JPH07110240B2 - Creatinine amide hydrolase preparation - Google Patents
Creatinine amide hydrolase preparationInfo
- Publication number
- JPH07110240B2 JPH07110240B2 JP60069752A JP6975285A JPH07110240B2 JP H07110240 B2 JPH07110240 B2 JP H07110240B2 JP 60069752 A JP60069752 A JP 60069752A JP 6975285 A JP6975285 A JP 6975285A JP H07110240 B2 JPH07110240 B2 JP H07110240B2
- Authority
- JP
- Japan
- Prior art keywords
- crn
- preparation
- amide hydrolase
- creatinine
- enzyme
- 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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- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 この発明はクレアチニンアミドヒドロラーゼ（以下CRN
という）を安定な状態で含有するCRN製剤に関するもの
である。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] This invention relates to creatinine amide hydrolase (hereinafter referred to as CRN).
") In a stable state.
クレアチニンは血液や尿中に存在し、正常人の１日当た
りの尿への排出量はほぼ一定であるため、これを測定す
ることによって腎障害、尿路閉塞等の疾患を診断するこ
とができる。そして、このクレアチニンの定量分析に供
されるCRN製剤は、近年注目されるようになったクレア
チニンを酵素的に分解する測定法に使用される診断用の
酸素製剤である。Since creatinine is present in blood and urine, and the amount of urinary excretion into the urine of a normal person per day is almost constant, diseases such as renal disorder and urinary tract obstruction can be diagnosed by measuring this. The CRN preparation used for the quantitative analysis of creatinine is an oxygen preparation for diagnosis used in a measuring method for enzymatically decomposing creatinine, which has been receiving attention in recent years.
その測定方法は、 の反応によってクレアチニンをクレアチンに分解し、生
成したクレアチンを更に他の酵素あるいは他の化学的方
法と組み合わせて測定し、クレアチニンの定量をするも
のである。The measuring method is The creatinine is quantified by decomposing creatinine into creatine by the reaction of 1) and measuring the produced creatine in combination with other enzyme or other chemical method.
しかし、CRNは酵素製剤の製造中や保存中に著しく酵素
活性が低下したり、他の酵素や色原体、補酵素等ととも
に溶液状態で保存中に著しく変性が生じて濁りを発生す
ることがあり、組成物に由来する過酸化物等により色原
体との反応時における自己発色があり測定精度に悪影響
を与える原因となっていた。However, CRN may have a marked decrease in enzyme activity during production or storage of enzyme preparations, or may cause turbidity due to significant denaturation during storage in solution with other enzymes, chromogens, coenzymes, etc. However, due to peroxides and the like derived from the composition, there is self-coloring during the reaction with the chromogen, which is a cause of adversely affecting the measurement accuracy.
そのため、CRNを含有する酵素製剤の安定化が重要な課
題となっていた。Therefore, stabilization of the enzyme preparation containing CRN has been an important issue.
そこで、本発明はCRNを含む酵素製剤の安定化を図るこ
とを目的として以下に述べる手段を講じた。Therefore, the present invention has taken the following means for the purpose of stabilizing the enzyme preparation containing CRN.
本発明はCRNを含有する酵素製剤を安定化させるため鋭
意研究を行った結果、CRN製剤に２価のマンガンイオ
ン、非イオン性界面活性剤及びアジ化物、特にアジ化ナ
トリウムを含有させると、酵素活性を著しく向上させる
ことを見出し、本発明をするに至ったものである。The present invention has conducted extensive studies to stabilize an enzyme preparation containing CRN. As a result, when the CRN preparation contains a divalent manganese ion, a nonionic surfactant and an azide, particularly sodium azide, The inventors have found that the activity is remarkably improved and have completed the present invention.
すなわち、本発明にクレアチニンアミドヒドロラーゼを
含有する酵素剤に２価のマンガンイオン、非イオン性界
面活性剤及びアジ化ナトリウム（以下活性化剤と総称す
る）を含有させたものである。That is, in the present invention, an enzyme agent containing creatinine amide hydrolase contains divalent manganese ions, a nonionic surfactant and sodium azide (hereinafter collectively referred to as activator).
以下、各構成について説明する。Each configuration will be described below.
クレアチニンアミドヒドロラーゼについて 本発明のクレアチニンアミドヒドロラーゼはその起源を
問わず全て活性化の対象となるが特に繁用されるのはア
ルカリゲネス属やシュードモナス属等に属する微生物起
源のCRNである。Creatinine amide hydrolase The creatinine amide hydrolase of the present invention is subject to activation regardless of its origin, but what is particularly frequently used is CRN of microbial origin belonging to the genera Alcaligenes or Pseudomonas.
クレアチニンアミドヒドロラーゼ製剤について 本発明のクレアチニンアミドヒドロラーゼ製剤はクレア
チニンアミドヒドロラーゼを単独で含有するものに限定
されず他の物質たとえばクレアチニンの定量に必要な酵
素並びに色原体更には補酵素またはこれらの安定化に必
要な安定化剤成分等を含有するものを総称する。Creatinine amide hydrolase preparation The creatinine amide hydrolase preparation of the present invention is not limited to those containing creatinine amide hydrolase alone, and other substances such as enzymes necessary for the quantification of creatinine and chromogens and coenzymes or their stabilization Those which contain the necessary stabilizer components and the like are collectively referred to.
また、CRN製剤は溶液状とは限らず、凍結乾燥や噴霧乾
燥等による乾燥粉末状等の状態であってもよい。Further, the CRN preparation is not limited to a solution form, and may be in a dry powder form such as freeze-dried or spray-dried form.
２価のマンガンイオンについて 本発明に用いられる活性化剤の最も代表的なものであ
り、水溶液で２価のマンガンイオンに解離するものであ
ればその種類は問わないが、代表的なものは塩化マンガ
ン、硫酸マンガン、酢酸マンガン、硝酸マンガン等であ
る。Divalent manganese ion The most representative activator used in the present invention is not particularly limited as long as it dissociates into divalent manganese ion in an aqueous solution. Examples include manganese, manganese sulfate, manganese acetate, and manganese nitrate.
２価のマンガンイオンの使用量はCRNの酵素活性を活性
化する範囲であればいかなる量でもよいが、もっとも効
果的な範囲は液状のCRN製剤の場合使用濃度が0.1mM〜1m
Mの範囲である。The amount of divalent manganese ion used may be any amount as long as it activates the enzymatic activity of CRN, but the most effective range is the use concentration of 0.1 mM to 1 m for liquid CRN preparations.
It is in the range of M.
非イオン性界面活性剤について 代表的なものとしてポリオキシエチレンアルキルエーテ
ル、ポリオキシエチレンアルキルフェニルエーテルが挙
げられ、アルキルとしては炭素数７以上のアルキル、例
えばオクチル、ノニル、ラウリル等が挙げられる。これ
らの非イオン性界面活性剤はHLBが10以上のものが望ま
しい。また、非イオン性界面活性剤は高感度な発色系に
影響を及ぼす過酸化物等の含有の少ない種類のものが望
ましい。Representative examples of the nonionic surfactant include polyoxyethylene alkyl ether and polyoxyethylene alkylphenyl ether, and examples of the alkyl include alkyl having 7 or more carbon atoms, such as octyl, nonyl and lauryl. It is desirable that these nonionic surfactants have an HLB of 10 or more. Further, it is desirable that the nonionic surfactant be of a type containing a small amount of peroxide or the like which affects a highly sensitive color forming system.
アジ化ナトリウムについて アジ化ナトリウムは酵素製剤中に混入される微生物にCR
N等の変質を防止する機能があり、微生物に対して静菌
作用あるいは殺菌作用を有したり、微生物の活性を不活
化するものである。About sodium azide Sodium azide is CR for microorganisms mixed in enzyme preparations.
It has a function of preventing alteration of N and the like, has a bacteriostatic action or a bactericidal action on microorganisms, and inactivates the activity of microorganisms.
非イオン性界面活性剤及びアジ化ナトリウムの配合割合 この割合は活性化作用の強弱あるいは活性化剤の化学的
安定性、更には各活性化剤の併用等を考慮して定めれば
よいが、一般にCRN製剤1ml当たり0.001〜10mgの範囲か
ら選択すれば確実な効果が得られる。Mixing ratio of nonionic surfactant and sodium azide This ratio may be determined in consideration of the strength of the activating action or the chemical stability of the activator, and further the combined use of each activator, Generally, a certain effect can be obtained by selecting from the range of 0.001 to 10 mg per 1 ml of CRN preparation.
CRN製剤の調製方法 CRN濃度を１〜500U/mlとなるように、pH6.0〜9.0程度の
緩衝液、例えば50mMグリシルグリシン緩衝液（pH7.8）
を用いて溶解し次に活性化剤を添加する。この際、上記
CRN製剤に活性化剤粉末を直接配合するか、その粉末を
一旦水あるいは所定の緩衝液に溶解してから添加し常法
に従って撹拌する。最後に、この混合液を乾燥させたい
時は凍結乾燥や噴霧乾燥等を行えばよい。Method for preparing CRN preparation A buffer solution having a pH of about 6.0 to 9.0, for example, a 50 mM glycylglycine buffer solution (pH 7.8) so that the CRN concentration is 1 to 500 U / ml.
To dissolve and then add activator. At this time,
The activator powder is directly added to the CRN preparation, or the powder is once dissolved in water or a predetermined buffer solution and then added and stirred according to a conventional method. Finally, when it is desired to dry this mixed solution, freeze drying, spray drying, or the like may be performed.
本発明に係るCRN製剤は活性化剤を添加して酵素活性を
著しく活性化したので、製剤中のCRN含量を減らすこと
ができる。このため、保存中に濁りを生ずることもなく
安定なCRN製剤を提供することができる。Since the CRN formulation according to the present invention markedly activated the enzyme activity by adding an activator, the CRN content in the formulation can be reduced. Therefore, a stable CRN preparation can be provided without causing turbidity during storage.
しかも、高価なCRNの使用量が減少するので経済的であ
る。Moreover, the amount of expensive CRN used is reduced, which is economical.
次に、本発明の実施例を説明する。実施例における酵素
活性の測定は下記に従った。Next, examples of the present invention will be described. The measurement of the enzyme activity in the examples was as follows.
（Ａ）試薬組成 （１）基質溶液 0.1Mクレアチニン溶液 〔クレアチニンを50mMグリシルグリシン緩衝液（pH8.
0）に溶解〕 （２）2mM−HgCl2水溶液 （３）発色液 ２％α−ナフトールエチルアルコール
溶液 （４）発色液 1.2％NaOH/3.2％Na2CO3水溶液 （５）発色液 0.05％ジアセチル水溶液 （Ｂ）測定方法 基質溶液0.9mlに酵素溶液0.1mlを加え、37℃で10分間反
応させた。次に、2mM−HgCl2水溶液2mlを添加して反応
を停止させ、その0.1mlを蒸留水0.9ml、発色液0.5m
l、発色液0.5ml及び発色液0.5mlと充分混合して25
℃で１時間放置して発色させた。2.5mlの蒸留水を添加
して希釈した後、この発色を525nmにおける吸光度で測
定し酵素力価を求めた。尚、酵素力価の表示は、上記条
件の下で１分間に１マイクロモルのクレアチンを精製す
る酵素量を１単位とした。(A) Reagent composition (1) Substrate solution 0.1M creatinine solution [creatinine was added to 50 mM glycylglycine buffer (pH 8.
Soluble in 0)] (2) 2mM-HgCl 2 aqueous solution (3) Color developing solution 2% α-naphthol ethyl alcohol solution (4) Color developing solution 1.2% NaOH / 3.2% Na 2 CO 3 aqueous solution (5) Color developing solution 0.05% diacetyl Aqueous solution (B) Measuring method 0.1 ml of enzyme solution was added to 0.9 ml of substrate solution and reacted at 37 ° C for 10 minutes. Then, the reaction was stopped by adding 2 ml of 2 mM-HgCl 2 aqueous solution, and 0.1 ml of the reaction was added to 0.9 ml of distilled water and 0.5 m of the coloring solution.
25, well mixed with 0.5 ml of coloring liquid and 0.5 ml of coloring liquid
Color was developed by leaving it at 1 ° C for 1 hour. After diluting by adding 2.5 ml of distilled water, the color development was measured by the absorbance at 525 nm to determine the enzyme titer. The enzyme titer was expressed as 1 unit of the amount of enzyme that purifies 1 micromol of creatine per minute under the above conditions.
実施例１ アルカリゲネス属に属する微生物から得られたCRNを用
い、50mlグリシルグリシン緩衝液（pH8.0）に溶解し
た。そしてこの溶液に第１表の各化合物を加えた。Example 1 CRN obtained from a microorganism belonging to the genus Alcaligenes was used and dissolved in 50 ml glycylglycine buffer (pH 8.0). Then, each compound shown in Table 1 was added to this solution.
上記の酵素活性の測定法によりCRNの酵素活性を測定し
た。The enzyme activity of CRN was measured by the above-mentioned assay method of enzyme activity.
第１表から明らかなように２価のマンガンイオン、非イ
オン性界面活性剤及びアジ化ナトリウムを添加したもの
は著しい酵素活性を示した。As is clear from Table 1, those to which the divalent manganese ion, the nonionic surfactant and sodium azide were added showed remarkable enzyme activity.
実施例２ 下記組成からなる酵素製剤を調製し、この酵素製剤を50
mMグリシルグリシン緩衝液（pH7.8）に溶解し全量を10m
lにした。 Example 2 An enzyme preparation having the following composition was prepared.
Dissolve in mM glycylglycine buffer (pH 7.8) to a total volume of 10 m
set to l.
CRN（アルカリゲネス属起源） 140単位 クレアチンアミジノヒドロラーゼ（バチルス属起源）18
0単位 ザルコシンオキシダーゼ（バチルス属起源） 120単位 ペルオキシダーゼ（西洋ワサビ起源） 100単位 ４−アミノアンチピリン 5mg ポリオキシエチレンノニルフェニルエーテル（HLB19.
0） 10mg アジ化ナトリウム 2mg MnCl2・4H2O 1mg 一方、この製剤からポリオキシエチレンノニルフェニル
エーテル（HLB19.0）、アジ化ナトリウム、MnCl2・4H2O
を除いた比較製剤を調製し、同様に50mMグリシルグリシ
ン緩衝液（pH7.8）に溶解し、全量を10mlにした。CRN (Alcaligenes origin) 140 units Creatine amidinohydrolase (Bacillus origin) 18
0 units Sarcosine oxidase (origin from Bacillus) 120 units Peroxidase (origin from horseradish) 100 units 4-aminoantipyrine 5 mg Polyoxyethylene nonyl phenyl ether (HLB19.
0) 10mg Sodium azide 2mg MnCl 2 .4H 2 O 1mg On the other hand, from this preparation polyoxyethylene nonylphenyl ether (HLB19.0), sodium azide, MnCl 2 .4H 2 O
A comparative preparation except that was prepared was dissolved in 50 mM glycylglycine buffer (pH 7.8) in the same manner to make the total volume 10 ml.
これらについて、37℃で４時間及び25℃で５日間の２方
法で保存した後、それぞれ3mlを採取し濁りの発生を600
mnにおける吸光度で測定し、第２表の結果を得た。After storing them for 2 hours at 37 ℃ for 4 hours and at 25 ℃ for 5 days, 3 ml of each was collected and turbidity of 600
The absorbance at mn was measured and the results shown in Table 2 were obtained.
第２表から明らかなように、２価のマンガンイオン、非
イオン性界面活性剤及びアジ化ナトリウムを添加したも
のは大した濁りは発生しないのに対して、無添加のもの
は著しい濁りが発生した。これにより、活性化剤の添加
による効果が確認された。As is clear from Table 2, the ones to which divalent manganese ion, nonionic surfactant and sodium azide were added did not produce much turbidity, while those without addition produced significant turbidity. did. This confirmed the effect of adding the activator.
フロントページの続き (72)発明者 中村 昭四郎 広島県広島市西区己斐大迫２−12―２ (56)参考文献 特開 昭55−23998（ＪＰ，Ａ) 特開 昭58−162294（ＪＰ，Ａ) 特開 昭59−85290（ＪＰ，Ａ) 特開 昭52−128288（ＪＰ，Ａ) 丸尾文治 外１名監修「酵素ハンドブッ ク」朝倉書店（1982−12−１）Ｐ．599Front Page Continuation (72) Inventor Shoshiro Nakamura 2-12-2, Kosui, Nishi, Nishi-ku, Hiroshima City, Hiroshima Prefecture (56) References JP-A-55-23998 (JP, A) JP-A-58-162294 (JP, A) JP-A-59-85290 (JP, A) JP-A-52-128288 (JP, A) Fumiharu Maruo "Enzyme Handbook" supervised by one person Asakura Shoten (1982-12-1) P. 599
Claims (2)
し、更に２価のマンガンイオン、非イオン性界面活性剤
及びアジ化ナトリウムを含有させてなることを特徴とす
るクレアチニンアミドヒドロラーゼ製剤。1. A creatinine amide hydrolase preparation comprising creatinine amide hydrolase and further containing a divalent manganese ion, a nonionic surfactant and sodium azide.
オキシエチレンアルキルエーテル及びHLB10以上のポリ
オキシエチレンアルキルフェニルエーテルのうちから選
択される少なくとも一種であることを特徴とする特許請
求の範囲第１記載のクレアチニンアミドヒドロラーゼ製
剤。2. The nonionic surfactant is at least one selected from a polyoxyethylene alkyl ether having an HLB of 10 or more and a polyoxyethylene alkylphenyl ether having an HLB of 10 or more. The creatinine amide hydrolase preparation according to 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60069752A JPH07110240B2 (en) | 1985-04-01 | 1985-04-01 | Creatinine amide hydrolase preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60069752A JPH07110240B2 (en) | 1985-04-01 | 1985-04-01 | Creatinine amide hydrolase preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61227799A JPS61227799A (en) | 1986-10-09 |
| JPH07110240B2 true JPH07110240B2 (en) | 1995-11-29 |
Family
ID=13411835
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60069752A Expired - Lifetime JPH07110240B2 (en) | 1985-04-01 | 1985-04-01 | Creatinine amide hydrolase preparation |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07110240B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2632391B2 (en) * | 1988-11-16 | 1997-07-23 | 和光純薬工業株式会社 | Method for stabilizing peroxidase |
| DK1889075T3 (en) | 2005-05-17 | 2013-09-02 | Radiometer Medical Aps | Method for stabilizing or reactivating a creatinine sensor with a solution of a divalent manganese ion |
| WO2006122552A1 (en) | 2005-05-17 | 2006-11-23 | Radiometer Medical Aps | Method of stabilising or reactivating a creatinine sensor with a solution of a divalent manganese ion |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS52128288A (en) * | 1976-04-15 | 1977-10-27 | Mitsubishi Chem Ind Ltd | Glucose isomerase solution |
| US4215197A (en) * | 1978-08-04 | 1980-07-29 | Miles Laboratories, Inc. | Test means and method for creatinine determination |
| JPS58162294A (en) * | 1982-03-18 | 1983-09-26 | Toyobo Co Ltd | Immobilized composite enzyme composition |
| JPS5985290A (en) * | 1982-11-06 | 1984-05-17 | Toyobo Co Ltd | Stable cleatine amidinohydrolase pharmaceutical |
-
1985
- 1985-04-01 JP JP60069752A patent/JPH07110240B2/en not_active Expired - Lifetime
Non-Patent Citations (1)
| Title |
|---|
| 丸尾文治外１名監修「酵素ハンドブック」朝倉書店（１９８２−１２−１）Ｐ．５９９ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61227799A (en) | 1986-10-09 |
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