Recombinant Glucose Dehydrogenase (GDH-FAD) 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.
D-Glucose + acceptor → D-Glucono-1,5-lactone + reduced acceptor
Specifications
Appearance: Yellow lyophilized powder
Activity: ≥700 U/mg lyophilized powder
Contaminants:
NAD Glucose Dehydrogenase < 1.0×10⁻²%
β-Glucosidase < 1.0×10⁻²%
Hexokinase < 1.0×10⁻²%
α-Glucosidase < 1.0×10⁻²%
Characteristics
Molecular weight: Approximately 85 kDa (determined by SDS-PAGE)
Structure: Monomer, 1 mol FAD per mol glycoprotein
Michaelis constant: 2.2×10⁻² m (D-glucose)
Optimal pH: 7.0-7.5 Fig. 1
pH stability: 4.5-8.0 Fig. 2
Optimal temperature: 40 - 50°C Fig. 3
Thermal stability (liquid form): Below 50°C Fig. 4
Thermal stability (powder form): stable at 30°C for at least 20 days Fig. 5
Specificity: Table 1
Inhibitor: Mn²⁺, Ag⁺
ASSAY PROCEDURE
Principle
The disappearance in the blue color of DCIP due to reduction is measured spectrophotometrically at 600 nm.
Reagents
A. D-Glucose solution, 2 M: 72 g D-glucose/200 ml distilled water.
B. Potassium phosphate buffer, 0.1 M, pH 7.0: Mix 0.1 m KH₂PO₄ solution and 0.1 M KH₂PO₄ solution to make a pH 7.0 solution.
C. 2,6-Dichloroindophenol (DCIP) solution, 1.8 mM: 58.7 mg DCIP/100 ml distilled water.
D. Phenazine methosulfate (PMS) solution, 30 mM: 91.9 mg PMS/10 ml distilled water.
E. 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°C for about 3 minutes.
3. Add 0.1 ml PMS solution (Reagent D) and mix.
4. Add 0.1 ml 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°C, 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
The activity can be calculated by 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
