GFP‐visualized immobilized enzymes: Degradation of paraoxon via organophosphorus hydrolase in a packed column

Abstract

A versatile gene-fusion technique for immobilizing and visualizing biologically active enzymes which includes from the N to C-termini, an affinity histidine tag, the green fluorescent protein (GFP), a proteolytic enzyme (enterokinase, EK) cleavage site and the enzyme of interest, were developed. Specifically, the organophosphorus hydrolase was bound to the affinity (His(6))-reporter(GFP)-EK fusion elements. Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. In the case of immobilized OPH, paraoxon was rapidly degraded when pumped through a packed column. In reaction mixtures containing CHES buffer at pH 6.9, a continual decay in OPH activity was observed and importantly, this was monitored by GFP fluorescence. This decay in activity was fully restored, along with fluorescence, upon washing with PBS buffer. Many subsequent experiments were performed at varied pH and in different background buffer solutions. In all cases when there was OPH activity there was also marked fluorescence from the GFP fusion partner. Likewise, when OPH activity was lost, so was GFP fluorescence and, importantly, both were regenerated when washed in the presence of the kosmotropic salt, phosphate. Recently, Waldo et al. (1999) showed that GFP fluorescence from whole cells indicated the extent of proper folding of normally aggregated proteins designed via directed evolution. The present work demonstrates an application wherein GFP fluorescence indicates stability and activity of its fusion partner.

Dried–Reswollen Immobilized Biocatalysts for Detoxification of Organophosphorous Compounds in the Flow Systems

“…The comparison of catalytic efficiency of the biocatalysts obtained via our method to the known analogs produced by other methods [7,[11][12] revealed the obvious advantages of our chosen immobilization technique and its components ( Table 1). The comparison of catalytic activities of known immobilized biocatalysts, based on OPH with genetically introduced affinity tags (440 U for OPH/GFP-OPH [14] and 0.0025 U for ELP-OPH [15]) to the same parameters of newly developed preparation (660 U) revealed the latter one's advantage.…”

“…The use of various amino acid sequences genetically introduced into OPH structure as ligands for enzyme immobilization proved to be an advanced and very effective method for biocatalyst production [14][15]. The use of specific amino acid sequences allows for combining the isolation, purification, and immobilization of the target protein on a carrier into a single step.…”

Functional Assembly of a Microbial Consortium with Autofluorescent and Mineralizing Activity for the Biodegradation of Organophosphates

“…GFP was used as a marker for expression of OPH in E. coli and later used as a fusion partner for the visualized degradation of paraoxon in a packed column (38)(39)(40). In this work, GFP was coexpressed with MPH in E. coli, and the fluorescence intensity in the coexpression system was 60-70% of that expressing EGFP alone.…”

Removal of neurotoxic ethyl parathion pesticide by two-stage chemical/enzymatic treatment system using Fenton’s reagent and organophosphorous hydrolase

“…is a homodimeric organophosphotriesterase that degrades a broad spectrum of neurotoxic OPs [15,16]. The use of OPH in bioremediation is of great interest due to its high turnover rate [17][18][19][20][21]. The OPH-expressing microorganism itself was also attempted to treat OPs as whole cell catalyst, but mass transport problems were not effectively solved yet [17,19,20].…”