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

Abstract

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%.

Cytochrome c covalently immobilized on mesoporous silicas as a peroxidase: Orientation effect

“…The orientation of the enzyme in the constricted pore space is important because the substrate's access to the active center of the enzyme is often through narrow pockets along certain directions. 17 Side-chainspecific chemical modification certainly can control to some extent the orientation of the enzyme. 18,19 Finally, selected covalent attachment of enzymes at certain local points can constitute a local perturbation of the protein structure which may be reflected in distortion of the environment near the active center.…”

A comparison of organophosphate degradation genes and bioremediation applications

“…Other avenues of potential research include the use of microbes and their assortment of OP degradative enzymes as components in biosensors to determine the level of contamination on site and therapeutically in the form of an encapsulated prophylactic to be administered in cases of OP poisonings or upon exposure to chemical agents (Mulchandani et al, 2001;Reeves et al, 2009;Petrikovics et al, 2012).…”

Designed Amphiphilic β-Sheet Peptides as Templates for Paraoxon Adsorption and Detection

“…The pesticide paraoxon is a commonly accepted model of organophosphate compounds, despite being less toxic and volatile than many other OPs. − Although paraoxon can be detected by various chromatographic techniques, − these are commonly time-consuming and require laboratory access. Biosensors based on AChE and organophosphate hydrolase (OPH) have been shown to detect OPs. − These enzyme-based biosensors are, however, limited by sensitivity, a loss of activity during enzyme immobilization, and the need to engineer the system to shorten the distance between the active site of the enzyme and the transducer device to which it is attached . For these reasons, there has been interest in devising biomimetic approaches using designed molecules, engineered to mimic enzyme activity.…”