JPH0728743B2 - Stable guanase composition - Google Patents
Stable guanase compositionInfo
- Publication number
- JPH0728743B2 JPH0728743B2 JP60251629A JP25162985A JPH0728743B2 JP H0728743 B2 JPH0728743 B2 JP H0728743B2 JP 60251629 A JP60251629 A JP 60251629A JP 25162985 A JP25162985 A JP 25162985A JP H0728743 B2 JPH0728743 B2 JP H0728743B2
- Authority
- JP
- Japan
- Prior art keywords
- guanase
- composition
- stable
- enzyme
- present
- 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 - Fee Related
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は安定なグアナーゼ（別名グアニンデアミナー
ゼ）組成物に関するものである。TECHNICAL FIELD The present invention relates to a stable guanase (also called guanine deaminase) composition.
最近、臨床検査薬の分野で血清中のグアナーゼを測定す
ることにより肝障害を診断することが注目されており、
その測定試薬の開発が期待されている。Recently, in the field of clinical diagnostics, attention has been focused on diagnosing liver damage by measuring guanase in serum,
Development of the measuring reagent is expected.
ここでいうグアナーゼは酸素番号（EC、３、５、４、
３）であって、動物の肝、腎、脳などに活性が認めら
れ、その他の臓器にも少ないが分布する。また酵母、細
菌にも存在する。Guanase is the oxygen number (EC, 3, 5, 4,
In 3), the activity is found in the liver, kidneys, brain, etc. of animals, and it is distributed to other organs, though it is small. It is also present in yeast and bacteria.
グアナーゼの反応は以下の通りである。The guanase reaction is as follows.
この酵素は正常血清、赤血球、白血球にはほとんど認め
られないが肝細胞障害をおこすと血液中に流出するの
で、肝障害のきわめて敏感な指標になるということで測
定試薬の開発が期待されている。しかしグアナーゼは正
常人では血液中でほとんど活性が認められないので、測
定困難な上に各測定法により測定値が種々異なるため、
安定な標準酵素（グアナーゼ）を用いての測定法の開発
が期待されている。 This enzyme is rarely found in normal serum, erythrocytes, and leukocytes, but it leaks into the blood when hepatocellular injury occurs, so it is expected to be a highly sensitive indicator of liver injury and therefore the development of a measurement reagent is expected. . However, since guanase has almost no activity in the blood of normal people, it is difficult to measure and the measured values vary depending on each measuring method.
It is expected to develop a measuring method using a stable standard enzyme (guanase).
（従来の技術） グアナーゼは血清中ではやや安定であるが、血清を凍結
させずに保存することは困難であり、また凍結させてし
まうと融解時に活性が低下する。しかし酵素標品に精製
してしまうと安定性が悪くなり、標品を硫安懸濁液やグ
リセロール水溶液の状態で保存しても10℃以上の高温に
なると活性の低下が著しく、また低温でも長期保存する
と活性の低下が見られた。(Prior Art) Although guanase is slightly stable in serum, it is difficult to store serum without freezing it, and if it is frozen, its activity decreases when it is thawed. However, if the enzyme preparation is purified, its stability will deteriorate, and even if the preparation is stored in the ammonium sulfate suspension or glycerol aqueous solution, the activity will remarkably decrease at high temperatures of 10 ° C or higher, and even at low temperatures it will remain for a long time. A decrease in activity was seen upon storage.
（発明の解決しようとする問題点） このようにグアナーゼは単位容量当りの比活性の高い酵
素標品である硫安懸濁液であっても、室温以上では酵素
活性の失活が著しいので事実上、グアナーゼの保存は、
困難であった。(Problems to be Solved by the Invention) As described above, guanase is an enzyme preparation having a high specific activity per unit volume, and even if ammonium sulfate suspension is used, the enzyme activity is significantly deactivated at room temperature or higher, so that it is practically impossible. , Preservation of guanase
It was difficult.
（問題点を解決するための手段） 本発明者等はグアナーゼの安定化剤について種々鋭意・
研究したところ本発明に到達した。すなわち本発明はグ
アナーゼにホウ酸、蛋白質、糖、糖アルコール、アミノ
酸及びその塩からなる群から選ばれた１種あるいは２種
以上の化合物を配合することを特徴とする安定なグアナ
ーゼ組成物である。(Means for Solving Problems) The inventors of the present invention have variously studied about a stabilizer for guanase.
After research, the present invention was reached. That is, the present invention is a stable guanase composition comprising guanase and one or more compounds selected from the group consisting of boric acid, proteins, sugars, sugar alcohols, amino acids and salts thereof. .
本発明のグアナーゼとは別名グアニンデアミナーゼ、グ
アニンアミノヒドロラーゼであり、動物の肝、腎、脳な
どに活性が認められ、その他の臓器にも少ないが分布
し、酵母、細菌にも存在する。グアナーゼ酵素標品は従
来は一番安定であるとされている硫安懸濁液の状態か又
はグリセロール水溶液に溶解した状態で保存されてい
た。しかし本発明ではホウ酸、ブトウ糖、ガラクトー
ス、サッカロース、乳糖、ソルビット、グルタミン酸又
はその塩を添加した溶液又はその凍結乾燥品は酵素標品
とする。The guanase of the present invention is also known as guanine deaminase or guanine aminohydrolase, which is found to be active in the liver, kidney, brain, etc. of animals, and is distributed in other organs, though it is present in yeast and bacteria. The guanase enzyme preparation has been stored in the state of ammonium sulfate suspension, which is considered to be the most stable in the past, or in the state of being dissolved in an aqueous glycerol solution. However, in the present invention, a solution to which boric acid, bute sugar, galactose, saccharose, lactose, sorbitol, glutamic acid or a salt thereof is added or a lyophilized product thereof is an enzyme preparation.
本発明に用いるホウ酸の添加量は酵素組成物中濃度0.02
M〜1Mである。ホウ酸はホウ酸カリウム、ホウ酸ナトリ
ウムあるいは有機物の塩であってもよい。The amount of boric acid used in the present invention is 0.02 in the enzyme composition.
It is from M to 1M. The boric acid may be potassium borate, sodium borate or an organic salt.
本発明に用いるブドウ糖、ガラクトース、サッカロー
ス、乳糖またはソルビットの添加量は酵素組成物中濃度
１％以上、好ましくは５〜10％である。上限はないが、
溶解度により限定される。１％未満では効果が期待でき
ない。The amount of glucose, galactose, sucrose, lactose or sorbitol used in the present invention is 1% or more, preferably 5 to 10%, in the enzyme composition. There is no upper limit,
Limited by solubility. If less than 1%, no effect can be expected.
グルタミン酸の塩としてはナトリウム、カリウム、アン
モニウム等の塩または塩酸塩などがある。グルタミン酸
又はその塩の添加量は酵素組成物中濃度10-3M以上、好
ましくは10-1以上である。10-3M未満では効果は期待で
きない。また上限はないが、グルタミン酸の溶解度によ
り限定される。Examples of the salt of glutamic acid include salts of sodium, potassium, ammonium and the like or hydrochlorides. The amount of glutamic acid or a salt thereof added is 10 -3 M or more, preferably 10 -1 or more, in the enzyme composition. No effect can be expected below 10 -3 M. There is no upper limit, but it is limited by the solubility of glutamic acid.
本発明の安定化剤の配合法は特に制限はない。例えばグ
アナーゼを含む緩衝液に安定化剤を配合する方法、安定
化剤を含む緩衝液にグアナーゼを配合する方法、あるい
はグアナーゼと安定化剤を緩衝液に同時に配合する方法
などがある。緩衝液としてはリン酸緩衝液、トリス緩衝
液、その他生化学で用いられる緩衝液なら何でも良い
が、pHは６〜９、望ましくは7.5〜8.5である。The method of blending the stabilizer of the present invention is not particularly limited. For example, there is a method of adding a stabilizer to a buffer solution containing guanase, a method of adding guanase to a buffer solution containing a stabilizer, or a method of simultaneously adding guanase and a stabilizer to the buffer solution. The buffer may be any of phosphate buffer, Tris buffer, and other buffers used in biochemistry, but the pH is 6 to 9, preferably 7.5 to 8.5.
また酵素組成物は安定化剤、緩衝液、グアナーゼの他に
他の酵素を含んでもよくまたこの他の酵素は１種でなく
多種混合してもよい。また酵素組成物は性状は液体でも
良いが、望ましくは凍結乾燥品にするとより安定性が向
上する。Further, the enzyme composition may contain other enzymes in addition to the stabilizer, the buffer solution, and the guanase, and the other enzymes may be mixed in various kinds instead of one kind. The enzyme composition may be liquid in nature, but is preferably freeze-dried to improve its stability.
（発明の効果） 本発明ではグアナーゼにホウ酸、ブドウ糖、ガラクトー
ス、サッカロース、乳糖、ソルビット、グルタミン酸及
びその塩からなる群から選ばれた１種あるいは２種以上
の化合物を配合することにより、従来の硫安懸濁液に比
較して、安定性を著しく改善した。これによりグアナー
ゼの長期保存が可能になった。(Effects of the Invention) In the present invention, guanase is blended with one or more compounds selected from the group consisting of boric acid, glucose, galactose, saccharose, lactose, sorbit, glutamic acid and salts thereof, to obtain a conventional composition. The stability was remarkably improved as compared with the ammonium sulfate suspension. This enabled long-term storage of guanase.
（実施例） 以下本発明を実施例を用いて説明する。(Example) Hereinafter, the present invention will be described using examples.
実施例中、グアナーゼ活性測定は以下の方法に従った。In the examples, guanase activity was measured according to the following method.
下記測定試薬3mlを37℃で５分間加温し、試料（グアナ
ーゼ酵素力価１〜５単位／程度）を50μ加え、１分
後より570nmにおける１分間当りの吸光度化（ΔOD/分）
を測定し、下記式に従い算出する。3 ml of the following measurement reagent was heated at 37 ° C for 5 minutes, 50 μl of sample (guanase enzyme titer 1-5 units / about) was added, and 1 minute later, absorbance at 570 nm per minute (ΔOD / min)
Is calculated and calculated according to the following formula.
測定試薬 50mM リン酸緩衝液 pH7.0 0.02U/ml キサンチンオキシダーゼ 0.3U/ml ウリカーゼ 0.1mM グアニン 0.1mM MBTH 1mM ADPS 2U/ml ペルオキシダーゼ 実施例 1. 下記第１表に示される添加剤を溶解した水溶液に、グア
ナーゼを溶解し、凍結乾燥した。Assay reagent 50 mM Phosphate buffer pH 7.0 0.02U / ml Xanthine oxidase 0.3U / ml Uricase 0.1mM Guanine 0.1mM MBTH 1mM ADPS 2U / ml Peroxidase Example 1. Guanase was dissolved in an aqueous solution in which the additives shown in Table 1 below were dissolved and freeze-dried.
得られた凍結乾燥品を30℃、４週間保存した後、再溶解
して酵素活性を測定した。凍結乾燥前の酵素活性に対す
る凍結乾燥後の酵素活性を残存活性（％）として第１表
に示す。The freeze-dried product thus obtained was stored at 30 ° C. for 4 weeks and then redissolved to measure the enzyme activity. The enzyme activity after freeze-drying with respect to the enzyme activity before freeze-drying is shown in Table 1 as residual activity (%).
参考のために、本発明の添加剤を溶解した水溶液に代え
て、硫安懸濁液、リン酸緩衝液、トリスアミノメタン−
塩酸緩衝液を溶解した場合を第１表に示す。For reference, instead of the aqueous solution in which the additive of the present invention is dissolved, ammonium sulfate suspension, phosphate buffer, trisaminomethane-
Table 1 shows the case where the hydrochloric acid buffer solution was dissolved.
第１表から明らかなように、ホウ酸、蛋白質、糖、糖ア
ルコール、アミノ酸を単独で、あるいは併用すると、グ
アナーゼを安定化することができる。 As is clear from Table 1, guanase can be stabilized by using boric acid, protein, sugar, sugar alcohol and amino acid alone or in combination.
Claims (1)
ース、サッカロース、乳糖、ソルビット、グルタミン酸
およびその塩からなる群から選ばれた１種または２種以
上の化合物を配合することを特徴とする安定なグアナー
ゼ組成物。1. A stable guanase comprising one or more compounds selected from the group consisting of boric acid, glucose, galactose, saccharose, lactose, sorbitol, glutamic acid and salts thereof in guanase. Composition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60251629A JPH0728743B2 (en) | 1985-11-08 | 1985-11-08 | Stable guanase composition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60251629A JPH0728743B2 (en) | 1985-11-08 | 1985-11-08 | Stable guanase composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62111686A JPS62111686A (en) | 1987-05-22 |
| JPH0728743B2 true JPH0728743B2 (en) | 1995-04-05 |
Family
ID=17225665
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60251629A Expired - Fee Related JPH0728743B2 (en) | 1985-11-08 | 1985-11-08 | Stable guanase composition |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0728743B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3081534B2 (en) * | 1995-12-22 | 2000-08-28 | 花王株式会社 | Enzyme-containing granules, method for producing the same, and compositions containing the same |
| JP6160094B2 (en) * | 2013-01-29 | 2017-07-12 | 東洋紡株式会社 | Diaphorase composition |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5635985A (en) * | 1979-08-30 | 1981-04-08 | Toyobo Co Ltd | Stabilization of glutamic acid dehydrogenase |
| JPS57118790A (en) * | 1981-01-14 | 1982-07-23 | Takeda Chem Ind Ltd | Freeze-dried substance containing beta-d-galactosidase |
-
1985
- 1985-11-08 JP JP60251629A patent/JPH0728743B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62111686A (en) | 1987-05-22 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |