Extracellular cholesterol oxidase from Rhodococcus sp.: isolation and molecular characterization.
BACKGROUND
Cholesterol oxidase (CHO) has various clinical and industrial applications. Recently, microbial CHO have received a great attention for their wide usage in medicine. Here, taxonomic characterizations of isolated strain from soil, optimization of the conditions for CHO production and biochemical characterizations of produced CHO enzyme were described. Finally, CHO gene was cloned into a cloning vector.


METHODS
Various samples were collected and cultivated in a screening medium consisting of cholesterol. For isolation of CHO-producing bacteria, well-grown colonies were inoculated into an optimized medium. Different biochemical and microbiological tests were performed on isolated bacteria to identify their properties. For phylogenic analysis, a partial sequence of l6s rRNA was amplified by PCR using universally conserved primers. A modified method was applied for determination of CHO activity. Then, extracellular CHO activity was assessed under different temperature, pH and cholesterol concentration conditions. Finally, CHO gene was amplified by PCR and cloned into STV28.


RESULTS
According to the morphological, cultural and biochemical tests, the isolated bacterium was identified as Rhodococcus sp. strain 501 and deposited in GenBank with accession number FN298676. Results showed that optimum temperature and pH for CHO activity were 35 degrees C and 7.5, respectively. Alignment of nucleotide sequence of CHO gene showed 99% homology with other bacterial CHO genes.


CONCLUSIONS
Rhodococcus sp. strain 501 produced significant levels of extracellular CHO in an optimized medium for a short period. CHO gene was cloned into cloning vector that can be a valuable tool for better identification and further studies on gene expression.
Lashkarian
Hamed
H
National Institute for Genetic Engineering and Biotechnology, Tehran, Iran.
Raheb
Jamshid
J
Shahzamani
Kiana
K
Shahbani
Hossein
H
Shamsara
Mehdi
M
D001483:Base Sequence; D002789:Cholesterol Oxidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008828:Microbiological Techniques; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D012988:Soil Microbiology
D003470:Culture Media; D002789:Cholesterol Oxidase