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
グルコースデヒドロゲナーゼ(FADGDH-AA)

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

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℃ protected from light

特性

Molecular weight ca. 90 kDa (gel filtration)
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

d-Glucono-1,5-lactone

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

Glucose Dehydrogenase (FADGDH-AB)

Glucose Dehydrogenase (FADGDH-AD)

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



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