LACTATE OXIDASE from Microorganism


L-Lactate : oxygen oxidoreductase (EC
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)


This enzyme is useful for enzymatic determination of L-lactate.



lactate oxidase

L-Lactate + O₂                                                   Pyruvate + H₂O₂


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.


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
(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


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.


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

: Total volume (3.05ml)
: Sample volume (0.05ml)
: Millimolar extinction coefficient of quinoneimine dye under the assay condition (㎠/micromole)
: Factor based on the fact that one mole of H₂O₂ produced half a mole of quinoneimine dye
: Reaction time (15minutes)
: Light path length (cm)
: Enzyme concentration in dissolution (C mg/ml)


  1. A. Toda, and Y. Nishiya; J. Ferment. Technol., 85, 507 (1998)
Table 1. Effect of Various Chemicals on Lactate oxidase
[The enzyme solution dissolved in 20mM ACES-NaOH, pH7.0 (50U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 IAA 2.0 90
Metal salt 2.0   Hydroxylamine
2.0 99
MgCl₂   100 EDTA 5.0 94
  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 NaN 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
2.0 94 DAC 0.05% 76
NEM 2.0 99 SDS 0.05% 0

Ac, CHCO; MIA; monoiodoacetate, NEM; N-ethylmaleimide, IAA; iodoacetate, EDTA; ethylenediaminetetraacetate, SDS; Sodium dodecyl sulfate, DAC; dimethylbenzylalkylammonium chloride.

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