Synergistic Antimicrobial Action of Lactoferrin-Derived Peptides and Quorum Quenching Enzymes.

Combined use of various antimicrobial peptides (AMPs) with enzymes that hydrolyze the signaling molecules of the resistance mechanism of various microorganisms, quorum sensing (QS), to obtain effective antimicrobials is one of the leading approaches in solving the antimicrobial resistance problem. Our study investigates the lactoferrin-derived AMPs, lactoferricin (Lfcin), lactoferampin and Lf(1-11), as potential partners for combination with enzymes hydrolyzing lactone-containing QS molecules, the hexahistidine-containing organophosphorus hydrolase (His6-OPH) and penicillin acylase, to obtain effective antimicrobial agents with a scope of practical application. The possibility of the effective combination of selected AMPs and enzymes was first investigated in silico using molecular docking method. Based on the computationally obtained results, His6-OPH/Lfcin combination was selected as the most suitable for further research. The study of physical-chemical characteristics of His6-OPH/Lfcin combination revealed the stabilization of enzymatic activity. A notable increase in the catalytic efficiency of action of His6-OPH in combination with Lfcin in the hydrolysis of paraoxon, N-(3-oxo-dodecanoyl)-homoserine lactone and zearalenone used as substrates was established. Antimicrobial efficiency of His6-OPH/Lfcin combination was determined against various microorganisms (bacteria and yeasts) and its improvement was observed as compared to AMP without enzyme. Thus, our findings demonstrate that His6-OPH/Lfcin combination is a promising antimicrobial agent for practical application.

Self-Assembling Enzymatic Nanocomplexes with Polypeptides and Low-Weight Organic Compounds: Preparation, Characterization, and Application of New Antibacterials.

The self-assembling of nanosized materials is a promising field for research and development. Multiple approaches are applied to obtain inorganic, organic and composite nanomaterials with different functionality. In the present work, self-assembling nanocomplexes (NCs) were prepared on the basis of enzymes and polypeptides followed by the investigation of the influence of low-molecular weight biologically active compounds on the properties of the NCs. For that, the initially possible formation of catalytically active self-assembling NCs of four hydrolytic enzymes with nine effectors was screened via molecular modeling. It allowed the selection of two enzymes (hexahistidine-tagged organophosphorus hydrolase and penicillin acylase) and two compounds (emodin and naringenin) having biological activity. Further, such NCs based on surface-modified enzymes were characterized by a batch of physical and biochemical methods. At least three NCs containing emodin and enzyme (His6-OPH and/or penicillin acylase) have been shown to significantly improve the antibacterial activity of colistin and, to a lesser extent, polymyxin B towards both Gram-positive bacteria (Bacillus subtilis) and Gram-negative bacteria (Escherichia coli).

Strategies for variable regulation of methanogenesis efficiency and velocity.

Combinations of various strategic approaches to the suppression of methanogenesis and the formation of biogas with a simultaneous decrease in the ratio of methane in its composition were investigated. Introduction of methanogenesis suppressors such as redox derivatives of humic acids, potassium persulfate (K2S2O8), possessing oxidizing and electron acceptor properties, enzyme hexahistidine-containing organophosphorus hydrolase with high lactonase activity and polypeptide antimicrobial agent bacitracin into the media with anaerobic consortia were studied. The effect of these substances was directed at various participants of the natural methanogenic consortium, as well as on the biochemical processes carried out by them. The use of K2S2O8 together with bacitracin provided maximum and almost complete suppression of CH4 production. The measured concentration of intracellular adenosine triphosphate has shown that viability of cells in the consortium remained almost the same, whereas their metabolic activity decreased. Various combinations of the above-mentioned suppressors provided different degrees of methanogenesis suppression, but redox agents played a key role in all the cases studied. Based on the accumulated data, combining suppressors in different concentrations can be used to manage the methanogenesis (efficiency and velocity of its decrease) in media with anaerobic consortia. KEY POINTS: • Various strategies for suppression of the methanogenesis were combined. • The enzyme His6-OPH was firstly used for quorum quenching in methanogenic consortium. • Velocity of methanogenesis decrease can be managed by combinations of suppressors.

"Universal" Antimicrobial Combination of Bacitracin and His6-OPH with Lactonase Activity, Acting against Various Bacterial and Yeast Cells.

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.

Recombinant organophosphorus hydrolase (OPH) expression in E. coli for the effective detection of organophosphate pesticides.

Accumulation and exposure of organophosphate pesticides are of great concern today owing to their abundant usage and potential health hazards. Harmful effects of organophosphate pesticide exposure and limitations of the available treatment methods necessitate the development of reliable, selective, cost-effective, and sensitive methods of detection. We developed a novel biosensor based on the enzymatic action of recombinant organophosphorus hydrolase (OPH) expressed in E. coli. We report the development of colorimetric biosensors made of His-Nus-OPH as well as His-Nus-OPH loaded alginate microspheres. The colorimetric detection method developed using solution-phase and alginate-encapsulated His-Nus-OPH exhibited detection limits of 0.045 and 0.039 mM, respectively, for ethyl paraoxon, and 0.101 and 0.049 mM, respectively, for methyl parathion. Additionally, fluorescence measurement using pH-sensitive fluorescein isothiocyanate (FITC) was used to sense the quantity of organophosphorus pesticides. The fluorometric detection method using solution-phase His-Nus-OPH, with ethyl paraoxon and methyl parathion as the substrate, reveals the lower limit of detection as 0.014 mM and 0.044 mM, respectively. Our results demonstrate the viability of His-Nus-OPH for OP detection with good sensitivity, LOD, and linear range. We report the first use of N-terminal His-NusA-tagged OPH, which enhances solubility significantly and presents a significant advance for the scientific community.

Overview of a bioremediation tool: organophosphorus hydrolase and its significant application in the food, environmental, and therapy fields.

In the past decades, the organophosphorus compounds had been widely used in the environment and food industries as pesticides. Owing to the life-threatening and long-lasting problems of organophosphorus insecticide (OPs), an effective detection and removal of OPs have garnered growing attention both in the scientific and practical fields in recent years. Bacterial organophosphorus hydrolases (OPHs) have been extensively studied due to their high specific activity against OPs. OPH could efficiently hydrolyze a broad range of substrates both including the OP pesticides and some nerve agents, suggesting a great potential for the remediation of OPs. In this review, the microbial identification, molecular modification, and practical application of OPHs were comprehensively discussed.Key points• Microbial OPH is a significant bioremediation tool against OPs.• Identification and molecular modification of OPH was discussed in detail.• The applications of OPH in food, environmental, and therapy fields are presented.

Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site.

Organophosphorus hydrolase (OPH) is a metalloenzyme that can hydrolyze organophosphorus agents resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified three hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. We then experimentally assayed single and double mutants involving these residues for paraoxon binding affinity. The binding free energy calculations and the experimental kinetics of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced substrate binding affinity over WT OPH. Interestingly, our experimental results show that the substrate binding affinity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.

Biodegradation of organophosphorus pesticide chlorpyrifos by Sphingobacterium sp. C1B, a psychrotolerant bacterium isolated from apple orchard in Himachal Pradesh of India.

A psychrotolerant Sphingobacterium sp. was isolated from the apple orchard situated in the Kufri region of Shimla, Himachal Pradesh, India using an enrichment culture technique having chlorpyrifos (CP) as the sole source of carbon and energy. Based on biochemical characterization and 16S rRNA analysis, the strain was identified as Sphingobacterium sp. C1B. The bacterium C1B was able to degrade chlorpyrifos ≥ 42 ppm and ≥ 36 ppm within 14 days at 20 °C and 15 °C, respectively. The strain was also able to degrade chlorpyrifos ≤ 35 ppm at 28 °C within 14 days. The enzyme organophosphorus hydrolase might be responsible for the initial degradation of CP by the strain C1B. Based on the HPLC and GCMS analysis, a probable degradation pathway has been proposed, which followed the path from chlorpyrifos to 3,5,6-trichloro-2-pyridinol to benzene, 1,3-bis (1,1-dimethylethyl) and then entered into the TCA cycle. Our current study revealed that the bacterium C1B was found to be a useful strain for the degradation of pesticide chlorpyrifos in the cold climatic environment.

Surface display of organophosphorus-degrading enzymes on the recombinant spore of Bacillus subtilis.

Organophosphorus-degrading enzymes show high hydrolysis efficiency and provide an environmentally friendly solution to the pollution of organophosphorus compound. However, poor enzyme stability and tedious purification process have limited practical applications. Spore-based display system can provide many advantages, such as safety, low cost, easy preparation and high resistance to harsh conditions. Recently, we have constituted the recombinant spore displaying organophosphorus hydrolase and organophosphorus acid anhydrolase. In the spore display systems, recombinant spores could be reliably produced and normal sporulation was not affected; the activities of recombinant spores were 15.81 and 10.67 U/mg spores (dry weight) respectively; furthermore, the recombinant spores exhibited significantly enhanced resistance to various harsh conditions compared to free-form enzymes. These results indicated that the spore display could contribute to the practical application of organophosphorus-degrading enzymes and provide a promising solution to bioremediation of organophosphorus compounds.

Advances in detection of hazardous organophosphorus compounds using organophosphorus hydrolase based biosensors.

Agricultural advancements focusing on increasing crop production have led to excessive usage of insecticides and pesticides, resulting in leaching and accumulation of these highly toxic chemicals in soil, water, and the food-chain. Organophosphorus (OP) compounds are the most commonly used insecticides and pesticides, which cause a wide range of long-lasting and life-threatening conditions. Due to the acute toxicity and long-term side effects of OP compounds, their timely, on-the-spot and rapid detection has gained importance, for efficient healthcare management. In this respect, several OP degrading enzymes have gained the spotlight in developing the enzyme-based biosensors, owing to their high activity and broad specificity. Among these enzymes, organophosphorus hydrolase (OPH) has emerged as a promising candidate for the detection of OP compounds, due to its ability to act on a broad range of substrates having a variety of bonds, like P─F, P─O, P─S, and P─CN. Various techniques employing OPH in free/immobilized/conjugated forms into sensing devices were reported to accurately detect OP compounds. The transduction mechanisms of bio-sensing are electrochemical, optical as well as novel methods like magnetoelastic/surface plasmon resonance. Furthermore, to improve the detection limits and sensitivity, nanoparticles and quantum dots are often employed in conjunction with OPH. Here, we highlight the recent advances in sensing OP compounds using OPH based biosensors, compare specifications of sensing methods, and evaluate the influence of different materials used in developing sensors. This review will also enable researchers to design and configure highly sensitive and accurate sensing systems, leading to the development of point-of-care devices for real-time analysis.

Novel approach to quorum quenching: rational design of antibacterials in combination with hexahistidine-tagged organophosphorus hydrolase.

N-acyl homoserine lactones (AHLs) are quorum sensing (QS) signal molecules used by most Gram-negative pathogenic bacteria. In this article the lactonase activity of the preparations based on hexahistidine-tagged organophosphorus hydrolase (His6-OPH) towards AHLs was studied. Initially, three of the most interesting ß-lactam antibiotics were selected from seven that were trialed during molecular docking to His6-OPH. Combinations of antibiotics (meropenem, imipenem, ceftriaxone) and His6-OPH taken in the native form or in the form of non-covalent enzyme-polyelectrolyte complexes (EPCs) with poly(glutamic acid) or poly(aspartic acid) were obtained and investigated. The lactonase activity of the preparations was investigated under different physical-chemical conditions in the hydrolysis of AHLs [N-butyryl-D,L-homoserine lactone, N-(3-oxooctanoyl)-D,L-homoserine lactone, N-(3-oxododecanoyl)-L-homoserine lactone]. An increased efficiency of catalytic action and stability of the lactonase activity of His6-OPH was shown for its complexes with antibiotics and was confirmed in trials with bacterial strains. The broadening of the catalytic action of the enzyme against AHLs was revealed in the presence of the meropenem. Results of molecular docking of AHLs to the surface of the His6-OPH dimer in the presence of antibiotics allowed proposing the mechanism of such interference based on a steric repulsion of the carbon chain of hydrolyzed AHLs by the antibiotics bounded to the enzyme surface.

High level expression of organophosphorus hydrolase in Pichia pastoris by multicopy ophcM assembly.

The residues of organophosphorus pesticides bring serious impact on the environmental safety and people's health. Biodegradation of organophosphorus pesticides is recognized as an ideal method. An organophosphorus hydrolase (OPHCM) from Pseudomonas pseudoalcaligenes was synthesized and expressed in Pichia pastoris. The yield reached approximately 470 mg/l after a 6-d induction in shake flasks. To improve the enzyme production, we describe a novel approach to express OPHCM efficiently with a biobrick assembly method in vitro. Four recombinant plasmids containing 1-4 copies of ophcM-expressing cassettes were constructed and transformed into P. pastoris. Increasing the copy number of ophcM gene enhanced the expression level of OPHCM. The maximum yield and specific activity in P. pastoris harboring two-copy tandem ophcM-expressing cassettes reached 610 mg/l after a 6-d induction in shake flasks and 7.8 g/l in high-density fermentation with specific activity of 13.7 U/mg. The optimum pH and temperature of the recombinant OPHCM activity were 11.0 and 50 °C, respectively. In addition, the enzyme activity of recombinant OPHCM enhanced 57.6% and 30.1% in the presence of 1 mM Cd(2+) and 5% glycerol, respectively. The high expression and good properties of recombinant OPHCM provide an effective solution to solve the pollution of organophosphorus pesticides in the environment. Moreover, the approach for generating multicopy gene expressing vectors here will benefit the study for enhancing the expression level of genes of interest.

Extensive hydrolysis of phosphonates as unexpected behaviour of the known His6-organophosphorus hydrolase.

The catalytic activity of hexahistidine-tagged organophosphorus hydrolase (His6-OPH) in hydrolytic reactions of methylphosphonic acid (MPA) and its monoesters and diesters being decomposition products of R-VX was demonstrated for the first time. The catalytic constants of enzyme in such reactions were determined. The mechanism of C-P bond cleavage in the MPA by His6-OPH was proposed. Such reaction was estimated to be carried out with the soluble and nanocapsulated forms of His6-OPH. His6-OPH was demonstrated to be capable of degrading the key organophosphorus components of reaction masses (RMs) that are produced by the chemical detoxification of R-VX and RMs are multi-substrate mixtures for this enzyme. The kinetic model describing the behaviour of His6-OPH in RMs was proposed and was shown to adequately fit experimental points during degradation of the real samples of RMs.

High-level extracellular secretion of organophosphorous hydrolase of Flavobacterium sp. in Escherichia coli BL21(DE3)pLysS.

Organophosphorus (OPs) compounds are widely used in many pesticides, insecticides, and chemical nerve agents. These compounds are hazardous for humans and the environment. There are many reports on detoxification of these compounds, among them enzymatic cleavage of these compounds with organophosphorus hydrolase (OPH) has been taken into more consideration. Several studies have been performed to improve OPH secretion in Escherichia coli by different signal peptides, but have not been successful. In this study, to achieve the extracellular secretion of OPH in E. coli, the complete opd gene along with its native signal peptide was codon optimized and expressed in E. coli BL21(DE3)pLysS. The culture medium showed OPH activity after 2, 4, and 6 H of induction time. The extracellular secretion of OPH was also confirmed by SDS-PAGE and Western blot analysis. The effects of different factors in growth medium were also investigated regarding expression and extracellular secretion of OPH. It appears that the secretion of OPH into the extracellular medium is highly affected by culture conditions. Therefore, our results revealed that the recombinant OPH was successfully secreted into the extracellular medium. This secretion system can be considered as a high efficiency biocatalyst for detoxification of OPs compounds.

Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551.

The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.

Covalent immobilization of recombinant organophosphorus hydrolase on spores of Bacillus subtilis.

AIMS: Organophosphorus pesticides are widely used in agriculture. Accordingly, decontamination of these pesticides and their residual in environment is an important aim of researchers. One of the best approaches is enzymatic detoxification of these compounds with organophosphorus hydrolase (OPH). The immobilization of OPH on environmentally friendly supports is of great importance for developing stabilized enzymes for degradation of organophosphorus compounds. METHODS AND RESULTS: In this study, Bacillus subtilis spores were applied as a new matrix for immobilizing OPH for the first time; this enzyme was covalently bound to the spores by using EDC-NHS as coupling reagents and the immobilization was confirmed by enzymatic activity, Western blot, flow cytometry and fluorescence microscopic analysis. The immobilization yield was about 55% and the immobilized OPH hydrolysed paraoxon, an organophosphate substrate, without significant loss of activity was six times. The spores with immobilized OPH on their surface were successfully characterized using FT-IR analysis and SEM imaging. Thermal and pH stability was improved by immobilization of OPH on the spore surface. CONCLUSIONS: Owing to safety, environmentally friendly and low cost of spores, these spores can be employed in biosensors for monitoring and biodegradation of organophosphate contaminants in the environment and detoxification processes in bioreactors with high reusability without decrease in the activity. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe that the spore, an environmentally friendly matrix, can be used for covalent immobilization of OPH efficiently and can be applied for detoxification of organophosphorus compounds under adverse environmental conditions.

Secretory expression of organophosphorus hydrolase OPHC2 in Yarrowia lipolytica Polg.

In the present study, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Yarrowia lipolytica and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses showed a major polypeptide band of 36 kDa. The purified enzyme was optimally active at 65°C and pH 8.5 and also displayed good thermal and pH stability using methyl parathion (O,O-dimethyl-O-4-p-nitrophenyl phosphorothioate) as a substrate. Moreover, as Y. lipolytica is a non-pathogenic, generally regarded as safe (GRAS) yeast, the cell culture supernatant can be used directly on vegetables and fruits that are contaminated by organophosphorus pesticides.

Cloning and expression of ophB gene encoding organophosphorus hydrolase from endophytic Pseudomonas sp. BF1-3 degrades organophosphorus pesticide chlorpyrifos.

Chlorpyrifos is an organophosphate pesticide that has adverse effect on animals and plants. We isolated endophytic bacterial strain, Pseudomonas sp. BF1-3, from balloon flower root which can hydrolyze chlorpyrifos. A gene (ophB) encoding a protein involved in chlorpyrifos degradation from this strain was cloned into Escherichia coli DH5α for confirming enzyme activity. After sequencing, total 1024bp nucleotide sequences were found in the open reading frame of ophB. The chlorpyrifos degradation patterns by E. coli DH5α (ophB) were observed. During incubation in minimal salt (M9) medium supplemented with chlorpyrifos (100mgL(-1)), the E. coli DH5α harboring ophB degraded about 97% initial chlorpyrifos (100mgL(-1)) and accumulated 86mgL(-1) 3,5,6-trichloro-2-pyridinol (TCP) within 9 days. In addition, optical density (OD) of E. coli DH5α (ophB) culture at 600nm was increased from 0.172 to 1.118 within 2 days of inoculation in the chlorpyrifos supplemented M9 medium. The estimated molecular weight of purified OphB protein was determined to be 31.4kDa by SDS-PAGE. The OphB enzyme was most active at pH 8 and an optimal temperature around 35°C. These results indicate that endophytic bacteria are supposed to be useful for biological control of environments contaminated with pesticides.

Active site engineering of Zn-doped mesoporous ceria toward highly efficient organophosphorus hydrolase-mimicking nanozyme.

Hydrolase-mimicking nanozymes have received increasing attention in recent years, but the effective rational design and development of these materials has not been realized, as they are not at present considered a critical research target. Herein, we report that Zn-doped mesoporous ceria (Zn-m-ceria) engineered to have an abundance of two different active sites with different functions-one that allows both co-adsorption binding of organophosphate (OP) and water and another that serves as a general base-has significant organophosphorus hydrolase (OPH)-like catalytic activity. Specifically, Zn-m-ceria exhibits a catalytic efficiency over 75- and 25-fold higher than those of m-ceria and natural OPH, respectively. First-principles calculations reveal the importance of Zn for the OPH-mimicking activity of the material, promoting substrate adsorption and proton-binding. The OPH-like Zn-m-ceria catalyst is successfully applied to detect a model OP, methyl paraoxon, in spiked tap water samples with excellent sensitivity, stability, and detection precision. We expect that these findings will promote research based on the rational engineering of the active site of nanozymes and efficient strategies for obtaining a diverse range of catalysts that mimic natural enzymes, and hence the utilization in real-world applications of enzyme-mimicking catalysts with properties superior to their natural analogs should follow.

Carrier Variety Used in Immobilization of His6-OPH Extends Its Application Areas.

Organophosphorus hydrolase, containing a genetically introduced hexahistidine sequence (His6-OPH), attracts the attention of researchers by its promiscuous activity in hydrolytic reactions with various substrates, such as organophosphorus pesticides and chemical warfare agents, mycotoxins, and N-acyl homoserine lactones. The application of various carrier materials (metal-organic frameworks, polypeptides, bacterial cellulose, polyhydroxybutyrate, succinylated gelatin, etc.) for the immobilization and stabilization of His6-OPH by various methods, enables creation of biocatalysts with various properties and potential uses, in particular, as antidotes, recognition elements of biosensors, in fibers with chemical and biological protection, dressings with antimicrobial properties, highly porous sorbents for the degradation of toxicants, including in flow systems, etc. The use of computer modeling methods in the development of immobilized His6-OPH samples provides in silico prediction of emerging interactions between the enzyme and immobilizing polymer, which may have negative effects on the catalytic properties of the enzyme, and selection of the best options for experiments in vitro and in vivo. This review is aimed at analysis of known developments with immobilized His6-OPH, which allows to recognize existing recent trends in this field of research, as well as to identify the reasons limiting the use of a number of polymer molecules for the immobilization of this enzyme.