Next Article in Journal
Three New Phytoecdysteroids Containing a Furan Ring from the Roots of Achyranthes bidentata Bl.
Next Article in Special Issue
Enzyme-Immobilized Microfluidic Process Reactors
Previous Article in Journal
Synthetic Organic Electrochemistry in Ionic Liquids: The Viscosity Question
Previous Article in Special Issue
Biocatalytic Resolution of Enantiomeric Mixtures of 1-Aminoethanephosphonic Acid
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

, ,  and *
Department of Chemical and Biochemical Engineering, Chair of Chemical Biotechnology, TU Dortmund, Dortmund 44221, Germany
* Author to whom correspondence should be addressed.
Received: 23 June 2011 / Accepted: 12 July 2011 / Published: 18 July 2011
(This article belongs to the Special Issue Enzyme-Catalyzed Reactions)
Download PDF [549 KB, uploaded 18 June 2014]

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 (CC BY 3.0).
SciFeed

Share & Cite This Article

Further Mendeley | CiteULike
Export to BibTeX |
EndNote |
RIS
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.

View more citation formats

Related Articles

Article Metrics

For more information on the journal, click here

Comments

[Return to top]
Molecules EISSN 1420-3049 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert