PMC:101390 / 3473-5267 JSONTXT

{"project":"2_test","denotations":[{"id":"11914155-8001397-8561956","span":{"begin":391,"end":393},"obj":"8001397"},{"id":"11914155-8001397-8561957","span":{"begin":961,"end":963},"obj":"8001397"},{"id":"11914155-1368208-8561958","span":{"begin":1193,"end":1195},"obj":"1368208"}],"text":"Due to the high cell-bound to extracellular ratio of cholesterol oxidase produced by Rhodococcus, even in those strains that also produce extracellular enzyme, the use of detergents is compulsory in the cost-effective extraction of this enzyme. The properties of protein extraction and purification combine in polyoxyethylene type detergents whose cloud point is in the biocompatible range [13]. For instance, Triton X-114 is as effective as Triton X-100 to extract membrane proteins, but its cloud point in semidiluted solutions (temperature at which the detergent solubility decreases sharply and a liquid-liquid phase separation is produced) is 23°C as compared to 65°C of Triton X-100. Therefore, extracted proteins partition between a detergent-rich phase and a detergent-depleted phase thus occurring protein purification. Protein purification has been accomplished successfully from either animal cells and organelles, plant tissues and microbial cells [13-15]. Triton X-114 at 1%w/v in buffers has been utilized to study the partitioning behavior of commercial cholesterol oxidase from several bacterial sources, resulting in partitioning toward the detergent rich-phase in all cases [16]. The polyoxyethylene detergent C12EO5 added the to a non-clarified culture of Nocardia rhodochrous was used for direct solubilization and extraction of the cell-bound cholesterol oxidase followed by phase separation [17]. A four-fold preconcentration and five-fold purification were achieved in optimal conditions. Due to the high cost of C12E05 these authors tried the cheaper four narrow range ethylene oxide surfactant C12-C18E05 [18] which was found equally suitable for direct solubilization and extraction of cell-bound cholesterol oxidase, thus this system was expanded to pilot scale [19]."}