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. (C) 2004 Wiley Periodicals, Inc.