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。血糖測定用酵素の中でも特に安定性に優れており、連続血糖測定センサに適しています。
| 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-AD) グルコースデヒドロゲナーゼ (FADGDH-AD)
- 臨床診断用酵素
仕様
| Appearance | yellow to brown lyophilizate |
|---|---|
| Activity | ≧700 U/mg |
| Contaminants | NAD glucose dehydrogenase <0.01 U/U% |
| Hexokinase <0.01 U/U% | |
| α-glucosidase <0.01 U/U% | |
| β-glucosidase <0.01 U/U% | |
| Storage condition | below -20℃ protected from light |
特性
| Molecular weight | ca. 90 kDa (SDS-PAGE) |
|---|---|
| Structure | monomer, one mole of FAD per mole of enzyme glycoprotein |
| Michaelis constant | 6.4×10-2M (D-glucose) |
| pH Optimum | 7.0–7.5 (Fig.1) |
| pH Stability | 2.5–9.5 (Fig.2) |
| Optimum temperature | 45℃ (Fig.3) |
| Thermal stability (liquid form) | below 60℃ (Fig.4) |
| Thermal stability (powder form) | stable at 30℃ for at least one month (Fig.5) |
| Inhibitors | Mn2+, Ag+ |
| Specificity (Table.1) | D-glucose (100%), maltose (0.2%), D-xylose (0.9%), D-galactose (0.8%) sucrose (<0.1%), D-mannose (0.4%) 2-deoxy-D-glucose (23.5%) |
アプリケーション
FADGDH-ADと電子メディエータ修飾ポリマーを用いることで、グルコースを電極法により連続測定できます。
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
Satake R, Ichiyanagi A, Ichikawa K, Hirokawa K, Araki Y, Yoshimura T, Gomi K (2015)
Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae
Biosci Bioeng., 120, 498-503
Masakari Y, Hara C, Araki Y, Gomi K, Ito K (2020)
Improvement in the thermal stability of Mucor prainii-derived FAD-dependent glucose dehydrogenase via protein chimerization
Enzyme Microb Technol., 132, 109387, doi: 10.1016/j.enzmictec.2019.109387.
https://doi.org/10.1016/j.enzmictec.2019.109387, (cited 2020-07-02)