Fructosyl-amino acid oxidase from Microorganism

Fructosyl-amino acid oxidase From Microorganism

Fructosyl-L-amino acid +HO +O                       Glucosone + L-amino acid+HO

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 + HO                                 ►L-Amino acid + Gluconosone

peroxidase

2HO+ 4-Aminoantipyrine + Phenol                                  ►Quinoneimine dye + 4HO

 

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 HO and, after adjusting the pH to 6.5±0.05 at 25℃
with 10N NaOH, fill up to 100ml with HO]
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 HO] (Store at
4℃ in a brownish bottle)
D. Phenol solution 1.5% [1.5g of phenol /100ml of HO] (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 HO]
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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