CE (Cholesterol esterase)
Researching and mass preparation of lipoprotein cholesterol reagent in high and low density.
Specification
|
Appearance |
White to pale brownish amorphous powder, lyophilized |
|
Purity |
≥90% |
|
Specific activity |
≥100 U/mg |
|
Catalase |
≤0.001% |
|
Protease |
≤0.001% |
|
Uricase |
≤0.01% |
|
ATPase |
≤0.005% |
|
Glycerol kinase |
≤0.01% |
|
Glucose oxidase |
≤0.01% |
Properties
|
Source |
Recombinant from microorganism |
|
|
EC number |
3.1.1.13 |
|
|
Molecular weight |
61 kDa (SDS-PAGE) |
|
|
Isoelectric point |
5. 15 |
|
|
Michaelis constants |
5.8× 10-5 M (Cholesterol linoleate) |
|
|
Inhibitors |
Hg2+, Ag+ |
|
|
Optimum pH |
5.0 |
Fig. 1 |
|
Optimum temperature |
55℃ |
Fig. 2 |
|
pH stability |
pH 4.5- 9.0 (37℃, 60mins) |
Fig. 3 |
|
Thermal stability |
Below 50℃ (pH 7.5, 30 min) |
Fig. 4 |
|
Storage stability |
Store at -25~-15°C for 12 months could maintain more than 90% activity |
Fig. 5 |
|
Protective agent |
Sodium cholate and BSA |
The amount of quinoneimine dye produced by the reaction can be detected by spectrophotometer at 555 nm.
Unit definition
One unit of cholesterol esterase is defined as the amount of enzyme producing 1 μmol H2O2 per minute under standard determination conditions.
Reagents preparation
Reagent I：0.2 M K3PO4 solution, pH 6.0.
Reagent II: Add 39 mg cholesterol linoleic acid ester to 2.0 ml isopropanol, heat by water bath for a complete dissolution. Add about 80 ml of 1% Genapol X-80 preheated at 72-74℃, water bath at 72-74℃ and stir for 30 min, the solution will become clear first and then turbid. Cool the solution to room temperature with water. Add 0.6 g sodium cholate, after dissolution, dilute to 100 ml with 1% Genapol X-80 solution.
Reagent III： 1 kU/mL POD.
Reagent IV：50 mM TOOS solution.
Reagent V：50 mM 4-AA solution.
Reagent VI：300 U/mL COX enzyme solution.
Enzyme diluent：20 mM K3PO4 solution, pH 6.0. 2 mM MgCl2, 0.5 mM EDTA-Na2 and 0.2% BSA.
Add the following components in the order specified:
|
Reagent I |
75 mL |
|
Reagent II |
50 mL |
|
Reagent III |
750 μL |
|
Reagent IV |
2.5 mL |
|
Reagent V |
2.5 mL |
|
|
Set to 135 mL |
Procedure
1. Add 2.75 mL of the reaction mixture to a 3 ml cuvette.
2. Add 0. 1 mL of reagent VI, water bath at 37℃ for 2 min.
3. Add 0. 1 mL of enzyme samples and mix well.
4. Measure absorbance change(As) in 1 minute at 555nm at 37°C. * Set a blank control with enzyme diluent, measure the blank absorbance change(∆Ab)
∆A= ∆As - ∆Ab
Calculation
2.95: Total volume of reaction solution (mL)
0. 10: Volume of enzyme solution (mL)
1.0: Optical Path (cm)
df: Dilution multiple
1/2: 1 mol of H2O2 yields 1/2 mol of quinoneimine dye
C: Enzyme concentration (mg/mL)
39.2: Millimolar extinction coefficient ofquinoneimine dye under 555 nm (cm2/μmol)
