Search Results (13)

Since the growing number of fungi resistant to the fungicides used is becoming a serious threat to human health, animals, and crops, there is a need to find other effective approaches in the eco-friendly suppression of fungal growth. One of the main mechanisms of the development of resistance in fungi, as well as in bacteria, to antimicrobial agents is quorum sensing (QS), in which various lactone-containing compounds participate as signaling molecules. This work aimed to study the effectiveness of action of enzymes exhibiting lactonase activity against fungal signaling molecules. For this, the molecular docking method was used to estimate the interactions between these enzymes and different lactone-containing QS molecules of fungi. The catalytic characteristics of enzymes such as lactonase AiiA, metallo-β-lactamase NDM-1, and organophosphate hydrolase His₆-OPH, selected for wet experiments based on the results of computational modeling, were investigated. QS lactone-containing molecules (butyrolactone I and γ-heptalactone) were involved in the experiments as substrates. Further, the antifungal activity of the enzymes was evaluated against various fungal and yeast cells using bioluminescent ATP-metry. The efficient hydrolysis of γ-heptalactone by all three enzymes and butyrolactone I by His₆-OPH was demonstrated for the first time. The high antifungal efficacy of action of AiiA and NDM-1 against most of the tested fungal cells was revealed. Full article

Paraoxonase-1 (PON1) is a calcium-dependent, HDL-bound serum hydrolase active toward a wide variety of substrates. PON1 displays three types of activities, among which lactonase, paraoxonase, arylesterase and phosphotriesterase can be distinguished. Not only is this enzyme a major organophosphate compound detoxifier, but it is also an important constituent of the cellular antioxidant system and has anti-inflammatory and antiatherogenic functions. The concentration and activity of PON1 is highly variable among individuals, and these differences can be both of genetic origin and be a subject of epigenetic regulation. Owing to the fact that, in recent decades, the exposure of humans to an increasing number of different xenobiotics has been continuously rising, the issues concerning the role and activity of PON1 shall be reconsidered with particular attention to growing pharmaceuticals intake, dietary habits and environmental awareness. In the following manuscript, the current state of knowledge concerning the influence of certain modifiable and unmodifiable factors, including smoking, alcohol intake, gender, age and genotype variation on PON1 activity, along with pathways through which these could interfere with the enzyme’s protective functions, is presented and discussed. Since exposure to certain xenobiotics plays a key role in PON1 activity, the influence of organophosphates, heavy metals and several pharmaceutical agents is also specified. Full article

The effect of Bacitracin as an antibiotic acting against Gram-positive bacterial cells was evaluated in combination with hexahistidine-containing organophosphate hydrolase (His₆-OPH), possessing lactonase activity against various N-acylhomoserine lactones produced by most Gram-negative bacteria as quorum-sensing molecules. The molecular docking technique was used to obtain in silico confirmation of possible interactions between molecules of His₆-OPH and Bacitracin as well as the absence of a significant influence of such interactions on the enzymatic catalysis. The in vitro experiments showed a sufficient catalytic efficiency of action of the His₆-OPH/Bacitracin combination as compared to the native enzyme. The notable improvement (up to 3.3 times) of antibacterial efficiency of Bacitracin was revealed in relation to Gram-negative bacteria when it was used in combination with His₆-OPH. For the first time, the action of the Bacitracin with and without His₆-OPH was shown to be effective against various yeast strains, and the presence of the enzyme increased the antibiotic effect up to 8.5 times. To estimate the role of the enzyme in the success of His₆-OPH/Bacitracin with yeast, in silico experiments (molecular docking) with various fungous lactone-containing molecules were undertaken, and the opportunity of their enzymatic hydrolysis by His₆-OPH was revealed in the presence and absence of Bacitracin. Full article

Acylpeptide hydrolase (APEH) is a serine protease involved in amino acid recycling from acylated peptides (exopeptidase activity) and degradation of oxidized proteins (endoproteinase activity). This enzyme is inhibited by dichlorvos (DDVP), an organophosphate compound used as an insecticide. The role of APEH in spermatogenesis has not been established; therefore, the aim of this study was to characterize the distribution and activity profile of APEH during this process. For this purpose, cryosections of male reproductive tissues (testis and epididymis) and isolated cells (Sertoli cells, germ cells, and spermatozoa) were obtained from adult rats in order to analyze the intracellular localization of APEH by indirect immunofluorescence. In addition, the catalytic activity profiles of APEH in the different male reproductive tissues and isolated cells were quantified. Our results show that APEH is homogeneously distributed in Sertoli cells and early germ cells (spermatocytes and round spermatids), but this pattern changes during spermiogenesis. Specifically, in elongated spermatids and spermatozoa, APEH was localized in the acrosome and the principal piece. The exopeptidase activity was higher in the germ cell pool, compared to sperm and Sertoli cells, while the endoproteinase activity in epididymal homogenates was higher compared to testis homogenates at 24 h of incubation. In isolated cells, this activity was increased in Sertoli and germ cell pools, compared to spermatozoa. Taken together, these results indicate that APEH is differentially distributed in the testicular epithelium and undergoes re-localization during spermiogenesis. A possible role of APEH as a component of a protection system against oxidative stress and during sperm capacitation is discussed. Full article

Patatin-like phospholipase domain-containing protein 6 (PNPLA6), originally called Neuropathy Target Esterase (NTE), belongs to a family of hydrolases with at least eight members in mammals. PNPLA6/NTE was first identified as a key factor in Organophosphate-induced delayed neuropathy, a degenerative syndrome that occurs after exposure to organophosphates found in pesticides and nerve agents. More recently, mutations in PNPLA6/NTE have been linked with a number of inherited diseases with diverse clinical symptoms that include spastic paraplegia, ataxia, and chorioretinal dystrophy. A conditional knockout of PNPLA6/NTE in the mouse brain results in age-related neurodegeneration, whereas a complete knockout causes lethality during embryogenesis due to defects in the development of the placenta. PNPLA6/NTE is an evolutionarily conserved protein that in Drosophila is called Swiss-Cheese (SWS). Loss of SWS in the fly also leads to locomotory defects and neuronal degeneration that progressively worsen with age. This review will describe the identification of PNPLA6/NTE, its expression pattern, and normal role in lipid homeostasis, as well as the consequences of altered NPLA6/NTE function in both model systems and patients. Full article

Organophosphate hydrolases are promising as potential biotherapeutic agents to treat poisoning with pesticides or nerve gases. However, these enzymes often need to be further engineered in order to become useful in practice. One example of such enhancement is the alteration of enantioselectivity of diisopropyl fluorophosphatase (DFPase). Molecular modeling techniques offer a unique opportunity to address this task rationally by providing a physical description of the substrate-binding process. However, DFPase is a metalloenzyme, and correct modeling of metal cations is a challenging task generally coming with a tradeoff between simulation speed and accuracy. Here, we probe several molecular mechanical parameter combinations for their ability to empower long simulations needed to achieve a quantitative description of substrate binding. We demonstrate that a combination of the Amber19sb force field with the recently developed 12-6 Ca²⁺ models allows us to both correctly model DFPase and obtain new insights into the DFP binding process. Full article

Phosphotriestease (PTE), also known as parathion hydrolase, has the ability to hydrolyze the triester linkage of organophosphate (OP) pesticides and chemical warfare nerve agents, making it highly suitable for environment remediation. Here, we studied the effects of various surfactants and commercial detergents on the esterase activity of a recombinant PTE (His₆-tagged BdPTE) from Brevundimonas diminuta. Enzymatic assays indicated that His₆-tagged BdPTE was severely inactivated by SDS even at lower concentrations and, conversely, the other three surfactants (Triton X-100, Tween 20, and Tween 80) had a stimulatory effect on the activity, especially at a pre-incubating temperature of 40 °C. The enzyme exhibited a good compatibility with several commercial detergents, such as Dr. Formula^® and Sugar Bubble^®. The evolution results of pyrene fluorescence spectroscopy showed that the enzyme molecules participated in the formation of SDS micelles but did not alter the property of SDS micelles above the critical micelle concentration (CMC). Structural analyses revealed a significant change in the enzyme’s secondary structure in the presence of SDS. Through the use of the intentionally fenthion-contaminated Chinese cabbage leaves as the model experiment, enzyme–Joy^® washer solution could remove the pesticide from the contaminated sample more efficiently than detergent alone. Overall, our data promote a better understanding of the links between the esterase activity of His₆-tagged BdPTE and surfactants, and they offer valuable information about its potential applications in liquid detergent formulations. Full article

Enzyme-catalyzed hydrolysis of echothiophate, a P–S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with K_m = 0.20 ± 0.03 mM and k_cat = 5.4 ± 1.6 min⁻¹. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (K_m = 2.6 ± 0.2 mM; k_cat = 53400 min⁻¹). With a k_cat/K_m = (2.6 ± 1.6) × 10⁷ M⁻¹min⁻¹, GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P–S bonded OPs by thiol-free OP hydrolases. Full article

Carboxylesterasesare an important class of detoxification enzymes involved in insecticide resistance in insects. A subgroup of Helicoverpa armigera esterases, known as Clade 001, was implicated in organophosphate and pyrethroid insecticide resistance due to their overabundance in resistant strains. In this work, a novel carboxylesterasegene 001D of H. armigera from China was cloned, which has an open reading frame of 1665 nucleotides encoding 554 amino acid residues. We used a series of fusion proteins to successfully express carboxylesterase 001D in Escherichia coli. Three different fusion proteins were generated and tested. The enzyme kinetic assay towards 1-naphthyl acetate showed all three purified fusion proteins are active with a Kcat between 0.35 and 2.29 s⁻¹, and a Km between 7.61 and 19.72 μM. The HPLC assay showed all three purified fusion proteins had low but measurable hydrolase activity towards β-cypermethrin and fenvalerate insecticides (specific activities ranging from 0.13 to 0.67 μM·min⁻¹·(μM⁻¹·protein)). The enzyme was stable up to 40 °C and at pH 6.0–11.0. The results imply that carboxylesterase 001D is involved in detoxification, and this moderate insecticide hydrolysis may suggest that overexpression of the gene to enhance insecticide sequestration is necessary to allow carboxylesterases to confer resistance to these insecticides in H. armigera. Full article

In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices. Full article

This review gives a survey on the state of the art of pesticide detection usingflow-based biosensing systems for sample screening. Although immunosensor systems havebeen proposed as powerful pesticide monitoring tools, this review is mainly focused onenzyme-based biosensors, as they are the most commonly employed when using a flowsystem. Among the different detection methods able to be integrated into flow-injectionanalysis (FIA) systems, the electrochemical ones will be treated in more detail, due to theirhigh sensitivity, simple sample pretreatment, easy operational procedures and real-timedetection. During the last decade, new trends have been emerging in order to increase theenzyme stability, the sensitivity and selectivity of the measurements, and to lower thedetection limits. These approaches are based on (i) the design of novel matrices for enzymeimmobilisation, (ii) new manifold configurations of the FIA system, sometimes includingminiaturisation or lab-on-chip protocols thanks to micromachining technology, (iii) the useof cholinesterase enzymes either from various commercial sources or genetically modifiedwith the aim of being more sensitive, (iv) the incorporation of other highly specificenzymes, such as organophosphate hydrolase (OPH) or parathion hydrolase (PH) and (v) thecombination of different electrochemical methods of detection. This article discusses thesenovel strategies and their advantages and limitations. Full article

This paper reports a first microbial biosensor for rapid and cost-effectivedetermination of organophosphorus pesticides fenitrothion and EPN. The biosensorconsisted of recombinant PNP-degrading/oxidizing bacteria Pseudomonas putida JS444anchoring and displaying organophosphorus hydrolase (OPH) on its cell surface asbiological sensing element and a dissolved oxygen electrode as the transducer. Surface-expressed OPH catalyzed the hydrolysis of fenitrothion and EPN to release 3-methyl-4-nitrophenol and p-nitrophenol, respectively, which were oxidized by the enzymaticmachinery of Pseudomonas putida JS444 to carbon dioxide while consuming oxygen,which was measured and correlated to the concentration of organophosphates. Under theoptimum operating conditions, the biosensor was able to measure as low as 277 ppb offenitrothion and 1.6 ppm of EPN without interference from phenolic compounds and othercommonly used pesticides such as carbamate pesticides, triazine herbicides andorganophosphate pesticides without nitrophenyl substituent. The applicability of thebiosensor to lake water was also demonstrated. Full article

The realisation of a miniaturised potentiometric enzyme biosensor is presented. The biosensor chip utilises the enzyme organophosphorus hydrolase (OPH) for the direct determination of pesticides. The transducer structure of the sensors chip consists of a pH-sensitive capacitive electrolyte-insulator-semiconductor (EIS) structure that reacts towards pH changes caused by the OPH-catalised hydrolysis of the organophosphate compounds. The biosensor is operated versus a conventional Ag/AgCl reference electrode. Measurements were performed in the capacitance/voltage (C/V) and the constant capacitance (ConCap) mode for the two different pesticides paraoxon and parathion. For the development of this new type of biosensor, different immobilisation strategies, influence of buffer composition and concentration, transducer material, detection limit, long-term stability and selectivity have been studied. Full article