EXPONENTIALLY FED-BATCH CULTURES AS AN ALTERNATIVE TO CHEMOSTATS - THE CASE OF PENICILLIN ACYLASE PRODUCTION BY RECOMBINANT ESCHERICHIA-COLI

Exponentially fed-batch cultures (EFBCs), fed with medium containing a highly concentrated carbon source, are commonly employed for attainme nt of high cell densities. However; large variations in environmental conditions occur, and quasi-steady-state is usually achieved only for the limiting substrate concentration restricting the use of such cultu res in kinetic characterization studies. In this work we report the pr oduction of recombinant penicillin acylase (PA) in EFBC of an E. coli JM101 transformed with the pPA102 plasmid, which includes the PA gene under regulation of the lacZ gene promoter and using isopropyl-beta-th io-galactopyranoside (IPTG) as inducer. The culture was fed with nonco ncentrated complete medium, resulting in the attainment of quasi-stead y-state conditions not only in substrate concentration, but also in ce ll concentration and in the specific rates of growth, product producti on, and substrate consumption. Similar transient behavior was observed between EFBC and chemostat results. At quasisteady-state, the dilutio n rate in the EFBC equaled the growth rate. Specific PA production rat e during the fed-batch phase remained relatively constant at each dilu tion rate and followed typical Luedeking-Piret kinetics, with growth-a ssociated and non-growth-associated constants of 142 U g(DCW)(-1) and 7.2 U g(DCW)(-1) h(-1), respectively. Specific glucose consumption rat e linearly increased from 0.025 to 0.6 g g(DCW)(-1) h(-1) as the dilut ion rate increased from 0.01 to 0.35 h(-1). The maximum specific PA ac tivity increased with decreasing dilution rate, reaching ifs highest v alue of 2.0 U mg(-1) at a dilution rate of 0.01 h(-1), the lowest dilu tion tested. Such an activity was 7-fold higher than in batch culture and, to our knowledge, represents the highest value reported in the li terature.