Extracellular cholesterol oxidase from Rhodococcus sp.: isolation and molecular characterization

Area of Science:

• Microbiology
• Biotechnology
• Enzymology

Background:

• Cholesterol oxidase (CHO) possesses significant clinical and industrial applications, particularly in medicine.
• Microbial sources of CHO are increasingly recognized for their utility.
• This study focuses on characterizing a novel soil-derived strain for CHO production.

Purpose of the Study:

• To taxonomically characterize a soil isolate with cholesterol oxidase (CHO) producing capabilities.
• To optimize fermentation conditions for enhanced CHO production.
• To biochemically characterize the produced CHO enzyme and clone its gene.

Main Methods:

• Bacterial isolation using cholesterol-based screening media.
• Identification via morphological, cultural, and biochemical tests, including 16S rRNA sequencing.
• Optimization of temperature, pH, and substrate concentration for extracellular CHO activity.
• PCR amplification and cloning of the CHO gene into the STV28 vector.

Main Results:

• The isolated bacterium was identified as Rhodococcus sp. strain 501 (GenBank Accession No. FN298676).
• Optimal conditions for CHO activity were determined as 35°C and pH 7.5.
• The cloned CHO gene exhibited 99% nucleotide sequence homology with known bacterial CHO genes.

Conclusions:

• Rhodococcus sp. strain 501 efficiently produces extracellular cholesterol oxidase (CHO) in a short period under optimized conditions.
• The successful cloning of the CHO gene provides a valuable tool for further genetic studies and expression analysis.
• This research contributes to the development of microbial CHO for biotechnological applications.