マルトース及びキシロースに対する反応性が低いFAD依存性グルコース脱水素酵素です。安定性が高く、低温下においても反応性が高いことが特徴です。
| 由来 | recombinant A. sojae |
|---|---|
| 系統名 | D-Glucose : acceptor 1-oxidoreductase |
| EC 番号 | 1.1.5.9 |
| 反応式 | D-Glucose + acceptor →→→ D-Glucono-1,5-lactone + reduced acceptor |
Glucose Dehydrogenase (FADGDH-AA) グルコースデヒドロゲナーゼ(FADGDH-AA)
- 臨床診断用酵素
仕様
| Appearance | yellow lyophilizate |
|---|---|
| Activity | ≧ 475 U/mg |
| Contaminants | NAD glucose dehydrogenase ≺1.0×10-2％ |
| Hexokinase ≺1.0×10-2 U/U% | |
| α-Glucosidase ≺1.0×10-2 U/U% | |
| β-Glucosidase ≺1.0×10-2 U/U% | |
| Storage condition | below -20℃ |
特性
| Molecular weight | ca. 90 kDa (SDS-PAGE) |
|---|---|
| Structure | monomer, one mole of FAD per mole of enzyme glycoprotein |
| Michaelis constant | 9.5×10-2M (D-glucose) |
| pH Optimum | 7.0–7.5 (Fig.1) |
| pH Stability | 2.5–7.5 (Fig.2) |
| Optimum temperature | 40–50℃ (Fig.3) |
| Thermal stability | below 50℃ (Fig.4) |
| Inhibitors | Ag+ |
| Specificity | D-glucose (100), maltose (≺1), |
| D-xylose (≺1), D-galactose (≺1) |
アプリケーション
FADGDH-AAとフェナジンメトサルフェート(PMS)等の電子メディエータを用いることで、グルコースを比色法や電極法により定量できます。
分析手順
Principle
The disappearance of the blue color of DCIP by the reduction is measured spectrophotometrically at 600 nm.
Definition of unit
One unit (U) causes the reduction of one micromole of DCIP per minute under the conditions described below.
Reagents
- D-Glucose solution, 2 M: 72 g of D-glucose/200 ml of distilled water.
- Potassium phosphate buffer, 0.1 M; pH 7.0: mix 0.1 M KH2PO4 solution and 0.1 M K2HPO4 solution to make a pH 7.0 solution.
- 2,6-Dichloroindophenol (DCIP) solution, 1.8 mM: 58.7 mg of DCIP/100 ml of distilled water.
- 5-Methylphenazinium methyl sulfate (PMS) solution, 30 mM: 91.9 mg of PMS/10 ml of distilled water.
- Enzyme dilution buffer: 10 mm potassium phosphate buffer, pH 6.0, containing 0.1% bovine serum albumin (BSA).
Sample: dissolve the lyophilized enzyme to final concentration about 0.4 μg/ml with enzyme dilution buffer (Reagent E) immediately before measurement.
Procedure
- Pipette the following reagents into a cuvette (light path: 1 cm).
600 μL D-Glucose solution (Reagent A)
2050 μL Potassium phosphate buffer pH 7.0 (Reagent B)
150 μL DCIP solution (Reagent C) - Equilibrate at 37℃ for about 3 min.
- Add 0.1 ml of PMS solution (Reagent D) and mix.
- Add 0.1 ml of sample and mix.
- Record the decrease of absorbance at 600 nm against water for 1 min. (30–90 sec) in a spectrophotometer thermostated at 37℃, and calculate the ΔA per min using the linear portion of the curve (ΔAS). The blank solution is prepared by adding Enzyme dilution buffer (Reagent E) instead of sample (ΔA0).
Calculation
Activity can be calculated by using the following formula:
20.4 : Millimolar extinction coefficient of DCIP under the assay condition (cm2/μmol)
1.0 : Light pass length (cm)
df : Dilution factor
実験データ
Line-up
参考文献