Glucose Dehydrogenase (FADGDH-AA)

  • Enzymes for Clinical Chemistry
Glucose Dehydrogenase (FADGDH-AA)

FADGDH-AA is an FAD-dependent glucose dehydrogenase with low reactivity toward maltose and xylose. It has high stability and maintains its reactivity even at low temperatures.

Origin recombinant A. sojae
Systematic name

D-Glucose : acceptor 1-oxidoreductase
EC Number 1.1.5.9
Reaction formula

D-Glucose + acceptor →→→ D-Glucono-1,5-lactone + reduced acceptor

SPECIFICATION

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

PROPERTIES

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) 

APPLICATIONS

Glucose can be determined by colorimetric and electrode methods by using FADGDH-AA and an electron mediator such as phenazine methosulfate (PMS).

ASSAY PROCEDURE

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

EXPERIMENTAL DATA

Line-up

Glucose Dehydrogenase (FADGDH-AB)

Glucose Dehydrogenase (FADGDH-AD)

REFERENCES

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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