Maksim Domnin

Pharmaceutical pollutants that contain lactone or lactam rings in their structure are frequently detected in wastewater. Enzymes with lactonase activity, synthesized by the microorganisms in aerobic activated sludge, are expected to be able to participate in the hydrolysis of these pharmaceutical pollutants, but the actual activity of many lactonases towards such compounds remains poorly characterized. In this work, a systematic molecular docking study of 12 enzymes and 39 pharmaceutical pollutants (β-lactam antibiotics, macrolides, coumarins, statins) was undertaken. The binding affinity, active-site occupancy, total enzyme surface occupancy and geometric characteristics of the molecules (the distance from the catalytic nucleophile to the carbonyl carbon of the lactone ring, and the attack angle formed between the nucleophilic atom of the enzyme and the C–N/C–O bond of the substrate) were evaluated in silico for a set of hydrolases displaying lactonase activity (AidC, AaL, hexahistidine-tagged organophosphate hydrolase (His₆-OPH), etc.) and metallo-β-lactamases (MβLs) (NDM-1, MIM-1, SAM-1). Lactamases exhibited favorable intermolecular interaction geometry for the majority of β-lactams. No enzyme was found that satisfied the geometric criteria corresponding to the biodegradation of macrolides. With coumarins, only Paraoxonase PON2 provided an optimal geometry of biocatalysis; however, this enzyme is absent from activated sludge but may be of interest for in vivo applications. For several statins widely used in practice, combinations with enzymes having virtually ideal geometry for biocatalysis were identified: AidC/rosuvastatin lactone, AaL/lovastatin, His₆-OPH/mevastatin, and PON2/simvastatin. For the first time, several pharmaceutical lactones were identified in silico and confirmed in vitro as inhibitors of MβL and His₆-OPH. Competitive inhibition of NDM-1 by lactone-containing signaling molecules of Gram-negative bacteria present in activated sludge was revealed for the first time both in silico and in vitro. The obtained results‘ may be taken into account when implementing monitoring of biological stages at water treatment stations and improve the efficiency of water purification. The revealed characteristics of MβL inhibition by macrolides may be taken into account as one of possible explanations for the effective application of β-lactams in combinations with macrolides known from multiple paratactical investigations.

Composites based on bacterial cellulose (BC) and synthetic polymers are attracting research interest as promising functional materials due to the ability to control their properties. In this study, the interactions between BC and synthetic polymers, poly(vinyl alcohol) (PVA), polylactide (PLA), and polycaprolactone (PCL), were investigated using computer modeling, and the biosynthesis, characteristics, and protein sorption capacity of the resulting composites were evaluated. In silico analysis using dissipative particle dynamics predicted a decrease in compatibility with BC in the order PVA > PLA > PCL. The calculated Flory–Huggins interaction parameters for BC/PVA, BC/PLA, and BC/PCL systems were 1.75, 3.93, and 6.03, respectively, indicating a gradual decrease in thermodynamic compatibility. These predictions were experimentally confirmed by in situ biosynthesis of BC/synthetic polymer composites under static and dynamic cultivation conditions. BC/PVA composites exhibited homogeneous morphology with pore size below 100 nm and improved structural integrity. BC/PLA and BC/PCL systems showed phase separation and broader pore size distributions, reaching up to 900 nm. The functionalization of the obtained composites via adsorption of different proteins (bovine serum albumin, lysozyme, and His₆-organophosphate hydrolase) revealed a high dependence of the results on the polymer type, the conditions applied for composite synthesis, and the molecule size of the proteins. Estimations of protein–composite interactions were conducted in silico and confirmed in vitro. The maximal sorption capacity was revealed for composites obtained during the cultivation of BC-producing microorganisms under static conditions, with the addition of synthetic polymers to the nutritional medium. In the case of enzymes used for the functionalization of composites, a partial activity loss after sorption was revealed. In BC/PVA composites, the maximal decrease in enzyme activity (~30% from the activity level of the same enzymes in the BC samples) was observed. BC/PLA and BC/PCL composites demonstrated preferences in the sorption of large protein molecules, making them attractive platforms for enzyme immobilization and biocatalytic applications of the obtained catalytically active composites.

Recently, we found that combining various antimicrobial polypeptides (AMPs) with enzymes exhibiting lactonase activity results in an antifungal agent with significantly enhanced stability and antimicrobial action efficiency. In this context, this study aims to investigate the catalytic and antifungal activity and physical-chemical properties of antifungal enzyme combinations hydrolyzing fungal cell wall components with various AMPs, comparing them with enzymes exhibiting lactonase activity (capable of hydrolyzing lactones by ring opening). Additionally, combinations of enzymes targeting the fungal cell wall and/or hydrolyzing fungal lactone-containing Quorum-sensing molecules with polyamino acids (PAAs) supplemented with fungicides (PAAF) were studied for comparison with AMP-containing combinations. Interaction models for these antifungal enzyme combinations were simulated in silico using the molecular docking method. The most promising variants, which were predicted to possess high catalytic activity, were selected, and their catalytic and physical-chemical characteristics were further evaluated in vitro. The antifungal activity of the selected combinations of enzymes with AMPs or PAAF was assessed against a number of fungi, leading to the identification of several combinations as potential candidates for inclusion in antifungals. Unexpectedly, antifungal enzyme combinations with lactonase activity were, in most cases, more effective than those with fungal-cell-wall-degrading enzymes.

Background: Various thiolactones are known as biologically active compounds, capable of stimulating the development of several human diseases and quorum sensing of Gram–positive bacteria. The enzymatic hydrolysis of thiolactones represents a promising approach to preventing their action. Methods: Thirteen enzymes, including various lactonases and serine hydrolases were studied in this work using several substrates including the homocysteine thiolactone (HTL), and its derivatives the N–acetylhomocysteine thiolactone (C₂–HTL) and the isobutyryl–homocystein thiolactone (i–but–HTL). The potential interactions of the ligands with the surface of enzymes molecules were predicted in silico using computational modeling and checked in wet experiments in vitro. Results: Based on the data obtained several enzymes were selected with localization of the thiolactones near their active sites, indicating the possibility of effective catalysis. The lactonase (AiiA), metallo-β-lactamase (NDM-1) and the organophosphate hydrolase with hexahistidine tag (His₆–OPH) were among them. Determination of catalytic characteristics of enzymes in the hydrolytic reactions with the HTL and the C₂–HTL revealed the maximal value of catalytic efficiency constant for the NDM-1 in the hydrolysis of the HTL (826 M⁻¹ s⁻¹). The maximal activity in the hydrolysis of C₂–HTL was established for AiiA (137 M⁻¹ s⁻¹). The polyaspartic (PLD₅₀) and the polyglutamic (PLE₅₀) acids were used to obtain polyelectrolyte complexes with enzymes. The further combination of these complexes with the clotrimazole and polymyxin B possessing antimicrobial properties resulted in notable improvement of their action in relation to Staphylococcus cells. Conclusions: It was revealed that the antimicrobial activity of the polymyxin B is enhanced by 9–10 times against bacteria and yeast when combined with the His₆–OPH polyelectrolyte complexes. The antimicrobial activity of clotrimazole was increased by ~7 times against Candida tropicalis cells in the case of the AiiA/PLE₅₀/Clotrimazole combination. These results make the obtained biology attractive and promising for their further advancement to practical application.

Accumulating information about the growing number of microorganisms that are resistant to the antibiotics used, on the one hand, and the emergence of information about the presence of laboratories in which closed work is carried out with microbial pathogens, the negative properties of which can be artificially enhanced.The aim of the work to analyze current trends in the development of antimicrobial drugs combining the use of wellknown and widely used antibiotics, as well as substances with antimicrobial properties, including metal nanoparticles, antimicrobial peptides, with various enzymes that allow to preserve and enhance the antimicrobial effect against the cells of various microorganismsThe source base of the research is mainly English–language scientific literature available via the global Internet network, as well as the authors’ own published experimental studies.The research method is analytical.Results. The work considers enzymes that, unlike classical antimicrobial agents, which more often enter into reactions inhibiting key biochemical reactions, are catalysts for processes aimed at oxidation and hydrolysis of molecules that are metabolically and structurally important for microorganisms.Conclusion. The enzyme repeatedly enters into reactions that significantly reduce the concentrations of substances important for the vital activity of microbial cells, which leads to their weakening and more effective action of antimicrobial agents. Most often, in this case, the enzymes themselves act as carriers for molecules with antimicrobial action, which avoids their non-specific interaction with other, primarily proteins, thus removing some, for example, antibiotics, from the active effect on pathogen cells. It has been demonstrated that the enzymatic enhancement of the bactericidal action of antimicrobial agents can be in relation to different cells, including the complex biosystems (biofilms) formed by them, both by several times and by several orders of magnitude. Such antimicrobial agents combined with enzymes may be of interest for carrying out sanitary treatments of various surfaces, containers, premises, including biological laboratories, for the introduction of protective and dressing materials.

The unresolved challenges in the development of highly efficient, stable and controlled synthetic microbial consortia, as well as the use of natural consortia, are very attractive for science and technology. However, the consortia management should be done with the knowledge of how not only to accelerate but also stop the action of such “little pots”. Moreover, there are a lot of microbial consortia, the activity of which should be suppressively controlled. The processes, catalyzed by various microorganisms being in complex consortia which should be slowed down or completely cancelled, are typical for the environment (biocorrosion, landfill gas accumulation, biodegradation of building materials, water sources deterioration etc.), industry (food and biotechnological production), medical practice (vaginitis, cystitis, intestinal dysbiosis, etc.). The search for ways to suppress the functioning of heterogeneous consortia in each of these areas is relevant. The purpose of this review is to summarize the general trends in these studies regarding the targets and new means of influence used. The analysis of the features of the applied approaches to solving the main problem confirms the possibility of obtaining a combined effect, as well as selective influence on individual components of the consortia. Of particular interest is the role of viruses in suppressing the functioning of microbial consortia of different compositions.

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.

Active research of metal-containing compounds and enzymes as effective antifungal agents is currently being conducted due to the growing antifungal resistance problem. Metals are attracting special attention due to the wide variety of ligands that can be used for them, including chemically synthesized and naturally obtained variants as a result of the so-called “green synthesis”. The main mechanism of the antifungal action of metals is the triggering of the generation and accumulation of reactive oxygen species (ROS). Further action of ROS on various biomolecules is nonspecific. Various hydrolytic enzymes (glucanases and proteases), in turn, exhibit antifungal properties by affecting the structural elements of fungal cells (cell walls, membranes), fungal quorum sensing molecules, fungal own protective agents (mycotoxins and antibiotics), and proteins responsible for the adhesion and formation of stable, highly concentrated populations in the form of biofilms. A wide substrate range of enzymes allows the use of various mechanisms of their antifungal actions. In this review, we discuss the prospects of combining two different types of antifungal agents (metals and enzymes) against mycelial fungi and yeast cells. Special attention is paid to the possible influence of metals on the activity of the enzymes and the possible effects of proteins on the antifungal activity of metal-containing compounds.

Active research of metal-containing compounds and enzymes as effective antifungal agents is currently noted. The interest in metals is due to the wide variety of ligands that can be used for metals, including chemically synthesized and naturally obtained variants as a result of the so-called "green synthesis". The main mechanism of antifungal action of metals is the triggering of generation and accumulation of reactive oxygen species (ROS). Further action of ROS on various biomolecules is nonspecific. This review highlights various hydrolytic enzymes (glucanases and proteases) that affect the structural elements of fungal cells (cell walls, membranes), fungal quorum sensing molecules, fungal own protective agents (mycotoxins and antibiotics), proteins responsible for the adhesion and formation of stable highly concentrated populations in the form of biofilms. A wide range of the substrates for enzymes allows the use of various mechanisms of their antifungal actions. The prospects of combining two different types of antifungal agents (metals and enzymes) for mycelial fungi and yeast cells are discussed in this review. Special attention is paid to the possible influence of metals on activity of the enzymes and the possible effects of proteins on antifungal activity of metal-containing compounds.

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.