Glucose Dehydrogenase (FADGDH-AD) 60102 グルコースデヒドロゲナーゼ (FADGDH-AD)

  • 臨床診断用酵素
グルコースデヒドロゲナーゼ (FADGDH-AD)

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

仕様

Appearance yellow to brown lyophilizate
Activity ≧700 U/mg lyophilizate
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℃

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

  1. D-Glucose solution, 2 M: 72 g of d-glucose/200 mL of distilled water.
  2. 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.
  3. 2,6-Dichloroindophenol (DCIP) solution, 1.8 mM: 58.7 mg of DCIP/100 mL of distilled water.
  4. 5-Methylphenazinium methyl sulfate (PMS) solution, 30 mM: 91.9 mg of PMS/10 mL of distilled water.
  5. 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

  1. 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)
  2. Equilibrate at 37℃  for about 3 min.
  3. Add 0.1 mL of PMS solution (Reagent D) and mix.
  4. Add 0.1 mL of sample and mix.
  5. 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

FAQ

FADGDH-ADの主な用途は何ですか？
FADGDH-ADの分子量を教えてください。
FADGDH-ADの至適pHと至適温度について教えてください。
FADGDH-ADの熱安定性について教えてください。
FADGDH-ADの安定化剤は何を使用していますか？
FADGDH-ADの基質特異性について教えてください。
FADGDH-ADの活性測定方法を教えてください。
FADGDH-ADの保存方法を教えてください。
開封後の取扱い方法に注意事項はありますか？

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 J. 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



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