Elena Efremenko

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.

Background: Interest in processing high-tonnage biodiesel production waste (crude glycerol and microalgal biomass after lipid extraction) into commercial products is steadily growing. Moreover, it is attractive to utilize these wastes for the microbiological production of fumaric acid (FA) using immobilized microorganisms, as it combines chemical and biological processes. Methods: The authors developed original approaches to FA production, which immobilize microalgal Chlorella vulgaris cells and the mycelium of the fungus Rhyzopus oryzae. Traditional techniques were employed for cultivating microorganisms, extracting lipids from accumulated microalgal biomass, and producing biomass hydrolysates through thermolysis under acidic conditions. The biochemical composition of C. vulgaris biomass obtained under different growth conditions (autotrophic, heterotrophic, and mixotrophic) was analyzed using standard methods. Results: The maximum accumulation of microalgal biomass was achieved under mixotrophic conditions using an inoculum immobilized in polyvinyl alcohol (PVA) cryogel in Tamiya medium supplemented with crude glycerol and bubbled with air containing 1% vol. CO2. A total of 4.8 ± 0.2 g DW/L of free C. vulgaris biomass was accumulated in five batch cycles. Two types of C. vulgaris hydrolysates were obtained: hydrolysates from whole cell C. vulgaris biomass (ChVH1) and hydrolysates from C. vulgaris biomass waste after lipid extraction (ChVH2), respectively. In combination with crude glycerol (10–50 g/L), microalgal hydrolysates were transformed to FA using fungi R. oryzae immobilized in PVA cryogel. The highest concentration and yield of FA from the converted substrate were obtained when 20 g/L of crude glycerol, in combination with C. vulgaris hydrolysates, was administered alongside the fungi for 40 h. Multiple uses (five cycles, 200 h total) of immobilized fungi added to the medium with 20 g/L of crude glycerol and ChVH2 produced 186 g of FA. The average FA yield was 309 ± 12 mg/g substrate. Conclusion: A combined approach to producing FA by bioconversion of crude glycerol and hydrolysates of lipid-free microalgae biomass waste is of practical importance. These results expand the potential of bioscience in utilizing microorganisms in an immobilized form, contributing to the integration of environmental biotechnology into bioenergy production.

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.

Environmental pollution continues to be an urgent global problem that requires innovative solution approaches, and biotechnology promises to address environmental problems and also serve as a scientific and practical basis for the remediation of various natural and artificial systems

In recent years, the sustained and even increasing interest in the development and application of novel composite materials based on the polysaccharide bacterial cellulose (BC) has been driven by the accumulation of experimental data and the emergence of analytical reviews that narratively summarize these findings. This review presents a comparative and critical analysis of various approaches to the fabrication of BC-based composites. Among them, in situ biosynthesis is highlighted as the most promising strategy. In this approach, different additives are introduced directly into the culture medium of BC-producing microorganisms, enabling the formation of materials with different mechanical and physicochemical properties. Such a method also allows imparting to the composites a range of properties that BC itself does not possess, including antibacterial and enzymatic activity, as well as electrical conductivity. During the so-called “cell weaving” stage, performed by BC-producing microorganisms, diverse substances and microorganisms can be incorporated into the cultivation medium. By varying the concentrations of the introduced compounds, their ratios to the synthesized BC, and by employing different BC-producing strains and substrates, it becomes possible to regulate the characteristics of the resulting composites. Special attention is given to the role of various polysaccharides that are either introduced into the medium during BC biosynthesis or co-synthesized alongside BC within the same environment. Depending on the mode of incorporation of these additional polysaccharides, the resulting materials demonstrate variations in Young’s modulus and tensile strength. Nevertheless, they almost invariably exhibit a decreased degree of BC crystallinity within the composite structure and an enhanced water absorption capacity compared to the pure polymer.

The current interest in microalgal biomass does not subside but continues to intensify due to the emergence of new trends in the use of this bioresource in various biotechnological and environmental processes. The rather slow growth rate of phototrophs compared to other microorganisms limits more active application of the biomass for various purposes. Stimulation of the Quorum Sensing formation in the cells due to the appearance of their own quorum molecules or those produced by other co-cultured microorganisms in the medium is one of the efficient approaches for overcoming this limitation. This review discusses the immobilization or co-immobilization of phototrophic cells with other microorganisms as an effective way to maintain accumulation of the target biomass for long-term period at improved rates. The 40% increase in the use of co-immobilized phototrophs for biomass obtaining and its use in wastewater treatment has been observed over the past five years. The level of investigations of co-immobilized microalgae cells is four times higher than that of the immobilized single cultures. Among the main trends in the new investigations of immobilized forms of microalgae, the predominant application of Chlorella genus cells in immobilized samples of individual cultures and the involvement of diatom microalgae and cyanobacteria, in addition to Chlorella cells, in co-immobilization with other microorganisms, was ascertained. The most significant increase in the rate of microalgal biomass accumulation uncovered in cases of co-immobilization of microalgae with bacteria. In several cases, in the presence of bacteria, co-immobilization has led to the emergence of new characteristics in microalgal cells (increased synthesis of pigments, polysaccharides, biofilm formation, etc.), which opens new directions for their further practical use as biopesticides, components of packaging and building materials, etc.

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.

The concentration of intracellular adenosine triphosphate (ATP) is one of the most important characteristics of the metabolic state of the cells of microorganisms and their viability. This indicator, monitored by bioluminescent ATP-metry, and accumulation of the suspension biomass in the medium were used to assess the effect of particles of different synthetic microplastics (MPs) (non-biodegradable and biodegradable) on the cells of yeast, filamentous fungi, bacteria and phototrophic microorganisms (microalgae and cyanobacteria) co-exposed with polymer samples in different environments and concentrations. It was found that the effect of MPs on microorganisms depends on the concentration of MPs (1–5 g/L), as well as on the initial concentration of cells (10⁴ or 10⁷ cells/mL) in the exposure medium with polymers. It was shown that the lack of a sufficient number of nutrition sources in the medium with MPs is not fatal for the cells. The study of the effect of MPs on the photobacteria Photobacterium phosphoreum, widely used as a bioindicator for assessing the ecotoxicity of various environments, demonstrated a correlation between the residual bioluminescence of these cells and the level of their intracellular ATP in media with biodegradable polycaprolactone and polylactide, which had an inhibitory effect on these cells. Marine representatives of phototrophic microorganisms showed the greatest sensitivity to the presence of MPs, which was confirmed by both a decrease in the level of intracellular ATP and the concentration of their biomass. Among the eight microorganisms studied, bacteria of the genus Pseudomonas turned out to be not only the most tolerant to the presence of the seven MP samples used in the work, but also actively growing in their presence.

This work was undertaken in order to identify the presence of changes in the characteristics of poly(vinyl alcohol) (PVA) cryogels that can contribute to the degradation of such polymer matrices under the influence of their contact with various microorganisms used in immobilized form in different biotechnological processes using various complex media and conditions. Immobilized cells of bacteria, yeasts, microalgae, fungi, and microbial consortia were involved in the investigations. It was established that the presence of microorganisms can indirectly (through media transformed by them, in particular, containing lipids) or directly (through high rates of metabolite production, in particular, the fast accumulation of gases in the pores of polymer matrices, or due to the colonization of cryogels (self)immobilization by fungi with the growing mycelium) decrease rheological characteristics of PVA cryogel. Such weakening of PVA cryogels can be expected as a result of the first stage of further degradation of polymer matrices. The values of both the modulus of elasticity and the shear modulus of PVA cryogels confirmed this. The effect of high pressure accumulated in the reactors with PVA cryogel-immobilized cells, as well as their use in flow systems, was not revealed. These factors can be taken into account for the sustainable use of matrices based on PVA cryogels as biocatalysts with microorganisms or soil-structuring elements in artificial or natural environments.