Degradation of organophosphorous nerve agents by enzyme-polymer nanocomposites: efficient biocatalytic materials for personal protection and large-scale detoxification

The biocatalytic destruction of organophosphates has become an important focus area, as efficient "clean" technologies are sought for chemical weapons decommissioning, counteracting nerve agent attacks, and protecting against organophosphate pesticide poisoning. A novel method is advanced for immobilizing the broad-spectrum enzyme organophosphorous hydrolase (OPH) from Pseudomonas diminuta, based on the formation of nanocomposite protein-silicone polymers. The resulting materials are highly active, stable, and versatile biocatalysts for the liquid and gas phase detoxification of organophosphates, and can be fabricated as monoliths, sheets, thick films, granulates, or macroporous foams. This approach offers an efficient avenue to robust, high-performance biocatalytic OPH-containing polymers that outperform immobilized OPH catalysts reported to date. The method provides for the first time a route to biocatalytic materials that may be suitable for "active" protective wear, as well as bulk catalysts for the destruction of large volumes of organophosphates. The preparation of OPH-silicone biocomposites, their performances in the liquid and gas phase detoxification of paraoxon, dichlorvos, and diisopropyl fluorophosphate, and their features are discussed.     

Bionanoconjugate‐based composites for decontamination of nerve agents

We have developed enzyme‐based composites that rapidly and effectively detoxify simulants of V‐ and G‐type chemical warfare nerve agents. The approach was based on the efficient immobilization of organophosphorus hydrolase onto carbon nanotubes to form active and stable conjugates that were easily entrapped in commercially available paints. The resulting catalytic‐based composites showed no enzyme leaching and rendered >99% decontamination of 10 g/m² paraoxon, a simulant of the V‐type nerve agent, in 30 minutes and >95% decontamination of diisopropylfluorophosphate, a simulant of G‐type nerve agent, in 45 minutes. The formulations are expected to be environmentally friendly and to offer an easy to use, on demand, decontamination alternative to chemical approaches for sustainable material self‐decontamination. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Activity and batch operational stability of Candida rugosa lipase immobilized in different hydrophilic polyurethane foams during hydrolysis in a biphasic medium

The aim of this study was to investigate eventual relationships between some physico-chemical properties (e.g. porosity, aquaphilicity, partition coefficient for oleic acid and drying curves) of relatively hydrophilic polyurethane foams and the activity and batch operational stabiliy of Candida rugosa lipase immobilized within these foams. Two biocompatible polyurethane pre-polymers ("HYPOL FHP 2002^TM" and "Hypol FHP X4300^TM" from Hampshire Chemical GmbH, Germany) were tested as immobilization supports. The model reaction was the hydrolysis of crude olive residue oil in a biphasic aqueous/n-hexane medium. Drying curves under normal and reduced pressures suggested that water molecules are more strongly bound to the "FHP 2002" than to "FHP X4300" foams. This is in agreement with the higher aquaphilicity value estimated for the "FHP 2002" foam (3.7 vs 2.8). For every enzyme loading tested, hydrolysis efficiency was considerably higher for the lipase in "FHP X4300" foam when compared to the other counterpart. However, internal mass transfer limitations seem to be more severe with "FHP X4300" foams. Operational stability was evaluated in 10 consecutive batches (1 batch = 23 hours) for both immobilized preparations. A fast deactivation was observed for both biocatalysts. However, a slightly higher operational stability was observed for the lipase in "FHP 2002" foam. For the lipase in "FHP X4300" foam, the activity decay can be explained by a dramatic lipase leakage from the foam observed along successive batches. For the lipase in "FHP 2002" foam, no significant enzyme loss was observed along the reutilizations probably due to a higher number of multi-point attachment between the lipase and its support. In fact, activity and operational stability of Candida rugosa lipase in "FHP 2002" and "FHP X4300" foams appear to be related with the strength and/or the number of covalent binding between the enzyme and the support rather than to the physico-chemical properties evaluated in this work.     

Microbial degradation of organophosphorus compounds

Synthetic organophosphorus compounds are used as pesticides, plasticizers, air fuel ingredients and chemical warfare agents. Organophosphorus compounds are the most widely used insecticides, accounting for an estimated 34% of world-wide insecticide sales. Contamination of soil from pesticides as a result of their bulk handling at the farmyard or following application in the field or accidental release may lead occasionally to contamination of surface and ground water. Several reports suggest that a wide range of water and terrestrial ecosystems may be contaminated with organophosphorus compounds. These compounds possess high mammalian toxicity and it is therefore essential to remove them from the environments. In addition, about 200,000 metric tons of nerve (chemical warfare) agents have to be destroyed world-wide under Chemical Weapons Convention (1993). Bioremediation can offer an efficient and cheap option for decontamination of polluted ecosystems and destruction of nerve agents. The first micro-organism that could degrade organophosphorus compounds was isolated in 1973 and identified as Flavobacterium sp. Since then several bacterial and a few fungal species have been isolated which can degrade a wide range of organophosphorus compounds in liquid cultures and soil systems. The biochemistry of organophosphorus compound degradation by most of the bacteria seems to be identical, in which a structurally similar enzyme called organophosphate hydrolase or phosphotriesterase catalyzes the first step of the degradation. organophosphate hydrolase encoding gene opd (organophosphate degrading) gene has been isolated from geographically different regions and taxonomically different species. This gene has been sequenced, cloned in different organisms, and altered for better activity and stability. Recently, genes with similar function but different sequences have also been isolated and characterized. Engineered microorganisms have been tested for their ability to degrade different organophosphorus pollutants, including nerve agents. In this article, we review and propose pathways for degradation of some organophosphorus compounds by microorganisms. Isolation, characterization, utilization and manipulation of the major detoxifying enzymes and the molecular basis of degradation are discussed. The major achievements and technological advancements towards bioremediation of organophosphorus compounds, limitations of available technologies and future challenge are also discussed.     

A cloned bacterial enzyme for nerve agent decontamination

Organophosphorus acid (OPA) anhydrolases offer considerable potential for safe, non-corrosive decontamination of chemical nerve agents. The Alteromonas sp. strain JD6.5 gene encoding an OPA anhydrolase (designated as OPAA-2), which hydrolyzes a wide variety of nerve agents, has been cloned in Escherichia coli. Employing agent-analog diisopropyl fluorophosphate (DFP) as a substrate, the effects of buffers, pH, temperature, and various protein stabilizing agents on OPAA-2 activity were studied. Ammonium carbonate, which is innocuous and inexpensive, proved to be a superior buffer for enzyme activity. Compared with enzyme assayed under standard conditions, enzyme activity with ammonium carbonate was six-fold greater. To evaluate effects of storage and reconstitution on enzyme activity, the cloned enzyme was lyophilized, rehydrated, and then assessed by measuring activity against DFP. Whereas almost 100% of the hydrolytic activity was recovered with enzyme reconstituted in (NH₄)₂CO₃-buffered distilled water or chlorinated drinking water, approximately 20% of the activity was recovered with ocean water. Enzyme stability in blast-containment foam or fire-fighting foam was also demonstrated by high activity in (NH₄)₂CO₃-buffered distilled water or drinking water. These findings suggest the potential of a foam-based enzyme system for field decontamination of chemical nerve agents.     

Crystal structures of human carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun

The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective, and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 A resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 10(4)-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure.     

The binding of substrate analogs to phosphotriesterase

Phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the detoxification of organophosphates such as the widely utilized insecticide paraoxon and the chemical warfare agent sarin. The three-dimensional structure of the enzyme is known from high resolution x-ray crystallographic analyses. Each subunit of the homodimer folds into a so-called TIM barrel, with eight strands of parallel beta-sheet. The two zinc ions required for activity are positioned at the C-terminal portion of the beta-barrel. Here, we describe the three-dimensional structure of PTE complexed with the inhibitor diisopropyl methyl phosphonate, which serves as a mimic for sarin. Additionally, the structure of the enzyme complexed with triethyl phosphate is also presented. In the case of the PTE-diisopropyl methyl phosphonate complex, the phosphoryl oxygen of the inhibitor coordinates to the more solvent-exposed zinc ion (2.5 A), thereby lending support to the presumed catalytic mechanism involving metal coordination of the substrate. In the PTE-triethyl phosphate complex, the phosphoryl oxygen of the inhibitor is positioned at 3.4 A from the more solvent-exposed zinc ion. The two structures described in this report provide additional molecular understanding for the ability of this remarkable enzyme to hydrolyze such a wide range of organophosphorus substrates.     

Enzymes hydrolyzing organophosphates as potential catalytic scavengers against organophosphate poisoning

Enzymes hydrolyzing organophosphates could be used as catalytic scavengers for treatment of organophosphate poisoning and for decontamination. Two organophosphorus hydrolases (OPH) were selected: the Flavobacterium sp./Pseudomonas diminuta phosphotriesterase (PTE) and human paraoxonase (HuPON). Genes encoding these enzymes were cloned and functional recombinant enzymes expressed. PTE was expressed in E. coli. Natural HuPON was purified from human plasma; recombinant HuPON was expressed in human embryonic kidney 293 T cells. Although HuPON displays interesting catalytic properties, a site-directed mutagenesis program was undertaken to improve its catalytic efficiency. PTE has high efficiency in hydrolysis of organophosphates, including nerve agents. PTE injected in rat has a half-life of 100 min. However, to overcome pharmacokinetic problems of injected OPH and/or immunological incompatibility, the model enzyme (recombinant PTE) was immobilized onto a hollow-fiber reactor. This reactor designed for extracorporeal blood circulation is under experimentation for post-exposure detoxification.     

Structural basis of heroin and cocaine metabolism by a promiscuous human drug-processing enzyme

We present the first crystal structures of a human protein bound to analogs of cocaine and heroin. Human carboxylesterase 1 (hCE1) is a broad-spectrum bioscavenger that catalyzes the hydrolysis of heroin and cocaine, and the detoxification of organophosphate chemical weapons, such as sarin, soman and tabun. Crystal structures of the hCE1 glycoprotein in complex with the cocaine analog homatropine and the heroin analog naloxone provide explicit details about narcotic metabolism in humans. The hCE1 active site contains both specific and promiscuous compartments, which enable the enzyme to act on structurally distinct chemicals. A selective surface ligand-binding site regulates the trimer-hexamer equilibrium of hCE1 and allows each hCE1 monomer to bind two narcotic molecules simultaneously. The bioscavenger properties of hCE1 can likely be used to treat both narcotic overdose and chemical weapon exposure.     

Bioremediation of organophosphorus pesticides by surface-expressed carboxylesterase from mosquito on Escherichia coli

The insecticide resistance-associated esterase, carboxylesterase B1 (CaE B1), from mosquito was used to degrade the organophosphorus compounds. To eradicate the need for enzyme purification and minimize the resistance to mass transport of the substrate and product across the cell membranes, the CaE B1 was displayed on the cell surface of Escherichia coli fused to the C-terminus of the ice nucleation protein (INP). The presence of CaE B1 on the bacterial cell surface was verified by SDS-PAGE, Western blotting analysis, and immunofluorescence microscopy. More than 50% of active CaE B1 is exported across the membrane and anchored onto the cell surface as determined by proteinase accessibility and cell fractionation experiments. In contrast, only a 6% drop in activity for proteinase K-treated cells was detected from E.coli cells containing pET-B1. From the degradation experiment, more than 80% of the malathion was degraded by whole cells containing plasmid pUC-NC-B1. Constitutive expression of CaE B1 on the surface using INPNC resulted in no cell lysis, and the suspended cultures also exhibited good stability. Because of their high biodegradation activity and superior stability, these "live biocatalysts" are promising for detoxification of organophosphorus pesticides.     

Structure and properties of starch-based foams prepared by microwave heating from extruded pellets

The physical properties of microwave-foamed starch-based pellets, including density, porosity, cell structure, water absorption characteristics and mechanical properties were characterized. It was found that the physical properties of these starch-based foams produced by microwave heating are highly dependent on the raw materials and additives. Foam density decreased significantly after addition of 5.5–10.5% w/w salts, while foams containing nucleation agent (talc) were denser than the control with reduced cell size. A proprietary blowing agent did not affect the foam density markedly. Addition of salts also increased the water sorption of foams and plasticized cell walls. Mechanical behaviour of foamed pellets can be adjusted effectively by controlling the cell structure through using different additives. Mechanical properties of the foamed pellets in the elastic region as well as under large deformation (up to 40% strain) all follow a power–law relationship with foam density.     

Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures

Prolidase isolated from the hyperthermophilic archaeon Pyrococcus furiosus has potential for application for decontamination of organophosphorus compounds in certain pesticides and chemical warfare agents under harsh conditions. However, current applications that use an enzyme-based cocktail are limited by poor long-term enzyme stability and low reactivity over a broad range of temperatures. To obtain a better enzyme for OP nerve agent decontamination and to investigate structural factors that influence protein thermostability and thermoactivity, randomly mutated P. furiosus prolidases were prepared by using XL1-red-based mutagenesis and error-prone PCR. An Escherichia coli strain JD1 (λDE3) (auxotrophic for proline [ΔproA] and having deletions in pepQ and pepP dipeptidases with specificity for proline-containing dipeptides) was constructed for screening mutant P. furiosus prolidase expression plasmids. JD1 (λDE3) cells were transformed with mutated prolidase expression plasmids and plated on minimal media supplemented with 50 μM Leu-Pro as the only source of proline. By using this positive selection, Pyrococcus prolidase mutants with improved activity over a broader range of temperatures were isolated. The activities of the mutants over a broad temperature range were measured for both Xaa-Pro dipeptides and OP nerve agents, and the thermoactivity and thermostability of the mutants were determined.     

Engineering and introduction of <Emphasis Type="Italic">de novo</Emphasis> disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement

The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the most frequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has been shown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineering of de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rational method of protein engineering. In this study, Disulphide by Design software, homology modelling and molecular dynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge to improve protein thermostability. The thermostability of the wild-type and three selected mutant enzymes were evaluated by half-life, ΔG inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Data analysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min, respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124C mutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results were correlated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsic fluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi also increased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results were submitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing a disulphide bridge after bioinformatics design, even though this design would not be always successful.     

Aquatic macroinvertebrate biodiversity associated with artificial agricultural drainage ditches

The formation of foams on lakes is a complex phenomenon whose origin is often hardly identifiable. Recently (2007, 2008, and 2010) foam episodes started to occur in Lake Maggiore, northern Italy. The present work aimed to verify the hypothesis of an endogenous-natural origin of these foams, driven by trophic or climatic changes. To this purpose, a long-term (2000–2013) analysis of phytoplankton biovolumes, meteorological, and hydrological data has been performed together with the chemical characterization of foams. Foams resulted of endogenous origin and likely related to phytoplankton biomass degradation. Data analysis highlighted atypical warm temperature and residual lake stratification in winter in two of the three years of foam events, coupled with exceptional Bacyllariophyceae blooms in spring. Tabellaria flocculosa mostly contributed in terms of biomass in 2007 and 2008, but not in 2010; thus overall algal biomass seemed a better predictor of the risk of foam formation. Foam events occurred from July to December, driven by atypically windy conditions, and congruently with the time needed to degrade biomass into surface-active compounds. A co-occurrence of different factors resulted essential to generate foams, and climate changes likely contribute to enhance their occurrence in Lake Maggiore.     

Mechanical properties of polyurethane foams prepared from liquefied corn stover with PAPI

Corn stover was liquefied by using ethylene carbonate (EC) as liquefying solvent and 97% sulfur acid as catalyst at 170 degrees C for 90 min. Polyurethane (PU) foams were prepared from liquefied corn stover (LCS) with variable amount of polymethylene polyphenylisocyanate (PAPI) by one-shot method, with water as blowing agent, silicone as surfactant and triethylamine and dibutyltine dilaurate as co-catalyst. The mechanical properties of LCS-PU foam with different [NCO]/[OH] ratio were studied on a universal tensile tester. With the increase of [NCO]/[OH] ratio from 0.4 to 1.0, the tensile strength and Young's modulus of the LCS-PU foam first raised, reached their maximum values at [NCO]/[OH] ratio of 0.8, and then declined; while the elongation at break decreased from 117% to 3.6%. The results indicated that by changing the [NCO]/[OH] ratio, mechanical properties of LCS-PU foams could be adjusted for various end uses.     

Preparation of crosslinked macroporous PVA foam carrier for immobilization of microorganisms

In this work, a new and economical way to prepare macroporous poly(vinyl alcohol) (PVA) foam was explored by adding calcium carbonate as a pore-forming agent and using epichlorhydrin as a chemical crosslinking agent to improve foam stability. The mixture for foam formation has been optimized to obtain macroporous PVA foam carriers with uniform apertures, narrow distribution of pore sizes, and good elasticity. The crosslinked PVA foam (CPVAF) carrier demonstrated better chemical and thermal stability, as well as larger specific surface area and diffusion coefficients than the traditional PVA (TPVA) carrier. Nitrifying bacteria were used to test the suitability of CPVAF and TPVA carriers for immobilized microorganisms. CPVAF carriers supported higher biomass density and microbial activity than TPVA carriers. At the same biomass density, the higher nitrification rate of CPVAF carriers was attributed to excellent mass transfer of the substrate (and oxygen) between the bulk solution and the immobilized microorganisms.     

Structural properties of starch-chitosan-gelatin foams and the impact of gelatin on MC3T3 mouse osteoblast cell viability

Background

This study examines the effects of adding gelatin to a starch-chitosan composite foam, focusing on the altered structural and biological properties. The compressive modulus of foams containing different gelatin concentrations was tested in dry, wet, and lyophilized states. MC3T3 mouse osteoblast cells were used to test the composite’s ability to support cell growth. The stability of the foams in α-MEM culture media with and without cells was also examined.

Results

It was found that for dry foams, the compressive modulus increased with increasing gelatin content. For foams tested in wet and lyophilized states, the compressive modulus peaked at a gelatin concentration of 2.5% and 5%, respectively. The growth of MC3T3 mouse osteoblast cells was tested on the foams with different gelatin concentrations. The addition of gelatin had a positive effect on the cell growth and proliferation.

Conclusion

The composite foam containing gelatin improved cell growth and is only dissolved by the growing cells at a rate influenced by the initial concentration of gelatin added to the foam.

     

The foams of fermentation broths. I. Some parameters of the foaming of fermentation media

The stability of foam formed during fermentation is decisively affect ed by the nature of the nutrient media used. In froth-flotation models, (a) the foam formation time, characteristic of the tendency to foam, and (b) foam subsistence time, characteristic of the stability of foams formed, have been studied. With the utilization of these two parameters, the stability of foam from aqueous solution of several surface active components of nutrient media has been noted as a function of concentrations. Further, but, without attempting completeness, the viscosity enhancing effect of carbohydrate components, and the effect of the subsistence time of their foam, upon the stability of foam have been studied together with the correlation between “standing” time after sterilization and tendency to foam. Taking soy-bean meal as a model, the stability of foam films in function of pi I, at constant concentration, has been studied. It seems that though a proper control of the factors mentioned, nutrient media with a low tendency of foaming can be formulated.     

Determination of the efficacy of two building decontamination strategies by surface sampling with culture and quantitative PCR analysis

The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus ("Bacillus subtilis subsp. niger," also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 10(5) to 10(6) CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.     

The Role of Copper in Protein Foams

Chefs have known that whipping egg white proteins (EWP) in a copper bowl will improve foam stability. The improved stability is attributed to a copper–conalbumin complex or alteration of sulfhydryl reactivity. Whey proteins bind copper and show copper-induced changes in disulfide bonds; therefore, they may also be responsive to whipping in a copper bowl. EWP and whey protein isolate (WPI) solutions were whipped in the presence of 1 mM CuSO₄ or in a copper bowl with and without sugar followed by overrun and yield stress measurements and angel food cake formation. Dilational elasticity and surface tension were also measured for WPI solutions. Whipping in a copper bowl or adding 1 mM CuSO₄ significantly improved stability of EWP foams while having no effect on WPI foams. Copper caused disulfide-linked dimer formation of β-lactoglobulin and decreased dilational elasticity and surface tension, but these modifications were insufficient to change the bulk properties of foams. The addition of 10 mM CuSO₄ to WPI solutions was sufficient to increase foam stability to levels similar to EWP; however, the more stable foams formed less stable cakes. It was concluded that the effect of whipping in a copper bowl on foam properties is mainly dependent on the specific proteins forming the foam. Paper no. FSR-07-29 of the Journal Series of the Department of Food Science, North Carolina State University, Raleigh, NC 27695-7624. Presented at the 2nd International Symposium: Delivery of Functionality in Complex Food Systems: Physically-inspired Approaches From Nanoscale to Microscale, October 8–10, 2007.