Cholesterol oxidase from Rhodococcus sp. is a key enzyme in the cholesterol degradation process. It catalyzes the isomerization and oxidation of cholesterol mediated by the coenzyme FAD. Research indicates that cholesterol oxidase plays a crucial role in the pathogenicity of Mycobacterium tuberculosis by modulating the host macrophage immune response through inhibition of the Toll-like receptor 2 (TLR2) signaling pathway, thereby enhancing the survival of Mycobacterium tuberculosis. Cholesterol oxidase from Rhodococcus sp., shows potential for research in the field of tuberculosis.
Preparation and Specification
Appearance: Yellowish amorphous powder, lyophilized
Specific activity: More than 15 U/mg solid
Properties
| Substrate specificity | -
| See Table 1 | | Molecular weight | 61.8 kDa (SDS-PAGE) | -
| | Isoelectric point | pH 4.5 | - | | Michaelis constant | 6.0 × 10⁻⁵ M (for Cholesterol) | -
| | Optimum pH | 7.0–7.5 | Figure 1 | | pH stability | 5.7–7.8 (65℃, 10 min) | Figure 2 | | Optimum temperature | 50℃ (Tris-HCl buffer) | Figure 3 | | Thermal stability | Stable at 65℃ and below (pH 7.0, 10 min) | Figure 4 and Figure 5 | | Effects of detergents | - | See Table 2 |
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Table 1. Substrate specificity
| Substrate (1 mM) | Relative activity (%) |
|---|
| Cholesterol | 100 | | β-Cholesterol | 93 | | Pregnenolone | 98 | | Dehydro-iso-androsterone | 10 | | β-Sitosterol | 94 | | Stigmasterol | 66 | | Androsterol | 2 | | Teststerone | 1 | | Cholic acid | 3 |
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Table 2. Effect of detergents on CHO activity
| Detergents (0.1%) | Relative activity (%) |
|---|
| Triton X-100 | 100 | | Emulgen 810 | 101 | | Emulgen 911 | 113 | | Emulgen 709 | 107 | | Emulgen 109P | 118 | | Adekatol SO-120 | 100 | | RHEODOL 460 | 63 | | SM 1080 | 122 |
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Applications
This enzyme is useful for enzymatic determination of total cholesterol, HDL-C, and LDL-C when coupled with cholesterol esterase.
Assay
Principle
The assay is based on the increase in absorbance at 240 nm as Δ⁴-cholesten-3-one is produced in the following reaction:
Unit definition
One unit is defined as the amount of enzyme which liberates 1 μmole of Δ⁴-cholesten-3-one per minute at 37℃ under the conditions specified in the assay procedure.
Reagents
1. Substrate solution (6 mM cholesterol solution) Dissolve 232 mg of cholesterol with isopropanol to make a total of 100 ml.
2. Enzyme dilution buffer
0.1 M KH₂PO₄–Na₂HPO₄ buffer pH 7.0 containing 0.05% (W/V) Triton X–100
※ Prepare the enzyme dilution buffer two days before use and keep it in the refrigerator until use.
Enzyme solution
Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. After 1–1.5 hour incubation at room temperature, dilute it with enzyme dilution buffer to adjust the concentration to within 0.1–0.2 U/ml.
Procedure
1.Pipette accurately 3.0ml of enzyme dilution buffer and 50 μl of enzyme solution and preincubate at 37℃.
※ In the case of a test blank, add 50 μl of enzyme dilution buffer in place of enzyme solution.
2. After 5 min, add 50 μl of substrate solution and mix to start the reaction at 37℃.
3. After starting the reaction, measure the rate of increase per minute in absorbance at 240nm. The rate must be measured within the linear portion of the absorbance curve.
Absorbance
sample : As/min
blank : Ab/min
0.010 Abs/min ≤ΔA/min = (As/min-Ab/min)
≤0.060 Abs/min
Calculation
Activity (U/mg) = (ΔA/min / 12.2) × (3.10 / 0.05) × (1 / X)
12.2 : millimolar extinction coefficient of Δ⁴-Cholesten-3-one at 240 nm (cm² / μmole)
3.10 : final volume (ml)
0.05 : volume of enzyme solution (ml)
X : concentration of the sample in enzyme solution (mg/ml)
ZJ26F0939028
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