On the feasibility of the microscale approach for a multistep biotransformation: Sitosterol side chain cleavage

Background: The millilitre/microlitre-scale approach is widely used for screening purposes and one-pot biotransformations, but it has seldom been applied to whole cell multistep biotransformations and two-liquid biphasic processes. The present study aims to contribute to filling this gap by using 24-well microtitre plates as reactors for the side chain cleavage of sitosterol with resting cells of Mycobacterium sp. NRRL B-3805 and comparing results with other set-ups. Results: High catalytic activity in microtitre plates and aqueous medium was observed at 250 rpm shaking frequency and 25 mm shaking diameter with 80% headspace and a mixture of Mg²⁺, Zn²⁺, Mn²⁺ and Co²⁺ salts. Data obtained with microtitre plates were reproduced in other set-ups when aqueous medium was used, whereas higher biotransformation rates were recorded in stirred reactors when biphasic systems were employed. Conclusion: Results suggest that microtitre plates may be used for the characterisation of complex bioconversion systems to establish representative screening systems, particularly if based in aqueous phase systems. Probing experiments also demonstrated the feasibility of using microlitre/millilitre shaken bioreactors to evaluate organic/aqueous two-liquid bioconversion systems, although the enhanced mass transfer patterns resulting from the biphasic system hampered the translation of the data to the more common stirred reactor configuration.