EXTRACTION OF CHOLESTEROL OXIDASE FROM NOCARDIA-RHODOCHROUS USING A NONIONIC SURFACTANT-BASED AQUEOUS 2-PHASE SYSTEM

The use of an aqueous two-phase system with the nonionic polyoxyethylene detergent C12EO5 (pentaethyleneglycol mono n-dodecyl ether) for direct solubilization and extraction of membrane-bound cholesterol oxidase from an unclarified culture of Nocardia rhodochrous was investigated. The potential of such nonionic detergent-based aqueous two-phase systems to simplify downstream processing of proteins is demonstrated. In only one step a clarification, preconcentration (up to 4-fold) and a partial purification (up to 5-fold) can be achieved. More than 90% of the enzyme could be extracted into the coazervate phase. Optimum conditions for the protein extraction from unclarified culture medium are shown in an analytical scale by investigation of the key parameters of the extraction process. These are detergent and biomass concentration as well as several additional agents for protein solubilization and stabilization.