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Integrated One-Pot Enrichment and Immobilization of Styrene Monooxygenase (StyA) Using SEPABEAD EC-EA and EC-Q1A Anion-Exchange Carriers

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Author(s): Reto Ruinatscha | Rohan Karande | Katja Buehler | Andreas Schmid

Journal: Molecules
ISSN 1420-3049

Volume: 16;
Issue: 7;
Start page: 5975;
Date: 2011;
Original page

Keywords: enzyme purification | enzyme immobilization | styrene monooxygenase | Sepabeads

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 mgStyA−1) as compared to Sepabeads EC-EA. This activity could be increased to 0.7 U mgStyA−1 by co-immobilizing StyB on Sepabeads EC-Q1A, which corresponds to 33% of the soluble StyA activity.

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