LACTATE OXIDASE from Microorganism
LCO-301
L-Lactate : oxygen oxidoreductase (EC 1.13.12.4)
L-Lactate + O₂ ► Pyruvate + H₂O₂
| Appearance: | Yellowish amorphous powder, lyophilized | ||
|---|---|---|---|
| Activity: | GradeⅢ 80U/mg-solid or more | ||
| Contaminants: | Pyruvate oxidase ≤1.0×10⁻³% Cholesterol oxidase ≤1.0×10⁻³% Uricase ≤1.0×10⁻³% Glucose oxidase ≤1.0×10⁻³% |
||
| Stability: | Stable at -20°C for at least One year (Fig.1) |
|---|---|
| Molecular weight : | approx. 160,000 (by gel filtration) |
| Isoelectric point: | 4.3±0.2 |
| Michaelis constant: | 1.0×10⁻³M (L-Lactate) |
| Inhibitors : | Fe⁺⁺⁺, SDS |
| Optimum pH : | 7.5(Fig.2) |
| Optimum temperature : | 35-40°C(Fig.3) |
| pH Stability : | 4.0-9.8 (25°C, 16hr)(Fig.4) |
| Thermal stability : | below 50°C (pH 7.0, 10min)(Fig.5) |
| Effect of various chemicals : | (Table 1) |
APPLICATIONS
This enzyme is useful for enzymatic determination of L-lactate.
ASSAY
Principle:
lactate oxidase
L-Lactate + O₂ ► Pyruvate + H₂O₂
peroxidase
2H₂O₂ + 4-Aminoantipyrine + EHSPT ► Quinoneimine dye + 4H₂O
The appearance of quinoneimine dye is measured at 555nm by spectrophotometry.
Unit definition:
One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per
minute under the conditions described below.
Method:
| A. DL-Lactate solution: | 0.125M [240mg of DL-lithium lactate (MW=96.01)/20ml of 50mM K-PB pH7.5] (Should be prepared fresh) |
|---|---|
| B. 4-AA solution: | 0.5% (500mg of 4-aminoantipyrine/100ml of H₂O) (Store at 4°C in a brownish bottle) |
| C. EHSPT(TOOS) solution: | 20mM [591mg N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (MW=295.3)/100ml of H₂O] (Store at 4°C in a brownish bottle) |
| D. Peroxidase solution: | 25U/ml [ca. 23mg of horseradish peroxidase (Toyobo GradeⅢ, 110 purpurogallin units/mg)/100 ml of H₂O] |
| E. SDS solution: | 0.25% (500mg sodium dodecyl sulfate/200ml of H₂O) |
| F. Enzyme diluent: | 20mM K-PB, pH7.0 containing 0.1%(w/v) sodium cholate |
Procedure
| Concentration in assay mixture | |
|---|---|
| K-phosphate buffer | 20 mM |
| DL-Lactate | 48 mM |
| 4-Aminoantipyrine | 1.2 mM |
| EHSPT | 0.76 mM |
| Peroxidase | 2.4 U/ml |
1. Prepare the following working solution (20 tests) in a brownish bottle, and store on ice.
8.0ml DL-Lactate solution (A)
1.2ml 4-AA solution (B)
0.8ml EHSPT solution (C)
2.0ml Peroxidase solution (D)
8.0ml distilled water
2. Pipette 1.0ml of working solution into a test tube and equilibrate at 37°C for about 5 minutes.
3. Add 0.05ml of the enzyme solution* and mix.
4. After exactly 15minutes at 37°C, add 2.0ml of SDS solution (E) to stop the reaction and measure the optical
density at 555nm against water (ODtest).
At the same time, prepare the blank by using the same method as the test except that the enzyme diluent
(F) is used instead of the enzyme solution (ODblank).
* Dissolve the enzyme preparation in ice-cold 20mM ACES-NaOH pH7.0 containing 1mM EDTA and 0.5%(w/v)
sodium cholate, and dilute to 0.04-0.1 U/ml with the enzyme diluent (F) immediately before assay.
Calculation
Activity can be calculated by using the following formula :
ΔOD (ODtest-ODblank)×Vt×df
Volume activity (U/ml) = = ΔOD×0.237×df
34.3×1/2× t ×1.0×Vs
Weight activity (U/mg)=(U/ml)×1/C
- Vt
- : Total volume (3.05ml)
- Vs
- : Sample volume (0.05ml)
- 34.3
- : 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
- t
- : Reaction time (15minutes)
- 1.0
- : Light path length (cm)
- C
- : Enzyme concentration in dissolution (C mg/ml)
REFERENCES
- A. Toda, and Y. Nishiya; J. Ferment. Technol., 85, 507 (1998)
| Chemical | Concn.(mM) | Residual activity(%) |
Chemical | Concn.(mM) | Residual activity(%) |
|---|---|---|---|---|---|
| None | − | 100 | IAA | 2.0 | 90 |
| Metal salt | 2.0 | Hydroxylamine |
2.0 | 99 | |
| MgCl₂ | 100 | EDTA | 5.0 | 94 | |
| CaCl₂ |
101 | o-Phenanthroline | 2.0 | 100 |
|
| Ba(OAc)₂ | 101 |
α,α′-Dipyridyl | 2.0 | 94 | |
| FeCl₃ | 5 |
Borate | 50.0 | 97 | |
| CoCl₂ | 100 | NaF | 2.0 | 99 |
|
| MnCl₂ | 100 | NaN₃ | 2.0 | 100 | |
| ZnCl₂ | 94 | Triton X-100 | 0.10% | 98 | |
| Cd(OAc)₂ | 91 | Brij 35 | 0.10% | 86 | |
| NiCl₂ | 99 | Tween 20 | 0.10% | 81 |
|
| CuSO₄ | 94 | Span 20 | 0.10% | 96 | |
| AgNO₃ | 54 |
Na-cholate | 0.10% | 101 | |
| MIA |
2.0 | 94 | DAC | 0.05% | 76 |
| NEM | 2.0 | 99 | SDS | 0.05% | 0 |
Ac, CH₃CO; MIA; monoiodoacetate, NEM; N-ethylmaleimide, IAA; iodoacetate, EDTA; ethylenediaminetetraacetate, SDS; Sodium dodecyl sulfate, DAC; dimethylbenzylalkylammonium chloride.
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