GDH (Glucose Dehydrogenase)
This enzyme is useful for clinical analysis of D-glucose determination in blood glucose meter.
Specification
|
Appearance |
Yellow amorphous powder, lyophilized |
|
Purity( SDS-PAGE) |
≥90.0% |
|
Enzyme powder specific activity |
≥90.0% |
|
Glucose dehydrogenase (NAD) |
≤0.02% |
|
Hexokinase |
≤0.02% |
|
α-glucosidase |
≤0.02% |
Properties
|
EC number |
1.1.5.9 (Recombinant from microorganism) |
|
Molecular weight |
66 kDa (SDS-PAGE) |
|
Isoelectric point |
5.04 |
|
Michaelis constant |
7.45 ×10-2 M (D-glucose) |
|
Inhibitor |
Ag+, Cu2+, SDS |
|
Optimum pH |
7.0 Fig.1 |
|
Optimum temperature |
50-55 °C Fig.2 |
|
pH stability |
pH 5.0 – 9.0 (25 °C, 16 h) Fig.3 |
|
Thermal stability |
Stable at 50 °C and blow (pH7.0, 30 min) Fig.4 |
|
Storage stability stability |
At least one year at -25～- 15 ℃ Fig.5 |
Figures
This enzyme is useful for clinical analysis of D-glucose determination in blood glucose meter.
Assay principle
Unit definition
One unit (U) is defined as the amount of enzyme which generates 1 μmol of DCIP per minute at 37 °C under the conditions described below.
Reagents preparation
Reagent I: 0.1 M potassium phosphate buffer, pH 7.0.
Reagent II: 2 M D-glucose solution.
Reagent III: 1.8 mM DCIP solution(be prepared when using).
Reagent IV: 30 mM PMS solution. Enzyme dilution buffer: 0.1 M potassium phosphate buffer pH 6.0, contains 0.1% BSA.
Sample: The enzyme was diluted to 0.1-0.9 U/mL with enzyme dilution buffer.
Procedure
1. Add 2.05 mL reagent I, 0.6 mL reagent II and 0.15 mL reagent III to the 3 mL cuvette at 37 ℃ for about 5 minutes;
2. Add 0.1 mL reagent IV to the 3mL cuvette and mix;
3. Add 0.1 mL enzyme solution to the 3 mL cuvette and mix;
4. Record the ΔAs at 600 nm in 1 minute in a spectrophotometer thermostated at 37 °C;
5. Replace the enzyme solution with enzyme dilution buffer, and record the change of blank absorbance (∆Ab) in the same steps;
∆A = ∆As - ∆Ab
Calculation
Vt: Total volume (3 mL);
Vs: Enzyme volume (0.1 mL);
1.0: Light path length (cm);
df: Dilution factor;
C: Enzyme concentration (mg/mL);
20.4: Millimolar extinction coefficient of quinoneimine dye under 600 nm (cm2 /μmol).
References
Satake, R. et al., J. Biosci. Bioeng., 120, 498–503 (2015).
