Fructosyl-amino Acid Oxidase (FAOD-E)

Enzymes for Clinical Chemistry

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 (N^ε-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	N^ε-fructosyl-L-lysine (100), fructosyl-L-valine (65)
Specificity	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

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.
TOOS solution, 0.5%: 125 mg of TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine)/25 ml of distilled water.
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.
Fructosyl-L-valine solution, 150 mM: 834 mg of fructosyl-l-valine/20 ml of distilled water
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

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
Equilibrate at 30℃ for about 5 min.
Add 0.1 ml of fructosyl-l-valine solution (Reagent D) and mix.
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 (ΔA_S).
The blank solution is prepared by adding distilled water instead of fructosyl-l-valine solution (Reagent D) (ΔA₀).

0.1 ml	TOOS solution	(Reagent B)
2.7 ml	POD-4-AA solution	(Reagent C)
0.1 ml	sample

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

FAQ

What precautions should be taken when handling the product after opening?: To avoid moisture absorption of the enzyme powder, please allow it to return to room temperature in a sealed state before use. (Recommended conditions: around 25°C, humidity below 55%)

How should FAOD-E be stored?: Please store at or below -20°C.

How is the activity of FAOD-E measured?: An example of activity measurement: The oxidase activity is assayed in a reaction mixture containing fructosyl valine as a substrate, peroxidase, 4-aminoantipyrine and TOOS. The prouction of hydrogen peroxide is colorimetrically evaluated by measuring absorbance at 555 nm.

What is the substrate specificity of FAOD-E?: It shows 100% activity toward ε-fructosyl lysine (εF-Lys), 65% toward fructosyl valine (F-Val), and 30% toward fructosyl glycine (F-Gly).

What stabilizers are used in FAOD-E?: Trehalose is used as a stabilizer.

How thermostable is FAOD-E?: It is stable in liquid form below 30°C (Fig.4) and remains stable at 25°C for at least two weeks (Fig.5). In powder form, it remains stable at 37°C for at least three weeks (Fig.6).

What are the optimal pH and temperature for FAOD-E?: The optimal pH is 8.0–8.5, and the optimal temperature is 35–40°C.

What is the molecular weight of FAOD-E?: The molecular weight is approximately 45 kDa (gel filtration), and it exhibits a monomeric structure of 50 kDa (SDS-PAGE).

What are the main applications of FAOD-E?: It is useful for the determination of glycated albumin, an indicator for diabetes diagnosis and blood glucose control, in clinical analysis.