Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase

A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents.     

Specific Adhesion to Cellulose and Hydrolysis of Organophosphate Nerve Agents by a Genetically Engineered Escherichia coli Strain with a Surface-Expressed Cellulose-Binding Domain and Organophosphorus Hydrolase

A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents.     

Generation of a mutagenized organophosphorus hydrolase for the biodegradation of the organophosphate pesticides malathion and demeton‐S

Aims: The bacterial organophosphorus hydrolase (OPH) enzyme hydrolyses and detoxifies a broad range of toxic organophosphate pesticides and warfare nerve agents by cleaving the various phosphorus‐ester bonds (P–O, P–F, P–CN, P–S); however, OPH hydrolyses these bonds with varying efficiencies. The aim of this study was to generate a variant OPH enzyme with improved hydrolytic efficiency against the poorly hydrolysed P–S class of organophosphates. Methods and Results: The gene encoding OPH was sequentially mutated at specific codons by saturation mutagenesis and screened for improved activity against the P–S substrates demeton‐S methyl and malathion. Escherichia coli lysates harbouring the variants displayed up to 177‐ and 1800‐fold improvement in specific activity against demeton‐S methyl and malathion, respectively, compared to the wild‐type lysates. The specificity constants of the purified variant proteins were improved up to 25‐fold for demeton‐S methyl and malathion compared to the wild‐type. Activity was associated with organophosphate detoxification as the hydrolysed substrate lost the ability to inhibit acetylcholinesterase. The improved hydrolytic efficiency against demeton‐S translated to the improved ability to hydrolyse the warfare agent VX. Conclusions: OPH variant enzymes were generated that displayed significantly improved ability to hydrolyse and detoxify organophosphates harbouring the P–S bond. Significance and Impact of the Study: The long‐term goal is to generate an environmentally‐friendly enzyme‐mediated bioremediation approach for the removal of toxic organophosphate compounds in the environment.     

Bacterial Cell Surface Display of Organophosphorus Hydrolase for Selective Screening of Improved Hydrolysis of Organophosphate Nerve Agents

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.     

Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.     

Simultaneous degradation of organophosphate and organochlorine pesticides by Sphingobium japonicum UT26 with surface-displayed organophosphorus hydrolase

A genetically engineered microorganism (GEM) capable of simultaneously degrading organophosphate and organochlorine pesticides was constructed for the first time by display of organophosphorus hydrolase (OPH) on the cell surface of a hexachlorocyclohexane (HCH)-degrading Sphingobium japonicum UT26. The GEM could potentially be used for removing the two classes of pesticides that may be present in mixtures at contaminated sites. A surface anchor system derived from the truncated ice nucleation protein (INPNC) from Pseudomonas syringae was used to target OPH onto the cell surface of UT26, reducing the potential substrate uptake limitation. The surface localization of INPNC–OPH fusion was verified by cell fractionation, western blot, proteinase accessibility, and immunofluorescence microscopy. Furthermore, the functionality of the surface-exposed OPH was demonstrated by OPH activity assays. Surface display of INPNC–OPH fusion (82 kDa) neither inhibited cell growth nor affected cell viability. The engineered UT26 could degrade parathion as well as γ-HCH rapidly in minimal salt medium. The removal of parathion and γ-HCH by engineered UT26 in sterile and non-sterile soil was also studied. In both soil samples, a mixture of parathion (100 mg kg^?1) and γ-HCH (10 mg kg^?1) could be degraded completely within 15 days. Soil treatment results indicated that the engineered UT26 is a promising multifunctional bacterium that could be used for the bioremediation of multiple pesticide-contaminated environments.     

Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae

The gene encoding organophosphorus hydrolase (OPH) from Flavobacterium species was expressed on the cell surface of Saccharomyces cerevisiae MT8-1 using a glycosylphosphatidylinositol (GPI) anchor linked to the C-terminal region of OPH. Immunofluorescence microscopy confirmed the localization of OPH on the cell surface, and fluorescence intensity measurement of cells revealed that 1.4 x 10(4) molecules of OPH per cell were displayed. Seventy percent of OPH whole-cell activity was detected on the cell surface by protease accessibility assay. The activity of OPH was highly dependent on cell growth conditions. The maximum activity was obtained when cells were grown in a synthetic dextrose medium lacking tryptophan (SD-W) buffered by 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, 200 mM, pH 7.0) at 20 degrees C, and cobalt chloride was added at 0.1 mM. S. cerevisiae MT8-1 displaying OPH which exhibited a higher activity than Escherichia coli displaying OPH using the ice nucleation protein (INP) anchor. The use of S. cerevisiae MT8-1, which has a "generally regarded as safe (GRAS)" status, as a host for the easy expression of the OPH gene provides a new biocatalyst useful for simultaneous detoxification and detection of organophosphorus pesticides.     

Enhanced biodegradation of toxic organophosphate compounds using recombinant Escherichia coli with sec pathway-driven periplasmic secretion of organophosphorus hydrolase

Although Escherichia coli can be genetically engineered to degrade environmental toxic organophosphate compounds (OPs) to nontoxic materials, a critical problem in such whole cell systems is limited substrate diffusion. The present work examined whether periplasmic expression of organophosphorus hydrolase (OPH) resulted in better whole cell enzymatic activity compared to standard cytosolic expression. Recombinant OPH periplasmic expression was achieved using the general secretory (sec) pathway with the pelB signal sequence. We found that while total OPH activity in periplasmic-expressing cell lysates was lower compared to that in cytosolic-expressing cell lysates whole cell OPH activity was 1.8-fold greater at 12 h post-induction in the periplasmic-expressing cells as a result of OPH translocation into the periplasmic space ( approximately 67% of whole cell OPH activity was found in the periplasmic fraction). These data suggest that E. coli engineered to periplasmically secrete OPH via the sec pathway may provide an improved whole cell biodegradation system for destruction of environmental toxic OPs.     

Functional periplasmic secretion of organophosphorous hydrolase using the twin-arginine translocation pathway in Escherichia coli

Recombinant Escherichia coli systems expressing organophosphorous hydrolase (OPH) have been proposed for biotransformation of toxic organophosphate compounds. However, whole cell biocatalyst systems are critically disadvantaged due to substrate diffusion limitations. To enhance whole cell biocatalytic efficiency, we engineered E. coli, for the first time, to secrete metal ion cofactor-requiring OPH into the periplasmic space using the twin-arginine translocation (Tat) pathway. In particular, the twin-arginine signal sequence of E. coli trimethylamine N-oxide (TMAO) reductase (TorA) was employed. Even though total OPH activity in the cell lysate fraction was lower in the periplasmic-secreting strain than in the control cytosolic-expressing strain, whole cell OPH activity was approximately 2.8-fold higher due to successful translocation of OPH into the periplasmic space. In addition, whole cell OPH activity in the periplasmic-secreting strain was far more stable than that in the cytosolic-expressing strain. Therefore, Tat-driven periplasmic-secreting E. coli can be successfully employed as efficient whole cell biocatalysts.     

Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.     

Whole cell-enzyme hybrid amperometric biosensor for direct determination of organophosphorous nerve agents with p-nitrophenyl substituent

In this paper, we reported the construction of a hybrid biosensor for direct, highly selective, sensitive, and rapid quantitative determination of organophosphate pesticides with p-nitrophenyl substituent using purified organophosphorus hydrolase (OPH) for the initial hydrolysis and Arthrobacter sp. JS443 for subsequent p-nitrophenol oxidation. The biocatalytic layer was prepared by co-immobilizing Arthrobacter sp. JS443 and OPH on a carbon paste electrode. OPH catalyzed the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon and methyl parathion to release p-nitrophenol that was oxidized by the enzymatic machinery of Arthrobacter sp. JS443 to carbon dioxide through electroactive intermediates 4-nitrocatechol and 1,2,4-benzenetriol. The oxidization current of the intermediates was measured and correlated to the concentration of organophosphates. The best sensitivity and response time were obtained using a sensor constructed with 0.06 mg dry weight of cell and 965 IU of OPH operating at 400 mV applied potential (vs. Ag/AgCl reference) in 50 mM citrate-phosphate pH 7.5 buffer at room temperature. Using these conditions, the biosensor measured as low as 2.8 ppb (10 nM) of paraoxon and 5.3 ppb (20 nM) of methyl parathion without interference from phenolic compounds, carbamate pesticides, triazine herbicides, and organophosphate pesticides that do not have the p-nitrophenyl substituent. The biosensor had excellent operational life-time stability with no decrease in response for more than 40 repeated uses over a 12-h period when stored at room temperature, while its storage life was approximately 2 days when stored in the operating buffer at 4 degrees C.     

Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source

Pseudomonas putida KT2442 was engineered to use the organophosphate pesticide parathion, a compound similar to other organophosphate pesticides and chemical warfare agents, as a source of carbon and energy. The initial step in the engineered degradation pathway was parathion hydrolysis by organophosphate hydrolase (OPH) to p-nitrophenol (PNP) and diethyl thiophosphate, compounds that cannot be metabolized by P. putida KT2442. The gene encoding the native OPH (opd), with and without the secretory leader sequence, was cloned into broad-host-range plasmids under the control of tac and taclac promoters. Expression of opd from the tac promoter resulted in high OPH activity, whereas expression from the taclac promoter resulted in low activity. A plasmid-harboring operons encoding enzymes for p-nitrophenol transformation to beta-ketoadipate was transformed into P. putida allowing the organism to use 0.5 mM PNP as a carbon and energy source. Transformation of P. putida with the plasmids harboring opd and the PNP operons allowed the organism to utilize 0.8 mM parathion as a source of carbon and energy. Degradation studies showed that parathion formed a separate dense, non-aqueous phase liquid phase but was still bioavailable.     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.     

Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k(cat)/K(m) value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli

Periplasmic secretion has been used in attempts to construct an efficient whole‐cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin‐arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole‐cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole‐cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL‐GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole‐cell OPH activity of the chaperone–coexpressing cells was ～5.5‐fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat‐based periplasmic translocation of OPH in the chaperone–coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole‐cell activity of periplasmic OPH‐secreting cells, yielding an effective whole‐cell biocatalyst system with highly reduced diffusion limitations. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 925–930, 2012     

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.     

Enhanced-rate biodegradation of organophosphate neurotoxins by immobilized nongrowing bacteria

Pesticide wastes generated from livestock dipping operations containing the organophosphate (OP) insecticide coumaphos (CP) are well suited for disposal by biodegradation since they are highly concentrated (approximately 1 g/L), generally contained, and lack additional toxic components. In this study, a significantly enhanced efficiency of degrading CP in cattle dip waste (CDW) is reported using a dense, nongrowing cell population that functions without the addition of nutrients required for growing cell cultures. A recombinant strain of Escherichia coli containing the opd gene for organophosphate hydrolase (OPH), which is capable of active hydrolysis of OP neurotoxins including CP, was cultivated in a rich medium containing all essential nutrients. Cells were harvested and utilized in lab scale experiments in the form of either freely suspended cells or cells immobilized within a macroporous gel matrix, poly(vinyl alcohol) (PVA) cryogel. Significantly higher degradation rates were achieved with either suspended or immobilized OPH(+) cells compared to rates with the microbial consortium naturally present in CDW. Of the two nongrowing cell systems, the detoxification rate with immobilized cells was approximately twice that of freely suspended cells, and kinetic studies demonstrated that a higher maximum reaction rate was achieved with the immobilized cell system. A comparative study using both the CDW and pure CP substrates with free cells indicated that the CDW contained one or more factors that reduced the bioavailability of CP. The immobilized cells retained their activity over a 4-month period of use and storage, demonstrating both sustained catalytic activity and long-term mechanical stability.     

Altering the Substrate Specificity of Organophosphorus Hydrolase for Enhanced Hydrolysis of Chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k_cat/K_m value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

Morphological and physiological modifications of cyanobacteria in experimental cyanobacterium-actinomycete associations

Associations of cyanobacteria and actinomycetes were formed experimentally from the cyanobacterium Anabaena variabilis ATCC 29413 and the streptomycetes isolated from apogeotropic roots of sago plants. Based on their phenotypic properties and the 16S rRNA gene sequencing, the streptomycetes were identified as representatives of Streptomyces pluricolorescens (strains 1 and 2). Cyanobacteria developing in monoculture and in association with an actinomycete were essentially different in their morphological and physiological-biochemical characteristics. In associations, cyanobacteria showed a higher (by tens of times) nitrogen-fixing activity compared to the monoculture and the morphological modifications of which were not observed in the monoculture (increase in cell size, increase in the portion of heterocysts among vegetative cells, appearance of the forms of unbalanced growth of cyanobacteria as giant, disc-shaped, curved, and rhomboid cells). At extremely low humidity (a_w 0.50), associated cyanobacterial cells remained viable, whereas in the monoculture, chlorophyll decomposition and cells death occurred. The methods of high-resolution (H¹ 600 MHz) nuclear magnetic resonance (NMR) and pulsed-gradient spin echo NMR revealed a fraction of mobile protons in lyophilized samples of the cyanobacterium-actinomycete association, which was evidence of the presence of free water. This fraction was not found in the lyophilized samples of cyanobacterial and streptomycete monocultures. The revealed differences can explain the survival of cyanobacterial cells in associations.     

Characterization of a novel organophosphorus hydrolase from Nocardiodes simplex NRRL B-24074

We characterized a novel organophosphorus hydrolase (OPH) activity expressed by Nocardiodes simplex NRRL B-24074, a member of a coumaphos-degrading microbial consortium from cattle dip waste. Like the previously characterized OPH from Nocardia sp. strain B- (NRRL B- 16944), OPH activity in N. simplex is located in the cytoplasm and is expressed constitutively. The purified enzyme is monomeric, has a native molecular size of 45,000 Da and has a specific activity toward ethyl parathion of 33 micromole/min x mg protein. Km constants for the enzyme with the structurally related organophosphate pesticides ethyl parathion and EPN were 100 microM and 345 microM, respectively. Although OPH activity in extracts did not require the addition of divalent cations, the purified enzyme lost activity during dialysis against phosphate buffer and this activity could be restored after incubation in buffer containing either CoSO4 or CuSO4. Our results suggest that OPH activity in N. simplex is distinct from other known OPHs and that the responsible gene is unrelated to known genes.