Background
Microbial cholesterol oxidases (EC 1.1.3.6) (COX) catalyze the oxidation and isomerization of cholesterol to 4-cholesten-3-one. Interest in these enzymes mostly relies in their utility in the determination of cholesterol in biological samples such as serum and foods [1], and also in the bioconversion of a number of 3β-hydroxysteroids in organic solvents [2] and in reverse micelles [3] (for a recent review see [5]). Since earliest reports on crude preparations from Mycobacterium sp.[4], cholesterol oxidases have been described in a number of bacteria and fungi [5]. Enzymatic properties of cholesterol oxidase from Rhodococcus strains (some of which named formerly as Nocardid) are particularly suitable for use in the analytical determination of cholesterol, in which the hydrogen peroxide formed is used in a chromogenic reaction catalyzed by horseradish peroxidase [6].
The Rhodococcus enzyme has been usually reported to be membrane bound, extractable from whole cells by treatment with detergents or trypsin, although no phospholipids are detected in the enzyme extracts [7]. More recent reports have demonstrated the production of both extracellular and cell-bound cholesterol oxidase by strains of this genus such as Rhodococcus sp. GK1 [8], R. erythropolis ATCC 25544 [9] and the pathogenic specie R. equi[10,11].
The kinetics of enzyme synthesis at both bench and large scale by Nocardia rhodochrous (renamed as Rhodococcus rhodochrous), a strain that produces only a cell-bound COX, has been studied and the growing conditions for bacterial enzyme synthesis in fermentor were defined [12].
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].
In a previous work [9] we described the cell-bound and extracellular cholesterol oxidase activities from R. erythropolis ATCC 25544, achieving in optimal conditions 55% cell-bound and 45% extracellular activity. Their enzymatic properties strongly supported the idea that the particulate and the extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular mass of 55 kDa. In this work we optimize the culture conditions in a 2-liter fermentor of this extracellular cholesterol oxidase producer strain and carry out the extraction, partial purification and concentration of both types of cholesterol oxidase by using Triton X-114 phase separation. The results obtained are very promising for the use of this strain and this technique in the industrial processing of bacteria to obtain cholesterol oxidase.