Keith E. LeJeune

Nerve agents degraded by enzymatic foams

The decontamination of large areas that have been exposed to chemical weapons is a critical component of defence and anti-terrorist strategies around the world. Until now, nerve agents could be decontaminated only with bleach treatment and/or ex situ incineration, which had severe environmental consequences. Small-scale methods of decontamination and demilitarization are available, but the problem of decontamination over a wide area has not been solved,. Here we incorporate organophosphorus hydrolase (OPH), an enzyme that hydrolyses nerve agents, into aqueous fire-fighting foams to catalyse surface decontamination. The performance of enzyme-containing foams is reproducible and predictable in detoxifying organophosphorus nerve toxins deposited onto surfaces. Such foams are an environmentally friendly alternative to current decontamination solutions, which are nonspecific in their action and contain significant amounts of hazardous organic solvents.

Dramatically stabilized phosphotriesterase—polymers for nerve agent degradation

Phosphotriesterase (EC 3.1.8.1) was immobilized within a polyurethane foam matrix during polymer synthesis using a prepolymer synthesis strategy. In addition to retaining greater than 50% of the enzyme specific activity, numerous benefits were incurred upon immobilization. Orders of magnitude increases in storage and thermal stability (net stabilization energy = 12.5 kJ/mol) were observed without the need for enzyme premodification. The immobilized enzyme system was protease resistant and seemed to display no adverse effects from immobilization, such as an alteration of enzyme function. The organic solvent, dimethyl sulfoxide, also exhibited a stabilizing effect on phosphotriesterase enzyme systems over a range of intermediate concentrations. We attribute these effects in part to direct interaction between the aprotic solvent and metal containing residues present at the enzymes active site. Our data demonstrate that just 2.5 kg of immobilized enzyme may be sufficient to degrade 30,000 tons of nerve agent in just 1 year.

Covalent binding of a nerve agent hydrolyzing enzyme within polyurethane foams.

A phosphotriesterase preparation, extracted from Escherichia coli DH5α cells, was immobilized within a polyurethane foam matrix during polymer synthesis. The enzyme‐foam interaction was shown to be covalent and analysis of the hydrolysis of paraoxon in aqueous solution demonstrated that more than 50% of the initial enzyme specific activity was retained after immobilization in the foam. Factors affecting the rate of paraoxon degradation include foam hydrophobicity, the degree of mixing applied to initiate polymerization, and foam pretreatment prior to use in substrate hydrolysis. The storage stability of the foam is significant, with phosphotriesterase‐foam activity profiles exhibiting a three month half‐life. Foams are currently being developed for biocatalytic air filtering, in which gaseous substrates will be simultaneously adsorbed and degraded by the immobilized enzyme system.

Organophosphate skin decontamination using immobilized enzymes

We previously demonstrated that a combination of cholinesterase (ChE) pre-treatment with an oxime is an effective measure against soman and sarin. We describe here a novel approach for the preparation of covalently linked ChEs which are immobilized to a polyurethane matrix. Such preparation of ChE-sponges enhances the stability and usefulness of the enzymes in non-physiological environments. The ChE-sponges, which can be molded to any form, can effectively be used to remove and decontaminate organophosphates (OPs) from surfaces, biological (skin or wounds) or otherwise (clothing or sensitive medical equipment), or the environment. The ChE-sponges retained their catalytic activity under conditions of temperature, time, and drying where the native soluble enzyme would rapidly denature, and can be reused in conjunction with oximes many times. The ChE-sponge in the presence of oxime repeatedly detoxified OPs such as DFP or MEPQ. These developments in ChE technology have extended the applicability of OP scavengers from in vivo protection, to a variety of external detoxification and decontamination schemes. In addition to treatment of OP-contaminated soldiers, the ChE-sponge could protect medical personnel from secondary contamination while attending chemical casualties, and civilians exposed to pesticides or highly toxic nerve agents such as sarin.

Fighting Nerve Agent Chemical Weapons with Enzyme Technology

Abstract: The extreme toxicity of organophosphorous‐based compounds has been known since the late 1930s. Starting in the mid‐1940s, many nations throughout the world have been producing large quantities of organophosphorous (OP) nerve agents. Huge stockpiles of nerve agents have since developed. There are reportedly more than 200,000 tons of nerve agents in existence worldwide. There is an obvious need for protective clothing capable of guarding an individual from exposure to OP chemical weapons. Also, chemical processes that can effectively demilitarize and detoxify stored nerve agents are in great demand. The new and widely publicized Chemical Weapons Treaty requires such processes to soon be in place throughout the world. Biotechnology may provide the tools necessary to make such processes not only possible, but quite efficient in reducing the nerve agent dilemma.

Increasing the tolerance of organophosphorus hydrolase to bleach

Organophosphorus hydrolase (OPH) has been incorporated within polyurethane foams during polymer synthesis as a means of reducing the enzymes environmental sensitivity to alterations in pH and bleach-induced enzyme denaturation. Unfavorable losses of enzyme activity upon altered pH are reduced by covalently incorporating OPH within polyurethane matrices. Also, the stability of the immobilized enzyme under alkaline conditions is significantly enhanced. The bleach compatibility of OPH is also increased upon enzyme polymerization. Although a fraction of the increased bleach compatibility results from polyurethane oxidation, the covalent linkages between OPH and polyurethane directly enhance enzyme stability in buffered solutions of calcium hypochlorite bleach. Copyright 1999 John Wiley & Sons, Inc.

Enzyme-polymer based environmental monitors

Enzymes are commonly used as the active element in chemical sensors because of their analyte specificity, sensitivity, and the speed with which they catalyze reactions. Their precision and reliability has them at the core of many FDAapproved medical diagnostic tests. Unfortunately, nature has evolved most enzymes to operate under a fairly narrow range of storage and operating conditions (i.e. pH, ionic strength, temperature, organic content, etc). The deployment of enzyme-based sensors in poorly controlled environments with fluctuating conditions can therefore be difficult. ICx Technologies has sought to minimize the impact of environmental parameters on enzyme catalysis through enzyme polymerization. Rather than being simply immobilized onto an existing substrate, enzymes are used as co-monomers with other conventional monomers in polymerization reactions. Enzymes are incorporated within the polymer through multiple covalent attachments. By essentially anchoring the enzymes tertiary structure, the polymerization process reduces enzyme sensitivity to many environmental factors. ICx has built a number of chemical sensors using enzyme polymers, some of which continuously monitor air or water in real time. The developed sensors have proven to operate well in many different environments.

Characterization of ChEs Immobilized on Polyurethane Foams

FBS-AChE and Equine-BChE were immobilized by covalently linking the enzymes to polyurethane foams (PUF), having the consistency of sponges. The enzymes attach to the inert foam at multiple points dependent upon the available free aliphatic amines (lysine and arginine residues) available on their surface. Based on molecular modeling, there are at least 1 Lys and 29 Arg water accessible residues on the surface of FBS-AChE, while 26 Lys and 26 Arg residues were found for Equine-BChE (truncated after residue 537). The majority of the Lys and Arg residues were found on the backside of the ChEs, although a few are close to the rim of the gorge. The covalently-bound ChEs were characterized and compared to their soluble counterparts. The reactions were monitored in a temperature controlled cuvette containing a stir bar and a small portion of sponge to be assayed. This is a two-phase system: Ellman components (acetylthiocholine for AChE and butyrylthiocholine for BChE) in the aqueous phase and the ChE-immobilized products in the solid phase. There was no apparent adsorption of the final reaction product of the Ellman assay on PUF lacking ChE (no added protein) or PUF synthesized with only bovine serum albumin.