme Protein Research Method pp. 187-195 (1976). Publisher: Kyoritsu Publishing Co., Ltd.). However, the main effect of the addition of these substances is the prevention of deactivation during purification and storage of the enzyme, and activation of the enzyme could not always occur.
一方ピルビン酸オキシダーゼは酵素安定性は良いが、
酵素活性は満足のいくものではなかった。その効果的な
活性化法は従来から知られておらず、効果的な活性化の
確立が望まれていた。On the other hand, pyruvate oxidase has good enzyme stability,
The enzyme activity was not satisfactory. The effective activation method has not been known so far, and establishment of effective activation has been desired.
（発明が解決しようとする問題点） 本発明者は上記の知見を踏まえ、ピルビン酸オキシダ
ーゼの活性化法を見い出そうとした。(Problems to be Solved by the Invention) Based on the above findings, the present inventor tried to find a method for activating pyruvate oxidase.
（問題点を解決するための手段） 本発明者らは、ピルビン酸オキシダーゼの１種、例え
ばラクトバチルス・エス・ピー・TE−6103株（微工研菌
寄第8886号）が生産する熱安定性に優れたピルビン酸オ
キシダーゼの反応液にアルコール類、有機溶媒類又はポ
リオール類を添加するとピルビン酸オキシダーゼの活性
が増大することを見い出し、本発明に到達した。(Means for Solving Problems) The present inventors have developed a thermostable product produced by one type of pyruvate oxidase, for example, Lactobacillus sp. TE-6103 strain (Ministry of Industrial Science and Technology No. 8886). The inventors have found that the activity of pyruvate oxidase is increased by adding alcohols, organic solvents or polyols to the reaction solution of pyruvate oxidase, which has excellent properties, and arrived at the present invention.
すなわち、本発明は、ピルビン酸塩、過酸化水素検出
試薬および緩衝液を含有するピルビン酸オキシダーゼ溶
液に、１価アルコール類、有機溶媒又は２価以上のポリ
オール類を含有させたことを特徴とするピルビン酸オキ
シダーゼ組成物である。That is, the present invention is characterized in that a pyruvic acid oxidase solution containing a pyruvate, a hydrogen peroxide detection reagent and a buffer contains a monohydric alcohol, an organic solvent or a divalent or higher valent polyol. It is a pyruvate oxidase composition.
本発明に使用されるピルビン酸オキシダーゼとして
は、好ましくはラクトバチルス・エス・ピー・TE−6103
株の生産するピルビン酸オキシダーゼが挙げられるが、
この菌の生産するピルビン酸オキシダーゼだけに限ら
ず、酵素含有液に１価アルコール類、有機溶媒類又は２
価以上のポリオール類を添加することにより活性化され
るピルビン酸オキシダーゼは、すべて本発明に使用でき
る。The pyruvate oxidase used in the present invention is preferably Lactobacillus sp. TE-6103.
Examples include pyruvate oxidase produced by the strain,
Not only pyruvate oxidase produced by this bacterium, but also monohydric alcohols, organic solvents or 2
Any pyruvate oxidase activated by adding a polyol having a valency or higher can be used in the present invention.
ピルビン酸塩とは、ピルビン酸オキシダーゼの基質と
なるピルビン酸塩であって、ピルビン酸カリウムなどが
例示される。過酸化水素検出試薬とは、ピルビン酸オキ
シダーゼ反応により生成する過酸化水素を検出試薬であ
って、４−アミノアンチピリン、TOOS（Ｎ−エチル−Ｎ
−（２−ヒドロキシ−３−スルホプロピル）−ｍ−トル
イジン）およびPOD（ペルオキシダーゼ）の組み合わせ
が例示される。緩衝液としては、Ｋ−リン酸緩衝液など
が例示される。Pyruvate is a pyruvate serving as a substrate for pyruvate oxidase, and examples thereof include potassium pyruvate. The hydrogen peroxide detection reagent is a detection reagent for hydrogen peroxide produced by a pyruvate oxidase reaction, and includes 4-aminoantipyrine and TOOS (N-ethyl-N
An example is a combination of-(2-hydroxy-3-sulfopropyl) -m-toluidine) and POD (peroxidase). Examples of the buffer solution include K-phosphate buffer solution and the like.
また本発明に使用される１価アルコール類、有機溶媒
類及び２価以上のポリオール類の好適な例としては以下
のものが挙げられる。The following are preferred examples of monohydric alcohols, organic solvents and dihydric or higher valent polyols used in the present invention.
１価アルコール類：メタノール，エタノール，プロ
パノール，イソプロパノール 有機溶媒類：アセトン,DMSO ２価以上のポリオール類：エチレングリコール，プ
ロピレングリコール，グリセリン さらにこれらの添加量としては、これらの添加により
ピルビン酸オキシダーゼが沈殿しない量であればよく、
通常0.1〜50w/v％、好ましくは10〜30w/v％程度であ
る。Monohydric alcohols: Methanol, ethanol, propanol, isopropanol Organic solvents: Acetone, DMSO Dihydric or higher polyols: ethylene glycol, propylene glycol, glycerin Furthermore, pyruvic oxidase precipitates by adding these If the amount is not,
It is usually 0.1 to 50 w / v%, preferably about 10 to 30 w / v%.
次にピルビン酸オキシダーゼの活性測定法を示す。 Next, a method for measuring the activity of pyruvate oxidase will be shown.
上記反応混合液を調製した後、2.5mlを分取し、0.3M
ピルビン酸カリウム水溶液を0.5ml加えて37℃で約５分
予備加温する。酵素溶液0.1mlを添加し、ゆるやかに混
和後、水を対照に37℃に制御された分光光度計で550nm
の吸光度変化を記録し、その初期直線部分から１分間当
り吸光度変化を求める（Δ0Dtest）。 After preparing the above reaction mixture, collect 2.5 ml and
Add 0.5 ml of potassium pyruvate aqueous solution and preheat at 37 ° C for about 5 minutes. Add 0.1 ml of enzyme solution, mix gently, and use water as a control at 370 nm with a spectrophotometer controlled at 37 ° C.
The change in absorbance is recorded and the change in absorbance per minute is obtained from the initial linear portion (Δ0Dtest).
盲検は酵素溶液の代りに50mMK−リン酸緩衝液pH5.7を
0.1ml加え、上記同様に操作を行なって１分間当りの吸
光度変化を求める（Δ0Dlank）。In the blind test, 50 mM K-phosphate buffer pH 5.7 was used instead of the enzyme solution.
Add 0.1 ml, and perform the same operation as above to obtain the change in absorbance per minute (Δ0Dlank).
ピルビン酸オキシダーゼ活性の表示は、上記条件下で
１マイクロモルの過酸化水素を生じる活性を１単位
（Ｕ）とした。In the display of pyruvate oxidase activity, 1 unit (U) was defined as the activity of producing 1 μmol of hydrogen peroxide under the above conditions.
（効果） 本発明では、上記活性測定法の反応液に１価アルコー
ル類、有機溶媒類又は２価以上のポリオール類を0.1〜5
0w/v％になるように添加することにより、ピルビン酸オ
キシダーゼが活性化され、活性が顕著に増大する。(Effect) In the present invention, monohydric alcohols, organic solvents or dihydric or higher polyols are added in an amount of 0.1 to 5 in the reaction solution of the above-mentioned activity measuring method.
By adding 0 w / v%, pyruvate oxidase is activated and the activity is remarkably increased.
（実施例） 以下実施例を挙げて本発明を説明するが、本発明は何
らこれらによって限定されるものではない。(Example) Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.
実施例 １ Ｋ−リン酸緩衝液、pH5.9M 50mM ４−AA 0.01％ TOOS 0.02％ FAD・Na2 10mM TPP 0.02mM POD 5U/ml EDTA・Na2 10mM MgSO4 10 mM エタノール ０〜30％ ピルビン酸カリウム 50mM 上記の組成よりなる反応液3mlを分取して37℃で約５
分間予備加温した。これにラクトバチルス・エス・ピー
・TE−6103株の生産するピルビン酸オキシダーゼ溶液
（0.152U/ml）0.1mlを加え、ゆるやかに混和した後、37
℃で550nmにおける吸光度変化を記録し、その初期直線
部分よりピルビン酸オキシダーゼの活性を求めた。Example 1 K-phosphate buffer, pH 5.9M 50 mM 4-AA 0.01% TOOS 0.02% FAD • Na 2 10 mM TPP 0.02 mM POD 5 U / ml EDTA • Na 2 10 mM MgSO 4 10 mM ethanol 0-30% pyruvate Potassium 50 mM 3 ml of the reaction solution having the above composition is collected and kept at 37 ° C for about 5 minutes.
Pre-warmed for minutes. To this, 0.1 ml of pyruvate oxidase solution (0.152U / ml) produced by Lactobacillus sp. TE-6103 strain was added, and after gently mixing, 37
The change in absorbance at 550 nm was recorded at ℃, and the activity of pyruvate oxidase was determined from the initial linear portion.
その結果は第１表に示した通りで、エタノールの添加
量に比例してピルビン酸オキシダーゼ活性は活性化さ
れ、30％エタノール存在下でピルビン酸オキシダーゼ活
性は約1.8倍に活性化された。The results are shown in Table 1, and the pyruvate oxidase activity was activated in proportion to the amount of ethanol added, and the pyruvate oxidase activity was activated about 1.8 times in the presence of 30% ethanol.
実施例 ２ Ｋ−リン酸緩衝液、pH5.9M 50mM ４−AA 0.01％ TOOS 0.02％ FAD・Na2 10mM TPP 0.02mM POD 5U/ml EDTA・Na2 1.0mM MgSO4 10 mM DMSO 0,10,30％ ピルビン酸カリウム 50mM 上記の組成よりなる反応液3mlを分取して37℃で約５
分間予備加温した。これにラクトバチルス・エス・ピー
・TE−6103株の生産するピルビン酸オキシダーゼ溶液
（0.152U/ml）0.1mlを加え、ゆるやかに混和した後、37
℃で550nmにおける吸光度変化を記録し、その初期直線
部分よりピルビン酸オキシダーゼ活性を求めた。 Example 2 K-phosphate buffer, pH 5.9M 50 mM 4-AA 0.01% TOOS 0.02% FAD • Na 2 10 mM TPP 0.02 mM POD 5 U / ml EDTA • Na 2 1.0 mM MgSO 4 10 mM DMSO 0,10,30 % Potassium pyruvate 50 mM 3 ml of the reaction solution having the above composition was collected and allowed to stand at 37 ° C. for about 5 minutes.
Pre-warmed for minutes. To this, 0.1 ml of pyruvate oxidase solution (0.152U / ml) produced by Lactobacillus sp. TE-6103 strain was added, and after gently mixing, 37
The change in absorbance at 550 nm was recorded at ℃, and pyruvate oxidase activity was determined from the initial linear portion.
その結果は第２表に示す通りでDMSOの添加量に比例し
てピルビン酸オキシダーゼ活性は活性化され、30％DMSO
存在下ではピルビン酸オキシダーゼ活性は約2.3倍に活
性された。The results are shown in Table 2. Pyruvate oxidase activity was activated in proportion to the amount of DMSO added, and 30% DMSO
In the presence, pyruvate oxidase activity was increased about 2.3 times.
実施例 ３ Ｋ−リン酸緩衝液、pH5.9M 50mM ４−AA 0.01％ TOOS 0.02％ FAD・Na2 10mM TPP 0.2mM POD 5U/ml EDTA・Na2 1.0mM MgSO4 10mM エチレングリコール 0,10,30％ ピルビン酸カリウム 50mM 上記の組成よりなる反応液3mlを分取して37℃で約５
分間予備加温した。これにラクトバチス・エス・ピー・
TE−6103株の生産するピルビン酸オキシダーゼ溶液（0.
152U/ml）0.1mlを加え、ゆるやかに混和した後、37℃で
550nmにおける吸光度変化を記録し、その初期直線部分
よりピルビン酸オキシダーゼ活性を求めた。 Example 3 K-phosphate buffer, pH 5.9M 50 mM 4-AA 0.01% TOOS 0.02% FAD • Na 2 10 mM TPP 0.2 mM POD 5 U / ml EDTA • Na 2 1.0 mM MgSO 4 10 mM ethylene glycol 0,10,30 % Potassium pyruvate 50 mM 3 ml of the reaction solution having the above composition was collected and allowed to stand at 37 ° C. for about 5 minutes.
Pre-warmed for minutes. Lactobacillus sp.
Pyruvate oxidase solution produced by the TE-6103 strain (0.
152U / ml) 0.1 ml, mix gently and then at 37 ℃
The change in absorbance at 550 nm was recorded, and pyruvate oxidase activity was determined from the initial linear portion.
その結果は第３表に示した通りでエチレングリコール
の添加量に比例してピルビン酸オキシダーゼ活性は活性
化され、30％エチレングリコール存在下ではピルビン酸
オキシダーゼ活性は約2.1倍に活性化された。The results are shown in Table 3, and the pyruvate oxidase activity was activated in proportion to the added amount of ethylene glycol, and in the presence of 30% ethylene glycol, the pyruvate oxidase activity was activated about 2.1 times.
実施例 ４ 一般的に酵素の安定化剤として知られている以下の物
質を実施例１と同じピルビン酸オキシダーゼの反応系に
含有させ、ピルビン酸オキシダーゼの活性を求めた。 Example 4 The following substances, which are generally known as stabilizers for enzymes, were added to the same reaction system of pyruvate oxidase as in Example 1 to determine the activity of pyruvate oxidase.
表示は無添加の場合の活性を100として相対活性で示
した。The display shows relative activity with the activity in the case of no addition as 100.
比較例 １ 一般的に酵素の安定化剤として知られている以下の物
質を実施例１と同じピルビン酸オキシダーゼの反応系に
含有させ、ピルビン酸オキシダーゼの活性を求めた。 Comparative Example 1 The following substances, which are generally known as stabilizers for enzymes, were added to the same reaction system of pyruvate oxidase as in Example 1 to determine the activity of pyruvate oxidase.
表示は無添加の場合の活性を100として相対活性で示
した。The display shows relative activity with the activity in the case of no addition as 100.
（発明の効果） 本発明では、ピルビン酸オキシダーゼを活性化するこ
とにより、従来のピルビン酸オキシダーゼの使用量を減
少させることができる。 (Effect of the Invention) In the present invention, by activating pyruvate oxidase, the amount of conventional pyruvate oxidase used can be reduced.
Claims (1)
緩衝液を含有するピルビン酸オキシダーゼ溶液に、１価
アルコール類、有機溶媒又は２価以上のポリオール類を
含有させたことを特徴とするピルビン酸オキシダーゼ組
成物。1. A pyruvate characterized by containing a monohydric alcohol, an organic solvent, or a dihydric or higher polyol in a pyruvate oxidase solution containing a pyruvate, a hydrogen peroxide detection reagent and a buffer solution. Acid oxidase composition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62154175A JP2546273B2 (en) | 1987-06-19 | 1987-06-19 | Pyruvate oxidase composition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62154175A JP2546273B2 (en) | 1987-06-19 | 1987-06-19 | Pyruvate oxidase composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63317080A JPS63317080A (en) | 1988-12-26 |
| JP2546273B2 true JP2546273B2 (en) | 1996-10-23 |
Family
ID=15578474
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62154175A Expired - Lifetime JP2546273B2 (en) | 1987-06-19 | 1987-06-19 | Pyruvate oxidase composition |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2546273B2 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5484090A (en) * | 1977-11-04 | 1979-07-04 | Toyo Jozo Co Ltd | Beta-galactosidase-activity enhancing medium |
-
1987
- 1987-06-19 JP JP62154175A patent/JP2546273B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5484090A (en) * | 1977-11-04 | 1979-07-04 | Toyo Jozo Co Ltd | Beta-galactosidase-activity enhancing medium |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63317080A (en) | 1988-12-26 |
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