From a technological point of view it is simpler to handle an extracellular enzyme than a cell-linked one. The amount of extracellular COX of the non-pathogenic strain R. erythropolis ATCC 25544 obtained in a 1.5 1 batch fermentation represents a 36% of the activity (130 out of 360 U/ml) when measured directly from the broth, but after the 6% Triton X-114 treatment and phase separation it represents a 65% of the total activity (442 out of 672 U/ml). In addition, active COX becomes 11.6 times purer and 20.3 times more concentrated. These results may make attractive and cost-effective the use of this bacterial strain and the Triton X-114 phase separation in a 6% w/v for the industrial production of COX used in serum and food cholesterol analysis. The purification step based on Triton X-114 phase separation should be followed by further steps, such as ion-exchange chromatography, which can combine non-ionic detergent removal and protein purification in one step, in order to obtain a preparation of COX suitable for analytical applications [16].