LOX (Lactate Oxidase)
Lactate oxidase is an enzyme which is predominantly used in the detection of Lactate. This has led to development of various biochemical sensors and devices utilizing Lactate oxidase.
Preparation and specification
|
Appearance |
Yellow amorphous powder,lyophilized |
|
Purity(SDS-PAGE) |
≥90.0% |
|
Enzyme powder specific activity |
≥45.0 U/mg |
|
Pyruvate oxidase |
≤0.001% |
|
Choline oxidase |
≤0.001% |
|
Uricase |
≤0.001% |
|
Glucose oxidase |
≤0.001% |
Properties bv
|
Source |
Microorganisms |
|
|
Classification |
EC 1.1.3.2 (Recombinant from microorganism) |
|
|
Molecular weight |
42kDa (SDS-PAGE) |
|
|
Isoelectric point |
pH 4.6 |
|
|
Km Value |
7.5 × 10-4 M (L-Lactate) |
|
|
Optimum pH |
6.0-7.0 |
Fig. 1 |
|
Optimum temperature |
50 °C |
Fig. 2 |
|
pH stability |
pH 6.0-8.5 (25 °C, 16 h) |
Fig. 3 |
|
Thermal stability |
Below 50 °C（pH 7.0, 30 min） |
Fig. 4 |
|
Stability |
Store at -25～- 15℃ for 12 months could maintain more than 90% activity |
Fig. 5 |
Storage conditions
Store at -25～- 15℃, valid for 1 year.
Figures
Assay Principle
Unit definition
One unit is defined as the amount of enzyme which produces 1 μmol of H2O2 per minute under the conditions described below.
Reagents preparation
Reagent I：0.2 M pH 6.5 potassium phosphate buffer.
Reagent II：1kU/mL peroxidase (POD) solution.
Reagent III：50 mM 4-AA solution.
Reagent IV：0.5 M DL- Lactic acid solution, pH 6.5.
Reagent V：50 mM TOOS solution.
Enzyme diluent ： 10 mM pH 7.0 potassium phosphate solution with 10 μM FAD.
Samples: dilute the enzyme with enzyme diluent to 0.05 – 0.2 U/mL.
Prepare reaction mixture as follows:
Reagent I 10 ml
Reagent II 0.25 mL
Reagent III 1.5 mL
Reagent IV 5 mL
Reagent V 1.5 mL
Add ddH2O to 50 ml
Procedure
1. Add 1 mL of the reaction mixture to 1 mL cuvette.
2. Heat the reaction mixture at 37 °C for 5 min.
3. Add 20 μL of the enzyme solution to the cuvette and mix.
4. Record the ΔAs at 555 nm in 1 minute in a spectrophotometer thermostated at 37 ℃.
* At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As - ∆Ab
1.02: Total volume of reaction solution (mL)
0.02: Volume of enzyme solution (mL)
1: Light path length (cm)
1/2: 1mol H2O2 will react to 1/2 mol Quinoneimine dye
df: Dilution multiple
C: Enzyme concentration (mg/mL)
39.2: Millimolar extinction coefficient of quinoneimine dye under 555nm (cm2/μmol)
