Cholesterol Esterase from Pseudomonas sp. is an enzyme that hydrolyzes cholesterol ester to cholesterol and free fatty acid in the intestinal lumen. Cholesterol synthesized in the acinar cells and is stored in zymogen granules. Cholesterol esterase is also known as bile salt-stimulated lipase and carboxy ester lipasea, acts function for acceleration of cholesterol absorption.
Cholesterol ester + H₂O → Cholesterol + Fatty acid
Preparation and Specification
| Appearance | : White to pale brownish amorphous powder, lyophilized |
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| Activity | : ≥100U/mg powder |
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Properties
| Molecular weight | 30 kDa (SDS-PAGE) | | Isoelectric point | pH 5.28 (estimated from amino acid sequence) | | Optimum pH | 6.5 | | pH Stability | 6.0–11.0 (37℃, 60 min, in 0.1% BSA) | | Optimum temperature | 35–40℃ (Phosphate Buffer) | | Thermal stability | Stable at 45℃ and below (pH 8.0, 30 min) | | Storage stability | At least one year at -20℃. | | Activator | Adekatol TN-100, Adekatol PC-8, Adelatol SO-120, Adekatol SO-135, Triton X-100 |
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Applications
This enzyme is useful for enzymatic determination of total cholesterol, HDL-C, and LDL-C coupled with cholesterol oxidase.
ASSAY
Principle:
The assay is based on the increase in absorbance at 493 nm as the formation of quinoneimine dye proceeds in the following reactions:
Unit definition:
One unit is defined as the amount of enzyme which liberates 1 μmole of cholesterol per minute at 37°C under the conditions specified in the assay procedure.
Reagents
1. Reaction mixture
| 0.2M KH₂PO₄-NaOH buffer, pH 6.8 | 0.60 ml |
| 0.35% (W/V) 4-AA solution | 0.30 ml |
| 0.2% (W/V) Phenol solution | 0.30 ml |
| 100 U/ml HRP (Horseradish Peroxidase) solution <sup>①</sup> | 0.30 ml |
| 3% (W/V) Triton X-100 solution | 0.30 ml |
| 0.2 U/ml CHO (Cholesterol Oxidase) solution <sup>②</sup> | 0.60 ml |
| Substrate solution <sup>③</sup> | 0.30 ml |
| Distilled water | 0.30 ml |
① 100 U/ml HRP solution: Dissolve 1000 U (PPU) of HRP with 10 ml of distilled water.
② 0.2 U/ml CHO solution: Dissolve 2 U of CHO with CHO dilution buffer <sup>※</sup>
※ CHO dilution buffer: 0.1 M KH₂PO₄-Na₂HPO₄ buffer, pH 7.0containing 0.05% (W/V) Triton X-100.
③ Substrate solution: Calf serum.
2. Enzyme dilution buffer: 10 mM KH₂PO₄–NaOH buffer pH 7.5 containing 0.1% (W/V) bovine serum albumin (BSA).
Enzyme solution
Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration to within 0.3–0.5 U/ml.
Procedure
1. Pipette accurately 3.0 ml of reaction mixture into a small test tube and preincubate it at 37°C.
2. After 10 min, add 50 μl of enzyme solution and mix to start the reaction at 37°C.
※ In the case of a test blank, add 50 μl of enzyme dilution buffer in place of enzyme solution. |
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3. After starting the reaction, measure the rate of increase per minute in absorbance at 493 nm. The rate must be measured within the linear portion of the absorbance curve.
| Absorbance sample : | As/min |
| blank : | Ab/min |
△A/min =(As/min−Ab/min)≤0.050 Abs/min
Calculation
Activity(U/mg of powder)= {(△A/min)/(12.0×1/2)} × 3.05/0.05 × 1/x
| 12.0 : | millimolar extinction coefficient of quinoneimine dye at 493 nm(cm²/μmole) |
| 1/2 : | a multiplier derived from the fact that 2 mole of H2O2 produce 1 mole of quinoneimine dye |
| 3.05 : | final volume(ml) |
| 0.05 : | volume of enzyme solution(ml) |
| X : | concentration of the sample in enzyme solution(mg/ml) |
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