Effect of phase composition on the whole-cell bioconversion of β-sitosterol in biphasic media

The multi-step bioconversion of β-sitosterol by Mycobacterium sp. NRRL B-3805 for the formation of androstenedione (AD) as major product, a key intermediate in the production of therapeutic steroids, is a well established industrial application of a biocatalytic process. In the present work, this sterol side-chain cleavage was selected as model system for a study of whole-cell biocatalyst operational stability in prolonged bioconversions in two-phase aqueous-organic media. The aim was to evaluate the effect of phase composition in the process yields by using four high logP_octanol phthalates as organic phases and phosphate buffer and nutritional media as aqueous phases. The effect of a non-ionic surfactant added to the aqueous phase was also studied. Results show no relation between bioconversion yields and solvent logP_octanol values, suggesting that, in the used range, solvent molecular structures and the resulting specific interactions with the cell envelope are determining for the biocatalyst behaviour. Different responses were obtained from the use of different aqueous media, the complex nutritional media leading to the highest conversion yields, while the lowest were observed with the mineral nutritional media. The effect of surfactant addition in the bioconversion yields was apparently not significant. In the systems with bis-(2-ethylhexyl) and bis-(3,5,5-trimethylhexyl) phthalates as the organic phases, the fed-batch operational mode allowed the maintenance of stable biocatalytic activity levels up to at least 6 days of operation, with high androstenedione yields.