Orientation Specific Positioning of Organophosphorus Hydrolase on Solid Interfaces for Biosensor Applications

Mechanical Engineering Department, 275 Wilmore Laboratories, Auburn University, Auburn, Alabama 36849, USA.
Langmuir (Impact Factor: 4.46). 09/2009; 25(16):9615-8. DOI: 10.1021/la9007526
Source: PubMed


Protein immobilization on solid interfaces is a crucial aspect of their successful application in technologies such as biosensing, purification, separation, decontamination, etc. Although immobilization can improve the long-term and operational stability of proteins, this is often at the cost of significant losses in the catalytic activity of the tethered enzyme. Covalent attachment methods take advantage of reactive groups on the amino acid side chains. The distribution of the solvent exposed side chains on an enzyme's molecular surface often results in an ensemble of orientations when the protein is immobilized on a surface or in a matrix through these side chain linkages. Depending on the attachment mechanism and resulting orientation, access to and from the active site could be restricted. This study describes a methodology for the design and implementation of an orientation specific attachment of an enzyme to a surface plasmon resonance sensor surface. The enzyme, organophosphorus hydrolase, was structurally analyzed to identify surface resides as candidates for modification to optimize active site accessibility and, thus, sensitivity of detection. A single surface lysine on the active site face of the enzyme dimer was selected for elimination, thus allowing for the immobilization of the catalyst in the preferred orientation. Kinetic evaluation of the enzymes determined that the surface lysine-to-alanine variant retained 80% of the wild-type activity with the neurotoxin substrates, paraoxon and demeton-S. After immobilization, surfaces bearing the variant were determined to be more active even though the enzyme coverage on the sensor surface was reduced by 17%.

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    ABSTRACT: IntroductionSynthesis and Characterization of Biosensor SupportsApplication of Enzyme-Based BiosensorsConclusions AcknowledgmentsReferences
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