Fructosyl-amino acid oxidase from Microorganism

Fructosyl-amino acid oxidase From Microorganism

Fructosyl-L-amino acid +H₂O +O₂ ► Glucosone + L-amino acid+H₂O₂

PREPARATION and SPECIFICATION
Appearance:	Yelowish amorphous powder,lyophilized
Activity:	3.0U/mg-solid or more
Contaminant:	Catalase ≤1.0%

PROPERTIES
Molecular weight:	approx. 48,000Da (by SDS-PAGE)
Isoelectric point:	6.6
Michaelis constant:	0.5mM(F-VH)
Substrate specificity:	0.01(F-K/F-VH) 1(F-V/F-VH)
Optimum temperature:	40~45℃(Fig.1)
Optimum pH::	pH 6.5(Fig.2)
Thermal stability:	below 55℃ (pH 6.5, 10min)(Fig.3)
pH Stability:	pH 5.0-9.0 (Fig.4)
Effect of various chemicals:	(Table 1)

FPO-301

ASSAY

Principle:

fructosylamino acid oxidase

Fructosylvalylhistidine + H₂O ►L-Amino acid + Gluconosone

peroxidase

2H₂O₂+ 4-Aminoantipyrine + Phenol ►Quinoneimine dye + 4H₂O

The appearance of quinoneimine dye is measured at 500nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of hydrogen peroxide (half a
micromoleofquinoneimine dye) per minute under the conditions described below.

Method:

Reagents
A. MES-NaOH buffer:	50mM MES buffer, pH 6.5 [Dissolve 1.07g of 2-(N-morpholino)-ethanesulfonic acid (MW=195.23) in ca. 80ml of H₂O and, after adjusting the pH to 6.5±0.05 at 25℃ with 10N NaOH, fill up to 100ml with H₂O]
B.F-VH solution:	1.2mg/ml [36mg of fructosylvalylhistidine (F-VH) /30ml of 50mM MES-NaOH buffer, pH 6.5. This solution should be used after 1 from 2 day of preparation. If phosphate content is more than 1%, calculate the concentration by the following formula; the F-VH concentration (mg/ml) = 1.2÷{1-(phosphate content(%)/100)}] (Store at 4℃)
C. 4-AA solution:	0.5% [500mg of 4-aminoantipyrine /100ml of H₂O] (Store at 4℃ in a brownish bottle)
D. Phenol solution:	1.5% [1.5g of phenol /100ml of H₂O] (Store at 4℃ in a brownish bottle)
E. Peroxidase solution:	500U/ml [ca. 45mg of horseradish perosidase (Toyobo Grade Ⅲ, 110 purpurogallin units/mg)/10ml of H₂O]
F. Enzyme diluent	50mM K-phosphate buffer, pH 6.5 containing 0.1%(w/v) Triton X-100.

Procedure

1. Prepare the following working solution (20 tests) in a brownish bottle, and store on ice.

7.4ml MES-NaOH buffer (A)
1.2ml 4-AA solution (C)
0.8ml Phenol solution (D)
0.6ml Peroxidase solution (E)

2. Pipette 0.5ml of working solution and 2.5ml of the F-VH solution (B) into a test tube and equilibrate at 37℃ for about 5 minutes.

3. Add 0.1ml of the enzyme solution* and mix so gently that the fluid level swings a
little.

4. Record the increase in optical density at 500nm against water for 2 to 3 minutes in a
spectrophotometer thermostated at 37℃ and calculate the ΔOD per minute from the
initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by using the same method
as the test except that the enzyme diluent is added instead of enzyme solution.

* Dissolve the enzyme preparation in 50mM K-phosphate buffer, pH 6.5, dilute to
0.21-0.93 U/ml with enzyme diluent (F) and store on ice.

Calculation

ΔOD (ΔOD test−ΔOD blank ) ×Vt × df

Volume activity (U/ml) = =ΔOD/min×4.7×df

13.3×1/2×1.0×Vs

Weight activity (U/mg) = (U/ml) × 1/C

Vt: : Total volume (3.1ml)
Vs: : Sample volume (0.1ml)
13.3: : Millimolar extinction coefficient of quinoneimine dye under the assay condition (㎠/micromole)
1/2: : Factor based on the fact that one mole of H2O2 produced half a mole of
quinoneimine dye
1.0: : Light path length (cm)
C: : Enzyme concentration in dissolution (c mg/ml)

**Table 1. Effect of Various Chemicals**
[The enzyme dissolved in 50mM K-phosphate buffer, pH6.5 (40U/ml) was incubated with each chemical for 1hr at 25 ℃.]
Chemical	Concn.(mM)	Residual activity	Chemical	Concn.(mM)	Residual activity
None	−	100%	MIA	2	98
MgCl₂	2	99	NaF	2	100
CaCl₂	2	98	EDTA	5	100
Ba(OAc)₂	2	98	o-Phenanthroline	2	100
FeCl₂	2	91	Borate	50	99
CoCl₂	2	99	IAA	2	96
MnCl₂	2	98	NEM	2	60
Zn(OAc)₂	2	100	Hydroxylamine	2	99
NiCl₂	2	100	Triton X-100	0.10%	100
CuSO₄	2	96	Brij 35	0.10%	101
AgNO₃	2	10	Tween20	0.10%	101
			Span20	0.10%	99
			Na-cholate	0.10%	99
			SDS	0.05%	49
			DAC	0.05%	97

MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, lodoacetamide;
NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethyl-benzyl-alkyl-ammoniumchloride

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