Assessment of alkaline cholesterol oxidase purified from Rhodococcus sp. PKPD-CL for its halo tolerance, detergent and organic solvent stability.
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 60 kDa 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(-4)M 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 60 days. The isoelectric point of the enzyme was 9.5. It showed 80% residual activity with 20% NaCl (3.42 M) and 83% relative activity with 12% NaCl (2.05 M) concentration. The metal ions like Zn(2+), Cu(2+), Ag+, Fe(3+), Ba(2+) inhibited the enzyme activity >60% while Hg(2+) served a potent inhibitor whereas Mg(2+) 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.
Department of Biochemistry, Shivaji University, Kolhapur 416004, Maharashtra, India.
Pramod J
PJ
Kasabe
Department of Biochemistry, Shivaji University, Kolhapur 416004, Maharashtra, India.
Geetanjali T
GT
Mali
Department of Biochemistry, Shivaji University, Kolhapur 416004, Maharashtra, India. Electronic address: pbd_biochem@unishivaji.ac.in.
Padma B
PB
Dandge
D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000737:chemistry; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D012240:Rhodococcus / Q000737:chemistry / Q000201:enzymology* / Q000254:growth & development; D054712:Salinity; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity
D003902:Detergents; D012997:Solvents; D002789:Cholesterol Oxidase
Alkaline cholesterol oxidase; Brackish water; Detergent and organic solvent tolerant; Halo tolerant