Glucose Dehydrogenase

Glucose Dehydrogenase (EC 1.1.5.9, from Microorganism)

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

Assay principle

D-Glucose + PMS ⇒ Glucose Dehydrogenase ⇒ D-Glucono-1,5-lactone +PMS (red) PMS (red) +DCIP ⇒ PMS + DCIP (red) 

The assay is based on the increase in absorbance at 600 nm as the formation of DCIP in the forward reactions. 

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 thermo-stated 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).

This enzyme is useful for clinical analysis of D-glucose determination in the blood glucose meter.