FPOX (Fructosyl peptide oxidase)
The enzyme is useful for the determination of fructosyl-peptide and fructosyl-L-amino acid.
Specification
|
Appearance |
Yellow amorphous powder, lyophilized |
|
Protein purity |
≥90% (from SDS-PAGE) |
|
Activity |
≥4 U/mg solid |
|
Catalase |
≤0.01% |
|
ATPase |
≤0.005% |
|
Glucose oxidase |
≤0.03% |
|
Cholesterol oxidase |
≤0.003% |
Properties
|
EC number |
1.5.3 (Recombinant from microorganism) |
|
Molecular weight |
60 kDa (SDS-PAGE) |
|
Isoelectric point |
6.4 |
|
Michaelis constants |
4.0×10-3M(fructosyl-Val-His) |
|
Inhibitors |
Hg2+, Pb2+ |
|
Optimum pH |
pH 6.5-7.5 Fig. 1 |
|
Optimum temperature |
37 ℃ Fig. 2 |
|
pH stability |
pH 6.5-9.5 (25 ℃, 16 h) Fig. 3 |
|
Thermal stability |
Below 40 ℃ (pH 8.0, 30 min) Fig. 4 |
|
Storage stability |
At least one year at -25 ～-15 ℃ Fig. 5 |
|
Stabilizers |
Glycerol, trehalose |
Figures
Assay principle
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 produces 1 μmol of H2O2 per min under the conditions described below.
Reagents preparation
Reagent I: 0.1 M potassium phosphate, pH 8.0.
Reagent II: 1 kU/mL POD.
Reagent III: 50 mM TOOS solution.
Reagent IV: 50 mM 4-AA solution.
Reagent V: 200 mM fructosyl-valine solution.
Enzyme diluent: 20 mM Tris-HCl，pH 8.0.
Sample: Dilute the sample to 0.2-0.5 U/mL by Enzyme diluent.
Reaction mixture:
Reagent I 10 mL
Reagent II 0.1 mL
Reagent III 1 mL
Reagent IV 1 mL
Reagent V 10 mL
Pure water Set to 100mL
Procedure
1. Add 0.98 mL reaction mixture to the cuvette.
2. Preincubate the reaction mixture at 37 ℃ for 5 min.
3. Add 0.02 mL the enzyme solution in the reaction mixture.
4. Record the ΔAs at 555 nm in 1 minute in a spectrophotometer thermostated at 37 ℃. At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As - ∆Ab
Calculation
Vt: Total volume (1.0 mL)
Vs: Enzyme volume (0.02 mL)
t: reaction time (1 min)
df: dilution factor
C: Enzyme concentration (mg/mL)
1.0: Light path length (cm)
1/2: 1mol H2O2 will react to 1/2 mol Quinoneimine dye
39.2: Millimolar extinction coefficient of quinoneimine dye under 555nm (cm2 /μmol)
References
1. Horiuchi, T. et al., Agric. Biol. Chem., 53, 103–110 (1989).
2. Hirokawa, K. et al., Arch. Microbiol., 180, 227–231 (2003).
3. Hirokawa, K. et al., Biochem. Biophys. Res. Commun., 311, 104–111 (2003).
