Assessment of alkaline cholesterol oxidase purified from Rhodococcus sp. PKPD-CL for its halo tolerance, detergent and organic solvent stability

• Published in: Protein expression and purification 2015-12, Vol.116, p.30-41
• Main Authors: Kasabe, Pramod J., Mali, Geetanjali T., Dandge, Padma B.
• Format: Article
• Language: English
• Subjects: Alkaline cholesterol oxidase; Brackish water; Cholesterol Oxidase - chemistry; Cholesterol Oxidase - isolation & purification; Cholesterol Oxidase - metabolism; Chromatography, Gel; Chromatography, Ion Exchange; Detergent and organic solvent tolerant; Detergents - chemistry; Enzyme Stability; Halo tolerant; Hydrogen-Ion Concentration; Rhodococcus - chemistry; Rhodococcus - enzymology; Rhodococcus - growth & development; Salinity; Solvents - chemistry; Substrate Specificity
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.08.011

•It’s a first report of halo tolerant, detergent and organic solvent stable, alkaline cholesterol oxidase.•Purified by using (NH4)2SO4 fractionation, anion exchange and size exclusion column chromatographies.•Enzyme was characterized for its physico-chemical, catalytic properties and its regulation by using certain metal ions, chemicals and detergents. The novel bacterium, Rhodococcus sp. PKPD-CL was isolated and identified from the ‘Chilika Lake’ located at Odisha state of India, which is a largest brackish water habitat in Asia. Rhodococcus sp. PKPD-CL produces extracellular halo tolerant, detergent and organic solvent stable alkaline cholesterol oxidase. It has apparent molecular weight of 60kDa and was purified 59 fold by using 60% saturated ammonium sulfate fractionation, anion exchange followed by size exclusion chromatographic techniques with 37% recovery. It showed substrate specificity for 3β-hydroxysteroids with Km of 1.1×10−4M for cholesterol. The pH, 8.0 and the temperature, 37°C were required for its optimum activity. Enzyme is considerably stable at pH 6.0–8.5 and temperature up to 50°C. At 4 and 30°C it maintained its 100% activity up to 60days. The isoelectric point of the enzyme was 9.5. It showed 80% residual activity with 20% NaCl (3.42M) and 83% relative activity with 12% NaCl (2.05M) concentration. The metal ions like Zn2+, Cu2+, Ag+, Fe3+, Ba2+ inhibited the enzyme activity >60% while Hg2+ served a potent inhibitor whereas Mg2+ found to be a good enhancer for it. The enzyme was stable in presence of chemical reagents (NaN3, EDTA), detergents (Tween-80, Tween-20, Triton X-100, sodium cholate) and various organic solvents (isopropanol, ethanol, benzene, chloroform, methanol, toluene, ethyl acetate, butanol and dimethylsulfoxide). Such a multi stress tolerant and versatile enzyme produced by Rhodococcus sp. PKPD-CL may serve as a good choice for industrial applications.