Ketoamine Oxidase

Ketoamine oxidase (EC 1.5.3, from Microorganism)

Fructosyl-L-amino acid + H2O + O2  ⇒     L-Amino acid + Glucosone + H2O2

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-Ala solution.

Enzyme diluent: 20 mM Tris-HCl, pH 8.0.

Sample: Dilute the sample to 0.02-0.2 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 ℃ for5 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

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. Sakaue, R. et al., Biosci. Biotechnol. Biochem., 66, 1256–1261 (2002).

3. Hirokawa, K. and Kajiyama, N., Biosci. Biotechnol. Biochem., 66, 2323–2329 (2002).

4. Sakaue, R. and Kajiyama, N., Appl. Environ. Microbiol., 69, 139–145 (2003).

5. Sakaue, R. et al., Acta Cryst., F61, 196–198 (2005)

The enzyme is useful for the determination of fructosyl-L-amino acid.