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Molecules 2011, 16(7), 5975-5988; doi:10.3390/molecules16075975
Article

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

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Received: 23 June 2011; Accepted: 12 July 2011 / Published: 18 July 2011
(This article belongs to the Special Issue Enzyme-Catalyzed Reactions)
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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.
Keywords: enzyme purification; enzyme immobilization; styrene monooxygenase; Sepabeads enzyme purification; enzyme immobilization; styrene monooxygenase; Sepabeads
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Ruinatscha, R.; Karande, R.; Buehler, K.; Schmid, A. Integrated One-Pot Enrichment and Immobilization of Styrene Monooxygenase (StyA) Using SEPABEAD EC-EA and EC-Q1A Anion-Exchange Carriers. Molecules 2011, 16, 5975-5988.

AMA Style

Ruinatscha R, Karande R, Buehler K, Schmid A. Integrated One-Pot Enrichment and Immobilization of Styrene Monooxygenase (StyA) Using SEPABEAD EC-EA and EC-Q1A Anion-Exchange Carriers. Molecules. 2011; 16(7):5975-5988.

Chicago/Turabian Style

Ruinatscha, Reto; Karande, Rohan; Buehler, Katja; Schmid, Andreas. 2011. "Integrated One-Pot Enrichment and Immobilization of Styrene Monooxygenase (StyA) Using SEPABEAD EC-EA and EC-Q1A Anion-Exchange Carriers." Molecules 16, no. 7: 5975-5988.



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