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Purification, characterization, and biological cytotoxic activity of the extracellular cholesterol oxidase produced by Castellaniella sp. COX
Author(s) -
Devi Sunita,
Sharma Bhupender,
Kumar Rakesh,
Singh Kanwar Shamsher
Publication year - 2020
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.201900365
Subject(s) - chemistry , chromatography , enzyme assay , enzyme , cholesterol oxidase , specific activity , acetone , chloroform , ethanol , extracellular , dimethyl sulfoxide , nuclear chemistry , biochemistry , organic chemistry
A new bacterial strain producing extracellular cholesterol oxidase (ChOx) was isolated and identified as Castellaniella sp. COX. The ChOx was purified by salting‐out and ion‐exchange chromatography up to 10.4‐fold, with a specific activity of 15 U/mg with a molecular mass of 59 kDa. The purified ChOx exhibited pH 8.0 and temperature 40°C for its optimum activity. The enzyme showed stability over a wide pH range and was most stable at pH value 7.0, and at pH 8.0, it retained almost 86% of its initial activity after 3 h of incubation at 37°C. The enzyme possessed a half‐life of 8 h at 37°C, 7 h at 40°C, and 3 h at 50°C. A Lineweaver–Burk plot was calibrated to determine its K m (0.16 mM) and V max (18.7 μmol·mg −1 ·min −1 ). The ChOx activity was enhanced with Ca 2+ , Mg 2+ , and Mn 2+ while it was inhibited by Hg 2+ , Ba 2+ , Fe 2+ , Cu 2+ , and Zn 2+ ions. Organic solvents like acetone, n ‐butanol, toluene, dimethyl sulfoxide, chloroform, benzene, and methanol were well tolerated by the enzyme while iso ‐propanol and ethanol were found to enhance the activity of purified ChOx. ChOx induced cytotoxicity with an IC 50 value of 1.78 and 1.88 U/ml against human RD and U87MG established cell lines, respectively, while broadly sparing the normal human cells.