Pyranose oxidase

The assay is based on the increase in absorbance at 555 nm as the formation of quinoneimine dye proceeds in the forward reactions.

UNIT DEFINITION

One unit (U) is defined as the amount of enzyme which hydrolyzes 1 μmol of D-glucose per minute at 37 °C under the conditions specified in the assay procedure.

REAGENTS PREPARATION

Reagent I: 100 mM, Na- phosphate buffer, pH 7.0.

Reagent II: 50 mM 4-AA solution.

Reagent III: 50 mM TOOS solution.

Reagent IV: 1 kU/mL peroxidase (POD) solution.

Enzyme dilution buffer: 50 mM PBS, pH 6.0.

Sample: The enzyme was diluted to 0.3-0.6 U/mL with enzyme dilution buffer.

Reaction mixture:

Reagent I                25 mL

D-glucose               2 g

Reagent II              1.5 mL

Reagent III             1.5 mL

Reagent IV             0.2 mL

pure water            to 50 mL

PROCEDURE

1. Add 1 mL reaction mixture to the 1 mL cuvette;

2. Preincubate the reaction mixture at 37 °C for 5 min;

3. Add 20 μL enzyme solution to the 1mL cuvette and mix;

4. Record the ΔAs at 555 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;

CALCULATION

1.02: Total volume (1.02 mL);

0.02: enzyme volume (0.02 mL);

1.0: light path length (cm);

1/2: 1mol H2O2 will react to 1/2 mol quinoneimine dye;

df: Dilution factor;

C: Enzyme concentration (mg/mL);

39.2: Millimolar extinction coefficient of quinoneimine dye under 555 nm (cm2/μmol).

REFERENCES

1. Ikuko, M. N. and Tomoyuki, M. (1999) Biotechnology Letters, 21, 203–207.

2. Artolozaga, M. J. and Kubatova E. (1997) Appl. Microbiol. Biotechnol. 47, 508–514.

3. Daniel, G. and Volc, J. (1994) Appl. Environment Microbiol. 60, 2524–2532.