Fructosyl-amino Acid Oxidase (FAOD-E)

  • Enzymes for Clinical Chemistry
Fructosyl-amino Acid Oxidase

CD : 60273

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

Origin recombinant E. coli
Systematic name

Fructosyl-L-amino acid : oxygen oxidoreductase
EC Number 1.5.3
Reaction formula

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

SPECIFICATION

Appearance yellow lyophilizate
Activity ≧4.0 U/mg
Stabilizer trehalose
Storage condition below -20℃ protected from light

PROPERTIES

Molecular weight ca. 45 kDa (gel filtration)
Structure monomer of 50 kDa (SDS-PAGE)
Michaelis constant 2.2×10-4M (Nε-fructosyl-L-lysine)
pH Optimum 8.0–8.5
pH Stability 6.0–8.5
Optimum temperature 35–40℃
Thermal stability below 30℃
Stability (liquid form) stable at 25℃ for at least two weeks
Stability (powder form) stable at 37℃ for at least three weeks
Inhibitors Ag+，Cu2+
Specificity Nε-fructosyl-L-lysine (100), fructosyl-L-valine (65)
fructosyl-glycine (30)

APPLICATIONS

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

ASSAY PROCEDURE

Principle

The appearance of quinoneimine dye is measured spectrophotometrically at 555 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of hydrogen peroxide per min at 30℃ and pH 8.0 under the conditions described below.

Reagents

  1. Potassium phosphate buffer, 0.1 M; pH 8.0: mix 0.1 M KH2PO4 solution and 0.1 M K2HPO4 solution to make a pH 8.0 solution.
  2. TOOS solution, 0.5%: 125 mg of TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine)/25 ml of distilled water.
  3. Peroxidase (POD)-4-aminoantipyrine (4-AA) solution: dissolve 5 mg of POD (200 guaiacol U/mg) in 800 ml of potassium phosphate buffer (Reagent A), then add 100 mg of 4-AA and dilute with potassium phosphate buffer (Reagent A) to 1000 ml.
  4. Fructosyl-L-valine solution, 150 mM: 834 mg of fructosyl-l-valine/20 ml of distilled water
  5. Enzyme dilution buffer: 10 mM potassium phosphate buffer, pH 8.0, containing 0.06% n-octhyl-β-D-thioglucoside.

Sample: dissolve the lyophilized enzyme to a volume activity of 0.05–0.18 U/ml in ice-cold enzyme dilution buffer (Reagent E) immediately before measurement.

Procedure

  1. Pipette the following reagents into a cuvette (light path: 1 cm).
    0.1 ml TOOS solution (Reagent B)
    2.7 ml POD-4-AA solution (Reagent C)
    0.1 ml sample
  2. Equilibrate at 30℃ for about 5 min.
  3. Add 0.1 ml of fructosyl-l-valine solution (Reagent D) and mix.
  4. Record the increase of absorbance at 555 nm in a spectrophotometer thermostated at 30℃, and calculate the ΔA
    per min using the linear portion of the curve (ΔAS).
    The blank solution is prepared by adding distilled water instead of fructosyl-l-valine solution (Reagent D) (ΔA0).

Calculation

Activity can be calculated by using the following formula:

39.2 : Millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm2/μmol)
1/2 : Factor based on the fact that 1 mol of hydrogen peroxide produces 1/2 mol of quinoneimine dye
df : Dilution factor
C : Content of fructosyl-amino acid oxidase preparation in sample (mg/ml)

EXPERIMENTAL DATA

REFERENCES



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