Materials and methods
Commercial cholesterol oxidase from R. erythropolis was purchased from Boehringer Mannheim (25 U/mg). Cholesterol from lanolin, cholesterol from human gallstones, Triton X-100 and Triton X-114 were purchased from Fluka. Triton X-114 was condensed three times in 5 mM sodium phosphate buffer pH 7.5 [30,31]. The detergent phase of the third condensation had a concentration of 25% w/v TX-114 and was used as the detergent stock solution for all the experiments. The Triton X-114 concentration was determined from its absorption at 278 nm (A278 = 1.25 for 0.05% w/v) [29]

Microorganism and culture conditions
The strain used in this work was R. erythropolis ATCC 25544 which was routinely maintained in the laboratory by periodic subculturing in GMP medium [20] consisting of 0.1 g/l glucose, 0.02 g/l yeast extract, 0.04 g/l peptone, 0.04 g/l meat extract, 0.05 g/l NaCl, 0.0025 g/l MgSO4 and 0.25 g/l agar.
The microbial production of cholesterol oxidase was assessed as previously described by us [9].
Cells were grown in GYS medium in a 2 1 reactor (BIOSTAT B from B. Braun Biotech Ltd.) with a working volume of 1.5 1. Air was supplied at 2.6 vol/vol/min; pH was set constant to 6.75 and temperature to 29°C. The GYS medium is a modification of the mineral medium described by Buckland [26] that consisted of 10 g/l glycerol, 20 g/l yeast extract, 2 g/l (NH4)2SO4, 2 g/l K2HPO4, 0.01 g/l CaCl2.2H2O, 0.01 g/l FeSO4.7H2O, 0.1 g/l MgSO4.7H2O. When this culture reached a dry weight of ca. 1.0 mg/ml, an aqueous suspension of Tween 80/cholesterol was added to a final concentration of 0.2% cholesterol and 0.1% Tween 80.
An aqueous suspension of cholesterol was prepared in two ways; at the flame and by a spray-dry method. (i) In the first method, cholesterol and Tween 80 were mixed by heating at the flame until total dissolution of solids, then water was added to form an emulsion by vigorous shaking for 1 hour. (ii) In the second, cholesterol and Tween 80 were co-dissolved in diethyl ether; the solvent was then removed by spray drying and the solid material was recovered and used to readily prepare an stable aqueous suspension.

Extraction and partial purification of cholesterol oxidase
The extraction of cell-linked COX by using Triton X-100 was as described previously [9]. For the cell linked COX extraction and purification by the Triton X-114 method, the extract obtained in cold after removal of cells by centrifugation was submitted to temperature-induced phase separation. The coalescence of the detergent was facilitated by warming up to 37°C for 15 min that was followed by the sharp separation of the two resulting phases by spinning at 4000 g for 15 min at 25°C. Both phases, the lower detergent-rich and the upper detergent-depleted were assayed for both enzyme activity (see below) and protein [32]. Using Triton X-114 also purified the extracellular COX. The cold culture broth was supplemented with Triton X-114 to the desired final concentration and detergent was completely dissolved at 4°C. Phase separation was induced as above.

Enzyme assay
Cholesterol oxidase activity was assayed by a modification of the method of Allain et al. [1] as described previously [9]. One unit of activity was defined as the amount of enzyme that converts 1 μmole of cholesterol/min at 37°C. All samples were diluted before enzyme assay to a final Triton X-114 concentration of 0.1% to avoid detergent interference with the assay [16].

SDS-PAGE
The protein extracts were prepared as described previously [9]. SDS-PAGE electrophoresis [33] was carried out at 200 volts at 25° in a Mini Protean cell (Bio-Rad, Richmond, California). The gels were developed by using the silver staining technique.