The establishment of a fermentation process for the production of pig liver esterase (PLE) in high yields is necessary for industrial applications. In our previous studies, we reported the recombinant expression of PLE in Escherichia coli Origami (TM) (DE3) in shake flask. Only a coexpression with chaperones GroEL/ES allowed the production of soluble and active enzyme. The optimization of the cultivation conditions, such as temperature, inducer concentrations, or media compositions to increase enzyme yield in a fermentation process is described here. Using fed-batch fermentation cell densities up to OD = 50 were obtained, but almost no active enzyme was expressed. Only batch fermentation was found suitable for production of active pig liver esterase and cell densities between OD = 7-13 and activities of 300-400 U L-1 for isoenzyme PLE-1 (gamma PLE) and 1,400 U L-1 for PLE-5 were obtained after 22 h total cultivation time or 18 h after induction of PLE expression, respectively.