Native Cholesterol Dehydrogenase from Microorganism

Products

Native Cholesterol Dehydrogenase from Microorganism

Product Specifications

Cat#	NATE-1004
Descriptions	Native microbial Cholesterol Dehydrogenase relies on coenzyme redox reactions to quantify cholesterol content without hydrogen peroxide byproducts, reducing background color interference in diagnostic assays. This enzyme adapts to simplified one-step cholesterol detection reagent formulas and is widely applied in cost-effective routine serum lipid diagnostic kits for grassroots medical testing institutions.
Applications	This product can be coupled with cholesterol esterase to achieve accurate enzymatic quantification of serum cholesterol within clinical diagnostic assays.
Synonyms	Cholesterol: NAD⁺ oxidoreductase
EC Number	EC 1.1.1.840
Catalysis	Cholesterol + NAD⁺ ⇆ Cholest-4-en-3-one + NADH + H⁺
Activity	≥ 18 U/mg (Cholesterol dehydrogenase activity)
Contaminant	≤ 5 × 10⁻²% (NADH oxidase activity); ≤ 5 × 10⁻²% (Glucose dehydrogenase activity)
Appearance	White to brown powder, lyophilized
Additive	Not added
Molecular Weight	37,000 (SDS-PAGE)
Isoelectric Point	4.5
Km	1.5 × 10⁻⁴ M (Cholesterol); 2.3 × 10⁻⁴ M (NAD⁺)
Optimum pH	7.0
pH Stability	7.0 (37°C, 15 min)
Optimum Temperature	30°C
Thermal Stability	up to 35°C (pH 7.0, 15 min)
Inhibitors	Ag⁺
Activator	Triton X-100
Unit Definition	1 unit is defined as the enzyme quantity which produces 1 μ mole of NADH per minute under the conditions described below.
Storage	36 months from the date of analysis when stored at -20°C or below in a dry place under sealed conditions.

General Properties

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Assay Principle

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Activity Assay

Unit Definition

1 unit is defined as the enzyme quantity which produces 1 μ mole of NADH per minute under the conditions described below.

Reagents

A. 50 mg/mL Triton X-100 solution (dissolved in deionized water)
B. 20 mg/mL Triton X-100 solution (dissolved in deionized water)
C. 0.3 M Tris-HCl buffer (pH 8.5)
D. 0.02 M Phosphate buffer (KH2PO4-Na2HPO4, pH 7.0)
E. NAD solution: Weigh 75 mg of β-NAD+ and dissolve in 0.3M Tris-HCl buffer (C). Fill up to 25 mL with 0.3 M Tris-HCl buffer (C). (Can be used for 5 days if kept refrigerated)
F. Substrate solution: Transfer 40 mL of 20 mg/mL Triton X-100 solution (B) in a beaker (50 mL) and incubate at 75-80℃ with stirring.: Weigh 50 mg of cholesterol and add 4 mL of 2-propanol and dissolve at 75-80℃. Add the solution into 20 mg/mL Triton X-100 solution (B) in the beaker, then keep the mixture at 75-80℃ in a water bath for 30 minutes. After cooling with running tap water, fill up to 50 mL with 20 mg/mL Triton X-100 solution (B). (Can be used for 10 days if kept refrigerated)
G. Diluent: Mix 0.02 M Phosphate buffer (D) and 5 mL of 50 mg/mL Triton X-100 solution (A), then fill up to 500 mL with 0.02 M Phosphate buffer (D).
H. Enzyme solution: Weigh out Cholesterol Dehydrogenase and dissolve in chilled Diluent (G). Enzyme solution should be prepared so that the value of ΔOD/minute becomes in the range of 0.020±0.005.

Procedure

Pipette 2.0 mL of Substrate Solution (F) and 1.0 mL of NAD solution (E) respectively into a quartz cell (d =10 mm) and keep at 25±0.5℃ for 5 minutes. Then pipette 0.10 mL of Enzyme solution (H) into the quartz cell and mix well immediately. Keep the reaction mixture at 25±0.5℃. Exactly at 1 minute and 3 minutes after the addition of Enzyme solution (H), measure the absorbances of the reaction mixture at 340 nm (A1 and A3). As a blank, pipette Diluent (G) into another quartz cell (d =10 mm) instead of Enzyme solution (H) and take the same procedure described above (Ab1 and Ab3).

Calculation

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Safe Handling Avoid inhalation; in the event of direct skin or eye contact, immediately flush the affected area with abundant water, and consult the SDS for further information.