title: Integrated one-pot enrichment and immobilization of styrene monooxygenase (StyA) using SEPABEAD EC-EA and EC-Q1A anion-exchange carriers.
abstract: A straightforward one-pot procedure combining enrichment and immobilization of recombinantely expressed FADH₂ dependent styrene monooxygenase (StyA) directly from Escherichia coli cell extracts was investigated. Sepabeads EC-EA and EC-Q1A anion-exchange carriers were employed to non-covalently adsorb StyA from the cell extracts depending on basic parameters such as varying initial protein concentrations and pH. The protein fraction of the cell extract contained around 25% StyA. At low initial protein concentrations (2.5 mg mL⁻¹) and pH 6, the enzyme could be enriched up to 52.4% on Sepabeads EC-EA and up to 46.0% on Sepabeads EC-Q1A, accounting for an almost complete StyA adsorption from the cell extracts. Higher initial protein concentrations were necessary to exploit the high loading capacity of the beads. At 20 mg mL⁻¹, up to 37.6% of the theoretical bead loading capacity could be utilized for StyA binding using Sepabeads EC-EA, and 34.0% using Sepabeads EC-Q1A. For both carriers, protein leakage under reaction conditions could be reduced to less than 2%. During assays, the FADH₂ cofactor necessary for StyA activity was supplied by the NADH-FAD reductase component styrene monooxygenase B (StyB). StyA immobilized on Sepabeads EC-Q1A displayed twice as high styrene epoxidation rates (0.2 U mg(StyA)⁻¹) as compared to Sepabeads EC-EA. This activity could be increased to 0.7 U mg(StyA)⁻¹ by co-immobilizing StyB on Sepabeads EC-Q1A, which corresponds to 33% of the soluble StyA activity.
authors.affiliation: Department of Chemical and Biochemical Engineering, Chair of Chemical Biotechnology, TU Dortmund, Dortmund 44221, Germany.
authors.forename: Reto
authors.initials: R
authors.lastname: Ruinatscha
authors.forename: Rohan
authors.initials: R
authors.lastname: Karande
authors.forename: Katja
authors.initials: K
authors.lastname: Buehler
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
mesh_terms: D000837:Anion Exchange Resins; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D010105:Oxygenases / Q000737:chemistry*
chemical_list: D000837:Anion Exchange Resins; D004800:Enzymes, Immobilized; D010105:Oxygenases; C026721:styrene monooxygenase