Glucose dehydrogenase (GDH-FAD) is an enzyme used for regenerating cofactors, converting glucose and NAD(P) into NAD(P)H and gluconolactone.
Technical Indicators
Appearance: Yellow amorphous lyophilized powder
Protein Purity: ≥90%
Activity: ≥450 U/mg enzyme powder
Glucose Dehydrogenase (NAD): ≤0.02%
Hexokinase: ≤0.02%
α-Glucosidase: ≤0.02%
Glucose Oxidase: ≤0.02%
Amyloglucosidase: ≤0.01%
Enzymatic Properties
Source:Microorganism
Classification:EC 1.1.5.9
Molecular Weight:72-135 kDa (SDS-PAGE)
Isoelectric Point:5.33
Km Value:2.8 ×10⁻² M (D-glucose)
Inhibitors:Ag⁺, Zn²⁺
Optimal pH:7.0Fig.1Optimal Temperature:45-60℃Fig.2pH Stability:pH 3.0 - 10.5 (25℃, 16 h)Fig.3Thermal Stability:Stable below 50℃ (pH 6.0, 30 min)Fig.4
Application
Used for the determination of D-glucose in clinical analysis and self-test blood glucose meters.
Assay Method
Principle
The amount of DCPIP can be detected at 600 nm using an ultraviolet-visible spectrophotometer.
Unit Definition
A unit of enzyme activity is defined as the amount of enzyme required to consume 1 μmol of DCPIP per minute under the following conditions.
Reagent Preparation
Reagent I: 0.1M pH 7.0 potassium phosphate buffer (dissolved in water).
Reagent II: 2M D-glucose solution (dissolved in water).
Reagent III: 1.8mM DCPIP (dissolved in water, prepared and used immediately).
Reagent IV: 30mM PMS (dissolved in water).
Enzyme Diluent: 0.1M pH 6.0 potassium phosphate buffer, containing 0.1% BSA.
Test Enzyme Solution: Dilute the enzyme solution to 0.1-0.9 U/mL with the enzyme diluent.
Operation Steps
1. In a 3mL cuvette, add 2.05mL Reagent I, 0.6mL Reagent II, and 0.15mL Reagent III, and preheat at 37°C for 5 min.
2. Add 0.1mL Reagent IV and mix well.
3. Add 0.1mL test enzyme solution and mix well.
4. Determine the absorbance change rate ΔAs within 1 minute at 37°C at 600 nm.
* Replace the enzyme solution with the enzyme diluent, follow the same steps, and the absorbance of the resulting solution is the blank absorbance (ΔAb).
ΔA = ΔAs - ΔAb
Calculation
Vt: Total volume of reaction solution (3mL);
Vs: Volume of enzyme solution (0.1mL);
1.0: Light path length (cm);df: Dilution factor;
C: Enzyme concentration (mg/mL);
20.4: Millimolar extinction coefficient of the chromophore at 600nm under standard reaction conditions (cm²/μmol).
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