A fermentor culture for production of recombinant phenol hydroxylase.

Fermentor cultures using the fed-batch technique produced the FAD-containing enzyme phenol hydroxylase (EC 1.14.13.7) originated in the lower eukaryote Trichosporon cutaneum, but expressed in Escherichia coli under the control of the tac promoter. At 30 degrees C and isopropyl beta-D-thiogalactopyranoside (IPTG) concentrations of 0.5-2 mM, the enzyme protein was expressed to high cellular content, but aggregated into inclusion bodies. At 25 degrees C similar levels of enzyme protein were synthesized after induction with 0.05 mM IPTG, but a soluble, active enzyme was obtained. The active enzyme was produced at up to 45% of total protein and constituted more than 50% of soluble protein. The total yield was 5 g x liter-1. The FAD content of the cells increased after induction at a rate not limiting the formation of active enzyme. The enzyme was purified in two chromatographic steps. The N-terminal amino acid residue and the kinetic properties of the purified recombinant enzyme were similar to those reported for the enzyme from T. cutaneum.