Glucose Dehydrogenase (FADGDH-AD)

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

FADGDH-AA is an FAD-dependent glucose dehydrogenase with low reactivity toward maltose and xylose. It is one of the most stable glucose dehydrogenases for blood glucose measurement and is suitable for continuous glucose monitoring sensor.

Origin recombinant Aspergillus 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 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

PROPERTIES

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%)

APPLICATIONS

By using FADGDH-AD and an electron mediator-modified polymer, glucose can be continuously measured by an electrode method.

ASSAY PROCEDURE

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

EXPERIMENTAL DATA

Line-up

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
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)



TOP