Search Results (10)

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. Full article

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. Full article

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). Full article

Mycotoxins are dangerous compounds and find multiple routes to enter living bodies of humans and animals. To solve the issue and degrade the toxicants, (bio)catalytic processes look very promising. Hexahistidine-tagged organophosphorus hydrolase (His₆-OPH) is a well-studied catalyst for degradation of organophosphorus neurotoxins and lactone-containing quorum-sensing signal molecules. Moreover, the catalytic characteristics in hydrolysis of several mycotoxins (patulin, deoxynivalenol, zearalenone, and sterigmatocystin) were studied in this investigation. The best Michaelis constant and catalytic constant were estimated in the case of sterigmatocystin and patulin, respectively. A possible combination of His₆-OPH with inorganic sorbents treated by low-temperature plasma was investigated. Further, enzyme–polyelectrolyte complexes of poly(glutamic acid) with His₆-OPH and another enzymatic mycotoxin degrader (thermolysin) were successfully used to modify fiber materials. These catalytically active prototypes of protective materials appear to be useful for preventing surface contact and exposure to mycotoxins and other chemicals that are substrates for the enzymes used. 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

In the present work, innovative composite biomaterials possessing bactericidal properties and based on the hexahistidine-tagged organophosphorus hydrolase (His₆-OPH) entrapped in the poly(vinyl alcohol) cryogel (PVA-CG)/bacterial cellulose (BC) were developed. His₆-OPH possesses lactonase activity, with a number of N-acyl homoserine lactones being the inducers of Gram-negative bacterial resistance. The enzyme can also be combined with various antimicrobial agents (antibiotics and antimicrobial peptides) to improve the efficiency of their action. In this study, such an effect was shown for composite biomaterials when His₆-OPH was entrapped in PVA-CG/BC together with β-lactam antibiotic meropenem or antimicrobial peptides temporin A and indolicidin. The residual catalytic activity of immobilized His₆-OPH was 60% or more in all the composite samples. In addition, the presence of BC filler in the PVA-CG composite resulted in a considerable increase in the mechanical strength and heat endurance of the polymeric carrier compared to the BC-free cryogel matrix. Such enzyme-containing composites could be interesting in the biomedical field to help overcome the problem of antibiotic resistance of pathogenic microorganisms. Full article

A biosensitive element in the form of bacterial Photobacterium phosphoreum cells immobilized in poly(vinyl alcohol) cryogel was tested for the determination of different mycotoxins under discrete and flow-through analysis conditions. The immobilized bioluminescent cells made it possible to quantify the presence of Ochratoxin A, Sterigmatocystin, Zearalenone, and Deoxynivalenon in aqueous media in a wide range of their concentrations (0.017–56 mg/L, 0.010–33 mg/L, 0.009–14 mg/L, and 0.026–177 mg/L, respectively) via measuring the quenching of cell luminescence. The flow conditions allowed the analysis sensitivity to be improved by an order of magnitude in terms of detected concentrations. Using the immobilized luminescent bacterial cells, we have shown the possibility of evaluating the efficiency of the mycotoxins’ hydrolysis under the action of enzymes. In this way, a 94 ± 4.5% efficiency of Zearalenone hydrolysis with hexahistidine-containing organophosphorus hydrolase for 1h-long treatment of the mycotoxin solution (100 mg/L) was shown. Full article

We have performed studies and comparative analysis of the biosynthesis characteristics of intracellular recombinant enzyme, such as hexahistidine-containing organophosphorus hydrolase (His₆-OPH) in Escherichia coli SG13009[pREP4] cells when various perfluorocarbon compounds (PFC) were introduced into the medium for cell cultivation. The PFC were found to facilitate the biosynthesis of His₆-OPH: increased levels of the total OPH-activity (up to 37%) were measured upon introduction of 1,1,1,2,2,3,3,4,4,5,5,6,6,6-tetradecafluorohexane (PFH) and 4,7,10,13,16,19,22,25,28,31-decaoxaperfluoro-5,8,11,14,17,18,21,24,27,30-decamethyl tetratriacontane (Polyether II) into culture medium. We have demonstrated the possibility of effective and multiple (at least five-fold) use of PFH for biosynthesis of intracellular recombinant protein His₆-OPH, which catalyzes the hydrolysis of organophosphorus pesticides (OP), is widely used in agriculture and can be applied as new antidote for OP-detoxification in vivo. The multiple use of PFH was achieved through recycling of this substance: sediment of Escherichia coli SG13009[pREP4] cell biomass was collected at the end of each culture growing step and disintegrated with ultrasound, and obtained residue containing almost all of the initially introduced PFC was then added to the medium at the start of the following culture growing step. Full article

Applying enzymatic biocatalysts based on hexahistidine-containing organophosphorus hydrolase (His₆-OPH) is suggested for the decomposition of chlorpyrifos, which is actively used in agriculture in many countries. The application conditions were optimized and the following techniques was suggested to ensure the highest efficiency of the enzyme: first, the soil is alkalinized with hydrated calcitic lime Ca(OH)₂, then the enzyme is introduced into the soil at a concentration of 1000 U/kg soil. Non-equilibrium low temperature plasma (NELTP)-modified zeolite is used for immobilization of the relatively inexpensive polyelectrolyte complexes containing the enzyme His₆-OPH and a polyanionic polymer: poly-l-glutamic acid (PLE₅₀) or poly-l-aspartic acid (PLD₅₀). The soil’s humidity is then increased up to 60–80%, the top layer (10–30 cm) of soil is thoroughly stirred, and then exposed for 48–72 h. The suggested approach ensures 100% destruction of the pesticide within 72 h in soils containing as much as 100 mg/kg of chlorpyrifos. It was concluded that using this type of His₆-OPH-based enzyme chemical can be the best approach for soils with relatively low humus concentrations, such as sandy and loam-sandy chestnut soils, as well as types of soil with increased alkalinity (pH 8.0–8.4). Such soils are often encountered in desert, desert-steppe, foothills, and subtropical regions where chlorpyrifos is actively used. Full article

Catalytic characteristics of hexahistidine-containing organophosphorus hydrolase (His₆-OPH) and its enzyme-polyelectrolyte complexes with poly-l-glutamic acid or poly-l-aspartic acid (His₆-OPH/PLD₅₀), hydrolyzing organophosphorous compounds, and N-acyl homoserine lactones were studied in the presence of various antibiotics (ampicillin, gentamicin, kanamycin, and rifampicin). The antibiotics at concentrations below 1 g·L⁻¹ had a negligible inhibiting effect on the His₆-OPH activity. Mixed inhibition of His₆-OPH was established for higher antibiotic concentrations, and rifampicin was the most potent inhibitor. Stabilization of the His₆-OPH activity was observed in the presence of antibiotics at a concentration of 0.2 g·L⁻¹ during exposure at 25–41 °C. Molecular docking of antibiotics to the surface of His₆-OPH dimer revealed the antibiotics binding both to the area near active centers of the enzyme subunits and to the region of contact between subunits of the dimer. Such interactions between antibiotics and His₆-OPH were verified with Fourier-transform infrared (FTIR) spectroscopy. Considering all the results of the study, the combination of His₆-OPH/PLD₅₀ with β-lactam antibiotic ampicillin was established as the optimal one in terms of exhibition and persistence of maximal lactonase activity of the enzyme. Full article