Recombinant Extracellular Cholesterol Oxidase from Nocardioides simplex.

Area of Science:

• Biochemistry
• Enzymology
• Microbial Biotechnology

Background:

• Cholesterol oxidase (ChO) is a vital enzyme with diverse applications in medicine, pharmacy, agriculture, chemistry, and biotechnology.
• It catalyzes the oxidation of steroids, producing hydrogen peroxide, a reaction crucial for various biological and industrial processes.

Purpose of the Study:

• To express and purify the extracellular cholesterol oxidase (ChO) from Nocardioides simplex VKM Ac-2033D in Escherichia coli.
• To characterize the biochemical properties and substrate specificity of the recombinant enzyme (ChO_Ns).

Main Methods:

• Expression of 6xHis-tagged mature ChO in E. coli.
• Purification of recombinant ChO_Ns using affinity chromatography.
• Enzyme activity assays, pH profiling, kinetic analysis, and storage stability tests.

Main Results:

• The recombinant ChO_Ns was successfully purified and demonstrated functionality towards various steroids including cholesterol, cholestanol, and phytosterols.
• Enzyme activity was influenced by the steroid's aliphatic side chain length and was lower for pregnenolone and dehydroepiandrosterone.
• ChO_Ns exhibited optimal activity at pH 6.0 and showed comparable or superior stability and kinetics to commercial cholesterol oxidase.

Conclusions:

• Cholesterol oxidase from N. simplex VKM Ac-2033D is a robust and versatile enzyme.
• The characterized ChO_Ns presents a promising alternative to existing commercial enzymes for industrial and biotechnological applications.