Formation of stable emulsions with the organic solvent is a general complication in the extractive workup of aqueous wholecell biotransformations. This hold-up has been overcome by biocatalytic lysis of emulsifying agents present in the medium through addition of living microorganisms. Of these, Bacillus subtilis and Rhodococcus erythropolis exhibited the most powerful de-emulsifying activity. As exemplified by microbial treatment of cell-free biotransformation media of Saccharomyces cereWisiae and Lactobacillus kefiri, phase separation time (t p ) was drastically reduced from one week to 20 s without significantly affecting product integrity. This practicable readyto-use method is appropriate to both fungal and bacterial biocatalysts. The highly efficient de-emulsification power and the considerably short phase separation time of this technique allow for cost-effective continuous extractions on a large-scale, for example with mixer-settler units.

Caffeine affects the stereoselectivity of microbial high cell density reductions with commercial grade Saccharomyces cerevisiae (Baker's yeast). Cyclic beta-keto esters ethyl 2-oxocyclopentanoate (1) and ethyl 2-oxocyclohexanoate (3) were shown to be reduced with increased diastereoselectivity (1: 90.1 --> 92.1% de, 3: 75.0 --> 90.0% de) after addition of caffeine. Effects on enantioselectivity were less pronounced (1: 97.3 --> 98.5% ee, 3: 90.1 --> 92.1% ee). The observations are ascribed to the action of caffeine on cellular calcium homeostasis. These effects are accompanied by caffeine-induced cell-death, which preferably takes effect on pre-stressed cells which were found to decrease diastereoselectivity.

Extractive work-up of whole-cell biotransformations generally suffers from the formation of stable gels and slimes upon addition of the organic solvent to the cell suspension and the cell-free solution, respectively. This problem has been overcome by enzymatic lysis of emulsifying agents present in the medium through addition of hydrolases. Of these agents, proteases have exhibited the most powerful de-emulsifying activity. Enzyme treatment of cell-free culture media of Saccharomyces cerevisiae with pronase E drastically reduced phase separation time (t(p)) from 1 week to 30 min without significantly affecting product integrity. Yeast cell suspensions were de-emulsified best with protease N-01, where phase separation was complete after 1 h. As was exemplified with cell-free culture media of Lactobacillus kefir, wherein addition of pronase E or protease N-01 reduced t(p) from 1 week to 2 h each, this practical, ready-to-use method is appropriate for both fungal and bacterial biocatalysts.

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