Luminescent Bacteria as Bioindicators in Screening and Selection of Enzymes Detoxifying Various Mycotoxins.

Interest in enzymes capable of neutralizing various mycotoxins is quite high. The methods used for the screening and selection of enzymes that catalyze the detoxification of mycotoxins should be sensitive and fast. However toxic compounds can be generated under the action of such enzymes. Thus, the assessment of the overall reduction in the toxic properties of reaction media towards bioluminescent bacteria seems to be the most reasonable control method allowing a quick search for the effective enzymatic biocatalysts. The influence of a wide range of mycotoxins and glucanases, which hydrolyze toxins with different chemical structures, on the analytical characteristics of luminescent photobacteria as a biosensing element has been studied. Different glucanases (ß-glucosidase and endoglucanase) were initially selected for reactions with 10 mycotoxins based on the results of molecular docking which was performed in silico with 20 mycotoxins. Finally, the biorecognizing luminescent cells were used to estimate the residual toxicity of reaction media with mycotoxins after their interaction with enzymes. The notable non-catalytic decrease in toxicity of media containing deoxynivalenol was revealed with luminous cells for both types of tested glucanases, whereas ß-glucosidase provided a significant catalytic detoxification of media with aflatoxin B2 and zearalenone at pH 6.0.

Advanced Situation with Recombinant Toxins: Diversity, Production and Application Purposes.

Today, the production and use of various samples of recombinant protein/polypeptide toxins is known and is actively developing. This review presents state-of-the-art in research and development of such toxins and their mechanisms of action and useful properties that have allowed them to be implemented into practice to treat various medical conditions (including oncology and chronic inflammation applications) and diseases, as well as to identify novel compounds and to detoxify them by diverse approaches (including enzyme antidotes). Special attention is given to the problems and possibilities of the toxicity control of the obtained recombinant proteins. The recombinant prions are discussed in the frame of their possible detoxification by enzymes. The review discusses the feasibility of obtaining recombinant variants of toxins in the form of protein molecules modified with fluorescent proteins, affine sequences and genetic mutations, allowing us to investigate the mechanisms of toxins' bindings to their natural receptors.

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).

Metal Nanomaterials and Hydrolytic Enzyme-Based Formulations for Improved Antifungal Activity.

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.

Combined Modification of Fiber Materials by Enzymes and Metal Nanoparticles for Chemical and Biological Protection.

To obtain fiber materials with pronounced chemical-biological protection, metal (Zn or Ta) nanoparticles were jointly applied with polyelectrolyte complexes of enzymes and polypeptides being their stabilizers. Computer modeling revealed the preferences between certain polyelectrolyte partners for N-acyl-homoserine lactone acylase and hexahistidine-tagged organophosphorus hydrolase (His6-OPH) possessing the quorum quenching (QQ) behavior with bacterial cells. The combinations of metal nanoparticles and enzymes appeared to function better as compared to the combinations of the same QQ-enzymes with antibiotics (polymyxins), making it possible to decrease the applied quantities by orders of magnitude while giving the same effect. The elimination of Gram-positive and Gram-negative bacterial cells from doubly modified fiber materials notably increased (up to 2.9-fold), whereas His6-OPH retained its hydrolytic activity in reaction with organophosphorus compounds (up to 74% of initially applied activity). Materials with the certain enzyme and Zn nanoparticles were more efficient against Bacillus subtilis cells (up to 2.1-fold), and Ta nanoparticles acted preferentially against Escherichia coli (up to 1.5-fold). Some materials were proved to be more suitable for combined modification by metal nanoparticles and His6-OPH complexes as antimicrobial protectants.

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions.

Organophosphorus compounds (OPCs) are able to interact with various biological targets in living organisms, including enzymes. The binding of OPCs to enzymes does not always lead to negative consequences for the body itself, since there are a lot of natural biocatalysts that can catalyze the chemical transformations of the OPCs via hydrolysis or oxidation/reduction and thereby provide their detoxification. Some of these enzymes, their structural differences and identity, mechanisms, and specificity of catalytic action are discussed in this work, including results of computational modeling. Phylogenetic analysis of these diverse enzymes was specially realized for this review to emphasize a great area for future development(s) and applications.

Enzymes for Detoxification of Various Mycotoxins: Origins and Mechanisms of Catalytic Action.

Mycotoxins are highly dangerous natural compounds produced by various fungi. Enzymatic transformation seems to be the most promising method for detoxification of mycotoxins. This review summarizes current information on enzymes of different classes to convert various mycotoxins. An in-depth analysis of 11 key enzyme mechanisms towards dozens of major mycotoxins was realized. Additionally, molecular docking of mycotoxins to enzymes' active centers was carried out to clarify some of these catalytic mechanisms. Analyzing protein homologues from various organisms (plants, animals, fungi, and bacteria), the prevalence and availability of natural sources of active biocatalysts with a high practical potential is discussed. The importance of multifunctional enzyme combinations for detoxification of mycotoxins is posed.

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.

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.

Using Fungi in Artificial Microbial Consortia to Solve Bioremediation Problems.

There is currently growing interest in the creation of artificial microbial consortia, especially in the field of developing and applying various bioremediation processes. Heavy metals, dyes, synthetic polymers (microplastics), pesticides, polycyclic aromatic hydrocarbons and pharmaceutical agents are among the pollutants that have been mainly targeted by bioremediation based on various consortia containing fungi (mycelial types and yeasts). Such consortia can be designed both for the treatment of soil and water. This review is aimed at analyzing the recent achievements in the research of the artificial microbial consortia that are useful for environmental and bioremediation technologies, where various fungal cells are applied. The main tendencies in the formation of certain microbial combinations, and preferences in their forms for usage (suspended or immobilized), are evaluated using current publications, and the place of genetically modified cells in artificial consortia with fungi is assessed. The effect of multicomponence of the artificial consortia containing various fungal cells is estimated, as well as the influence of this factor on the functioning efficiency of the consortia and the pollutant removal efficacy. The conclusions of the review can be useful for the development of new mixed microbial biocatalysts and eco-compatible remediation processes that implement fungal cells.

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.

Combination of Enzymes with Materials to Give Them Antimicrobial Features: Modern Trends and Perspectives.

Multidrug-resistant bacteria form serious problems in many areas, including medicine and the food industry. At the same time, great interest is shown in the transfer or enhancement of antimicrobial properties to various materials by modifying them with enzymes. The use of enzymes in biomaterials with antimicrobial properties is important because enzymes can be used as the main active components providing antimicrobial properties of functionalized composite biomaterials, or can serve as enhancers of the antimicrobial action of certain substances (antibiotics, antimicrobial peptides, metal nanoparticles, etc.) against cells of various microorganisms. Enzymes can simultaneously widen the spectrum of antimicrobial activity of biomaterials. This review presents the most promising enzymes recently used for the production of antibacterial materials, namely hydrolases and oxidoreductases. Computer modeling plays an important role in finding the most effective combinations between enzymes and antimicrobial compounds, revealing their possible interactions. The range of materials that can be functionalized using enzymes looks diverse. The physicochemical characteristics and functionalization methods of the materials have a significant impact on the activity of enzymes. In this context, fibrous materials are of particular interest. The purpose of this review is to analyze the current state of the art in this area.

Quorum Sensing as a Trigger That Improves Characteristics of Microbial Biocatalysts.

Quorum sensing (QS) of various microorganisms (bacteria, fungi, microalgae) today attracts the attention of researchers mainly from the point of view of clarifying the biochemical basics of this general biological phenomenon, establishing chemical compounds that regulate it, and studying the mechanisms of its realization. Such information is primarily aimed at its use in solving environmental problems and the development of effective antimicrobial agents. This review is oriented on other aspects of the application of such knowledge; in particular, it discusses the role of QS in the elaboration of various prospective biocatalytic systems for different biotechnological processes carried out under aerobic and anaerobic conditions (synthesis of enzymes, polysaccharides, organic acids, etc.). Particular attention is paid to the biotechnological aspects of QS application and the use of biocatalysts, which have a heterogeneous microbial composition. The priorities of how to trigger a quorum response in immobilized cells to maintain their long-term productive and stable metabolic functioning are also discussed. There are several approaches that can be realized: increase in cell concentration, introduction of inductors for synthesis of QS-molecules, addition of QS-molecules, and provoking competition between the participants of heterogeneous biocatalysts, etc.).

Progressive Biocatalysts for the Treatment of Aqueous Systems Containing Pharmaceutical Pollutants.

The review focuses on the appearance of various pharmaceutical pollutants in various water sources, which dictates the need to use various methods for effective purification and biodegradation of the compounds. The use of various biological catalysts (enzymes and cells) is discussed as one of the progressive approaches to solving problems in this area. Antibiotics, hormones, pharmaceuticals containing halogen, nonsteroidal anti-inflammatory drugs, analgesics and antiepileptic drugs are among the substrates for the biocatalysts in water purification processes that can be carried out. The use of enzymes in soluble and immobilized forms as effective biocatalysts for the biodegradation of various pharmaceutical compounds (PCPs) has been analyzed. Various living cells (bacteria, fungi, microalgae) taken as separate cultures or components of natural or artificial consortia can be involved in biocatalytic processes under aerobic or anaerobic conditions. Cells as biocatalysts introduced into water treatment systems in suspended or immobilized form are used for deep biodegradation of PCPs. The potential of combinations of biocatalysts with physical-chemical methods of wastewater treatment is evaluated in relation to the effective removing of PCPs. The review analyzes recent results and the main current trends in the development of biocatalytic approaches to biodegradation of PCPs, the pros and cons of the processes and the biocatalysts used.

Destruction of Mycotoxins in Poultry Waste under Anaerobic Conditions within Methanogenesis Catalyzed by Artificial Microbial Consortia.

To reduce the toxicity of modern feeds polluted by mycotoxins, various sorbents are added to them when feeding animals. A part of the mycotoxins is excreted from the body of animals with these sorbents and remains in the manure. As a result, bulk animal wastes containing mixtures of mycotoxins are formed. It is known that it is partially possible to decrease the initial concentration of mycotoxins in the process of anaerobic digestion (AD) of contaminated methanogenic substrates. The aim of this review was to analyze the recent results in destruction of mycotoxins under the action of enzymes present in cells of anaerobic consortia catalyzing methanogenesis of wastes. The possible improvement of the functioning of the anaerobic artificial consortia during detoxification of mycotoxins in the bird droppings is discussed. Particular attention was paid to the possibility of effective functioning of microbial enzymes that catalyze the detoxification of mycotoxins, both at the stage of preparation of poultry manure for methanogenesis and directly in the anaerobic process itself. The sorbents with mycotoxins which appeared in the poultry wastes composed one of the topics of interest in this review. The preliminary alkaline treatment of poultry excreta before processing in AD was considered from the standpoint of effectively reducing the concentrations of mycotoxins in the waste.

Artificial Humic Substances as Biomimetics of Natural Analogues: Production, Characteristics and Preferences Regarding Their Use.

Various processes designed for the humification (HF) of animal husbandry wastes, primarily bird droppings, reduce their volumes, solve environmental problems, and make it possible to obtain products with artificially formed humic substances (HSs) as analogues of natural HSs, usually extracted from fossil sources (coal and peat). This review studies the main characteristics of various biological and physicochemical methods of the HF of animal wastes (composting, anaerobic digestion, pyrolysis, hydrothermal carbonation, acid or alkaline hydrolysis, and subcritical water extraction). A comparative analysis of the HF rates and HS yields in these processes, the characteristics of the resulting artificial HSs (humification index, polymerization index, degree of aromaticity, etc.) was carried out. The main factors (additives, process conditions, waste pretreatment, etc.) that can increase the efficiency of HF and affect the properties of HSs are highlighted. Based on the results of chemical composition analysis, the main trends and preferences with regard to the use of HF products as complex biomimetics are discussed.

Decarboxylases as hypothetical targets for actions of organophosphates: Molecular modeling for prediction of hidden and unexpected health threats.

The rise of various neurodegenerative disorders are somewhat correlating with the worldwide application of multiple anthropogenic toxicants. Though different possible targets were revealed to date, for example, for organophosphorus compounds (OPs), plenty of questions remain. Several decarboxylases (aromatic amino acid decarboxylase, AADC; histidine decarboxylase, HDC; glutamate decarboxylase, GAD) catalyze the biosynthesis of neurotransmitters and neuromodulators and contain pyridoxal phosphate (PLP) as a cofactor. In the current work, 18 OPs which have different neurotoxicity (chemical warfare agents and pesticides) and can penetrate through the blood-brain barrier, were selected. Then, their possible interaction with these decarboxylases in both apo- and holoforms was revealed using computer modeling methods (molecular docking and dynamics). The main amino acid residues of the enzymes responsible for binding OPs have been identified. Individual substances that are most dangerous from the point of view of a possible negative effect on the activity of several decarboxylases were revealed among studied OPs. Glyphosate should be of special interest, since it is not highly toxic towards serine hydrolases, but may prove to be a strong inhibitor for decarboxylases. Holo-AADC could be the most inhibition-prone enzyme among all those investigated.

Degradation of mycotoxins in mixtures by combined proteinous nanobiocatalysts: In silico, in vitro and in vivo.

New combined proteinous (enzymatic) nanobiocatalysts capable of destructing mycotoxins in mixtures were developed and investigated in vitro and in vivo. Candidate enzymes for such combined biocatalysts were computationally screened using molecular docking of mycotoxins to the proteins. Catalytic characteristics of the 7 selected enzymes were estimated in the potential reactions with various mycotoxins (aflatoxin B1, citrinin, deoxynivalenol, ergotamine, fumonisin B1, gliotoxin, ochratoxin A, patulin, sterigmatocystin, T-2 toxin, zearalenone) at different pH values. To stabilize the enzymes hydrolyzing the mycotoxins, special biopolymers were selected using computer modeling. The poly(glutamic acid) was revealed as universal partner for the polyelectrolyte complexes with the selected enzymes. Finally, Sprague-Dawley rats were used for in vivo feeding experiments with feed contaminated by mycotoxin mixture at doses being up to orders of magnitude higher than maximum allowable limits. The treatment of contaminated feed by novel combined enzyme nanocomplexes significantly decreased negative effects of mycotoxin mixture on blood biochemical parameters which indicated huge damage to liver and kidneys of intoxicated animals. Such nanobiocatalysts and enzymatic treatment itself seem to be promising way for ensuring both food and feed chemical safety.

"Unity and Struggle of Opposites" as a Basis for the Functioning of Synthetic Bacterial Immobilized Consortium That Continuously Degrades Organophosphorus Pesticides.

This work was aimed at the development of an immobilized artificial consortium (IMAC) based on microorganisms belonging to the Gram-positive and Gram-negative bacterial cells capable of jointly carrying out the rapid and effective degradation of different organophosphorus pesticides (OPPs): paraoxon, parathion, methyl parathion, diazinon, chlorpyrifos, malathion, dimethoate, and demeton-S-methyl. A cryogel of poly(vinyl alcohol) was applied as a carrier for the IMAC. After a selection was made between several candidates of the genera Rhodococcus and Pseudomonas, the required combination of two cultures (P. esterophilus and R. ruber) was found. A further change in the ratio between the biomass of the cells inside the granules of IMAC, increasing the packing density of cells inside the same granules and decreasing the size of the granules with IMAC, gave a 225% improvement in the degradation activity of the cell combination. The increase in the velocity and the OPP degradation degree was 4.5 and 16 times greater than the individual P. esterophilus and R. ruber cells, respectively. Multiple uses of the obtained IMAC were demonstrated. The increase in IMAC lactonase activity confirmed the role of the cell quorum in the action efficiency of the synthetic biosystem. The co-inclusion of natural strains in a carrier during immobilization strengthened the IMAC activities without the genetic enhancement of the cells.

His6-OPH enzyme-based bio-hybrid material for organophosphate detection.

In this work, we report on the development of a bio-sensing film for the detection of organophosphorous compounds using sol-gel technology. A novel sol-gel immobilization method employing tetraethoxysilane/3-glycidoxypropyltrimethoxysilane/water hybrid material was developed and used to immobilize the hexahistidine-tagged organophosphorous hydrolase enzyme (His(6)-OPH). Bio-sensing layers with encapsulated His(6)-OPH of various structures (water/silane, precursor ratios) have been prepared. The optimal (P = 5:1, R = 188) bio-sensing layers retained 90% of the initial enzyme activity. Furthermore, the bio-sensing layer prepared by this method was able to maintain its activity at or above 80% of its initial activity for 2 weeks. The bio-hybrid film also showed excellent reusability and improved activity at neutral pH in comparison to the same enzyme in solution.