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.
Department of Biochemistry and Biochemical Technology, Royal Institute of Technology, Stockholm, Sweden.
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Waters
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Neujahr
D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics*; D005182:Flavin-Adenine Dinucleotide / Q000096:biosynthesis; D002479:Inclusion Bodies / Q000201:enzymology; D007544:Isopropyl Thiogalactoside; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D014250:Trichosporon / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D007544:Isopropyl Thiogalactoside; D006899:Mixed Function Oxygenases; C022336:phenol 2-monooxygenase