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.
Waters
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Department of Biochemistry and Biochemical Technology, Royal Institute of Technology, Stockholm, Sweden.
Neujahr
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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