JPH0430834B2 - - Google Patents
Info
- Publication number
- JPH0430834B2 JPH0430834B2 JP62096072A JP9607287A JPH0430834B2 JP H0430834 B2 JPH0430834 B2 JP H0430834B2 JP 62096072 A JP62096072 A JP 62096072A JP 9607287 A JP9607287 A JP 9607287A JP H0430834 B2 JPH0430834 B2 JP H0430834B2
- Authority
- JP
- Japan
- Prior art keywords
- gpo
- solution
- added
- freeze
- 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 - Lifetime
Links
- 239000000203 mixture Substances 0.000 claims description 12
- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 claims description 5
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 claims description 5
- 239000011714 flavin adenine dinucleotide Substances 0.000 claims description 5
- 229940093632 flavin-adenine dinucleotide Drugs 0.000 claims description 5
- 108010041921 Glycerolphosphate Dehydrogenase Proteins 0.000 claims description 4
- 102000000587 Glycerolphosphate Dehydrogenase Human genes 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 239000006172 buffering agent Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- GNGACRATGGDKBX-UHFFFAOYSA-N dihydroxyacetone phosphate Chemical compound OCC(=O)COP(O)(O)=O GNGACRATGGDKBX-UHFFFAOYSA-N 0.000 description 2
- 108010054790 glycerol-3-phosphate oxidase Proteins 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- AWUCVROLDVIAJX-VKHMYHEASA-N sn-glycerol 1-phosphate Chemical compound OC[C@H](O)COP(O)(O)=O AWUCVROLDVIAJX-VKHMYHEASA-N 0.000 description 1
- HFQQZARZPUDIFP-UHFFFAOYSA-M sodium;2-dodecylbenzenesulfonate Chemical compound [Na+].CCCCCCCCCCCCC1=CC=CC=C1S([O-])(=O)=O HFQQZARZPUDIFP-UHFFFAOYSA-M 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Description
本発明は安定化されたグリセロリン酸オキシダ
ーゼ組成物に関するものである。
近年、Ｌ−α−グリセロリン酸を酸化してジヒ
ドロキシアセトンリン酸と過酸化水素を生成する
Ｌ−α−グリセロリン酸オキシダーゼ（以下
GPOと略称する）は診断用試薬として使用され、
その重要性がますます増加してきた。ところが
GPOは不安定であり、特にGPOを水溶液にした
場合、該水溶液中のGPO濃度が１mg／ml以下の
低濃度では極端に不安定である。さらにGPOを
含有する組成物は凍結乾燥に極めて敏感で弱く、
GPO活性は凍結乾燥の間、あるいは凍結乾燥後、
相当低下する。したがつて、低濃度のGPO水溶
液では長時間に亘る品質の維持が困難である。こ
のような理由からGPOを用いる利点が失われる
場合が多い。
本発明者等はこのような欠点を有しない安定な
GPO含有組成物を得るべく、種々鋭意検討した
結果、安定化剤として、フラビンアデニンジヌク
レオチドを使用すると所期の目的を達成すること
を見出し、本発明に到達した。すなわち本発明は
フラビンアデニンジヌクレオチドとGPOから本
質的になる凍結乾燥物であることを特徴とする安
定化されたグリセロリン酸オキシダーゼ組成物で
ある。
本発明では、上記安定剤を使用することによ
り、凍結乾燥によつてもGPOの活性低下が見ら
れない。さらに、安定化効果は長期間に亘つて、
持続することができる。
本発明の凍結乾燥物はまず緩衝液にGPOとフ
ラビンアデニンジヌクレオチドを含有させた水性
組成物を得る。緩衝剤としては通常のものが使用
され、該水性組成物のPHを通常５〜10とするもの
が好ましい。
前記の水性組成物には必要により、他の成分を
添加してもよい。
前記の水性組成物において、フラビンアデニン
ジヌクレオチドの含有量は１×10-6〜１×10-2M
である。
水性組成物を調整する場合、緩衝液への各物質
の添加順序は特に制限されない。
本発明の凍結乾燥物は通常の方法に従い、前記
水性組成物を凍結乾燥して得る。
本発明では凍結乾燥状態（凍結乾燥物）である
場合に、GPOの安定性は著しく高く、長期間に
亘つて安定性が維持される。
次に本発明を実施例により説明する。
なおGPOの酵素活性の測定は次の方法に従つ
た。0.2Mトリス塩酸緩衝液（PH8.0）0.2ml、パー
オキシダーゼ（340単位／ml）0.1ml、４−アミノ
アンチピリン（0.3％水溶液）0.1ml、フエノール
（0.2％水溶液）0.1ml、グリセロール−３−リン
酸（0.5MPH8.0）0.2ml、トリトンＸ−100（0.5％
水溶液）0.1mlおよび水0.2mlを混合し、37℃で５
分間加温した後、20μの酵素溶解液を加え、37
℃で10分間加温した。
次いで１％ラウリルイベンゼンスルホン酸ソー
ダ水溶液0.5mlを加えて反応を停止させ、さらに
水1.5mlを加えて500nｍの吸光度を測定した。
比活性（Ｕ／mg）＝ΔA／10／13.3×１／２
×0.03／0.02×１／Ｘ
Ｘ：酵素溶解液中の酵素濃度
ΔA：AS−AC
AS：酵素溶解液を用いたときの吸光度
AC：酵素溶解液に代えて水を用いたときの吸光
度
実施例 １
ペデイオコツカス属由来のGPOを0.05Mリン酸
緩衝液（PH7.0）に溶解した。溶液中のGPO濃度
は0.5mg／mlであつた。下記表の添加物を該溶液
に加え、凍結乾燥し、安定性を検討した。その結
果を第１表に示す。溶液調製直後の酵素活性を
100％とした。
The present invention relates to stabilized glycerophosphate oxidase compositions. In recent years, L-α-glycerophosphate oxidase (hereinafter referred to as L-α-glycerophosphate oxidase), which oxidizes L-α-glycerophosphate to produce dihydroxyacetone phosphate and hydrogen peroxide, has been developed.
GPO) is used as a diagnostic reagent,
Its importance has been increasing. However
GPO is unstable, and especially when GPO is made into an aqueous solution, it is extremely unstable at a low concentration of GPO in the aqueous solution of 1 mg/ml or less. Furthermore, compositions containing GPO are extremely sensitive and weak to lyophilization;
GPO activity was determined during or after lyophilization.
It decreases considerably. Therefore, it is difficult to maintain quality over a long period of time with a low concentration GPO aqueous solution. For these reasons, the benefits of using GPOs are often lost. The inventors have proposed a stable method that does not have these drawbacks.
As a result of various intensive studies in order to obtain a GPO-containing composition, it was discovered that the intended purpose could be achieved by using flavin adenine dinucleotide as a stabilizer, and the present invention was achieved. That is, the present invention is a stabilized glycerophosphate oxidase composition characterized by being a lyophilizate consisting essentially of flavin adenine dinucleotide and GPO. In the present invention, by using the above-mentioned stabilizer, no decrease in GPO activity is observed even after freeze-drying. Furthermore, the stabilizing effect lasts for a long time,
can last. For the lyophilized product of the present invention, an aqueous composition is first obtained by containing GPO and flavin adenine dinucleotide in a buffer solution. As the buffering agent, a conventional buffering agent is used, and it is preferable that the buffering agent adjusts the pH of the aqueous composition to usually 5 to 10. Other components may be added to the aqueous composition as necessary. In the aqueous composition, the content of flavin adenine dinucleotide is 1×10 −6 to 1×10 −2 M
It is. When preparing an aqueous composition, the order of adding each substance to the buffer solution is not particularly limited. The lyophilizate of the present invention is obtained by lyophilizing the aqueous composition according to a conventional method. In the present invention, the stability of GPO is extremely high when it is in a freeze-dried state (freeze-dried product), and the stability is maintained for a long period of time. Next, the present invention will be explained by examples. The enzymatic activity of GPO was measured according to the following method. 0.2M Tris-HCl buffer (PH8.0) 0.2ml, peroxidase (340 units/ml) 0.1ml, 4-aminoantipyrine (0.3% aqueous solution) 0.1ml, phenol (0.2% aqueous solution) 0.1ml, glycerol-3- Phosphoric acid (0.5MPH8.0) 0.2ml, Triton X-100 (0.5%
Mix 0.1 ml of aqueous solution and 0.2 ml of water and heat at 37℃ for 5 minutes.
After heating for 3 minutes, add 20μ of enzyme lysate and
Warmed at ℃ for 10 minutes. Next, 0.5 ml of a 1% sodium laurylbenzenesulfonate aqueous solution was added to stop the reaction, and 1.5 ml of water was further added to measure the absorbance at 500 nm. Specific activity (U/mg ) = ΔA/10/13.3×1/2 ×0.03/ 0.02 ×1/ X Absorbance A C : Absorbance when water is used instead of enzyme solution Example 1 GPO derived from the genus Pedeiococcus was dissolved in 0.05M phosphate buffer (PH7.0). The GPO concentration in the solution was 0.5 mg/ml. Additives listed in the table below were added to the solution, freeze-dried, and examined for stability. The results are shown in Table 1. Enzyme activity immediately after solution preparation
It was set as 100%.
【表】【table】
【表】
上記表のごとく、フラビン含有物質を添加して
凍結乾燥した場合、無添加に比較して著しく失活
が少ない。
実施例 ２
下記表の添加物を加えたペデイオコツカス属由
来のGPO溶液を凍結乾燥した凍結乾燥品の、40
℃、１週間での安定性を検討した。その結果を第
２表に示す。凍結乾燥直後の酵素活性を100％と
した。[Table] As shown in the table above, when a flavin-containing substance is added and freeze-dried, there is significantly less deactivation than when no substance is added. Example 2 A lyophilized product obtained by lyophilizing a GPO solution derived from the genus Pedeiococcus to which the additives listed in the table below were added.
The stability at 1 week was examined at ℃. The results are shown in Table 2. Enzyme activity immediately after freeze-drying was defined as 100%.
【表】【table】
【表】
フラビン含有物質を添加した凍結乾燥品は無添
加に比べ著しく安定化された。[Table] Freeze-dried products to which flavin-containing substances were added were significantly more stable than those without additives.
Claims (1)
リン酸オキシダーゼから本質的になる凍結乾燥物
であることを特徴とする安定化されたグリセロリ
ン酸オキシダーゼ組成物。1. A stabilized glycerophosphate oxidase composition characterized in that it is a lyophilizate consisting essentially of flavin adenine dinucleotide and glycerophosphate oxidase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9607287A JPS63263082A (en) | 1987-04-17 | 1987-04-17 | Stabilized glycerophosphoric acid oxidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9607287A JPS63263082A (en) | 1987-04-17 | 1987-04-17 | Stabilized glycerophosphoric acid oxidase |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14588180A Division JPS5768788A (en) | 1980-10-17 | 1980-10-17 | Stablized glycerophosphate oxidase composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63263082A JPS63263082A (en) | 1988-10-31 |
| JPH0430834B2 true JPH0430834B2 (en) | 1992-05-22 |
Family
ID=14155205
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9607287A Granted JPS63263082A (en) | 1987-04-17 | 1987-04-17 | Stabilized glycerophosphoric acid oxidase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63263082A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5768788A (en) * | 1980-10-17 | 1982-04-27 | Toyobo Co Ltd | Stablized glycerophosphate oxidase composition |
-
1987
- 1987-04-17 JP JP9607287A patent/JPS63263082A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5768788A (en) * | 1980-10-17 | 1982-04-27 | Toyobo Co Ltd | Stablized glycerophosphate oxidase composition |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63263082A (en) | 1988-10-31 |
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