%0 Journal Article
%T Integrated One-Pot Enrichment and Immobilization of Styrene Monooxygenase (StyA) Using SEPABEAD EC-EA and EC-Q1A Anion-Exchange Carriers
%A Reto Ruinatscha
%A Rohan Karande
%A Katja Buehler
%A Andreas Schmid
%J Molecules
%D 2011
%I MDPI AG
%R 10.3390/molecules16075975
%X 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.
%K enzyme purification
%K enzyme immobilization
%K styrene monooxygenase
%K Sepabeads
%U http://www.mdpi.com/1420-3049/16/7/5975