The aim of the work was to study the effect of Bacillus subtilis strains VKM B‑2604D and VKM B‑2605D which form the basis of the Vitaplan biological product, and their combinations with chitosan salicylate on antioxidant defense enzymes in the process of formation of resistance of wheat plants to infection with the dark brown blotch pathogen Bipolaris sorokiniana. In wheat plants treated with B. subtilis strains and their combinations with chitosan salicylate, upon subsequent infection, catalase and peroxidase are activated, which regulate the intensity of oxidative stress induced by the introduction of the pathogen. Taking into account the data obtained by us, it can be assumed that the increase in plant resistance to the pathogen is realized through the control of the activity of antioxidant enzymes (inparticular, catalase, peroxidase), which maintain the concentration of H2O2 at the level necessary for the neutralization of the phytopathogen in tissues, including the direct destruction of the invading pathogen and / or activation of crosslinking and lignification of the cell wall. These processes strengthen the cell wall and help contain the spread of the pathogen in the plant. At the same time, protective reactions are also switched on, leading to the development of induced resistance in wheat plants to dark brown spotting, which manifests itself in a decrease in the development of the disease by 25–45% relative to the infected control in the treated plants. The obtained results indicate that the combination of active strains of microorganisms-antagonists of plant pathogens and chitosan salicylate is promising for increasing the biological efficiency and expanding the spectrum of action of the developed prescription forms of biological products.
In derivatives of the Escherichia coli strain MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, devoid of mixed-acid fermentation pathways, the expression of native L-1,2-propanediol oxidoreductase and NADPH-dependent aldehyde reductase genes, fucO and yqhD, was enhanced, and the butyraldehyde dehydrogenase gene of Clostridium saccharoperbutylacetonicum, bld, was expressed. The ability to biosynthesize 1,3-butanediol from glucose resulting from a functional reversal of fatty acid β-oxidation was ensured in the recombinants due to the increased expression of the atoB and fadB genes encoding acetyl-CoA C-acetyltransferase and bifunctional (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase. Anaerobic substrate to target product conversion of 0.2 mol/mol was achieved with 4 mM 1,3-butanediol accumulation. When the intracellular availability of NADH equivalents was increased due to constitutive expression of genes of the pyruvate dehydrogenase complex, aceEF-lpdA, the conversion of glucose to 1,3-butanediol increased up to 0.3 mol/mol with accumulation of the target product at the level of 7 mM. Enhanced expression of the membrane-bound transhydrogenase genes, pntAB, led to the synthesis of 9.5 mM 1,3-butanediol by the yqhD-overexpressing strain with a yield of 0.4 mol/mol.
In recent years modified bacteriophage lysins are widely investigated for the purposes of antibacterial therapy development. Thus, effective and precise methods for the quantitative analysis of these enzymes are of high demand. The enzyme-linked immunosorbent assay (ELISA) method has been developed for the detection of recombinant modified endolysin LysAm24-SMAP in biological samples. The optimal parameters for protein detection were determined, particularly, the influence of salt and the composition of the buffer system for samples preparation was studied. The applicability of the immunodetection system of the genetically engineered endolysin LysAm24-SMAP in various biological samples with enzyme concentrations from 0.4 ng/ml was demonstrated. Also, the influence of matrix effects in animals’ organs and tissues homogenates samples, producer strain lysates and their individual components during the analysis was assessed and it was shown that 0.65 M NaCl addition in the ELISA buffer is crucial for achieving correct results and reduces non-specific interactions in the case of LysAm24-SMAP. The effectiveness of the developed system in the immunochemical control of the bacteriolytic enzyme was confirmed.
The widespread use of antibiotics, leading to antibiotic resistance and the appearance of antibiotics in the environment and food, stimulates the development of new methods for monitoring antibacterial drugs in environmental objects. One of the promising areas for the development of methods for determining antibiotics belongs to sensor technologies. The key point in the development of sensory systems is the selection of a sensitive (recognizing) element. One of the most popular methods for recognizing antibiotics is the use of antibodies. The work presents the main immunosensory systems based on recording the “antigen-antibody” interaction and shows the advantages and disadvantages of polyclonal and monoclonal antibodies. The possibility of using phage antibodies to determine of antibiotics is described separately.
The work investigated the effect of four enzyme preparations (EP): Bacillolysin, Agroprot, Protozyme and Protozyme C (Russia), on smell, taste, as well as protein and peptide profiles of protein isolate isolated from Focor peas. It has been shown that enzyme treatment improves the odor characteristics of the isolate. Thus, it was possible to significantly reduce the severity of bean and herbal smell. At the same time, enzyme treatment also improved the taste of the isolate: it was possible to significantly reduce the severity of such disturbing flavors as legume, astringent, bitter and herbal. The results obtained made it possible to select EP (fungal acidic aspartate proteinase) to improve the organoleptic parameters of pea protein isolates intended for the manufacture of analogues of meat and dairy products.
The effect of the PGPB strain of bacteria Pseudomonas sp. was studied. OBA 2.4.1, resistant to NiCl2 (up to 3 mM), Pb(CH3COO)2 (up to 5 mM) and glyphosate (up to 8 mg/ml), on Pisum sativum L. plants at different concentrations of HMs and herbicide. It was found that the strain under study had a positive effect on the length of the roots of pea plant seedlings in the presence of HM, which indicates an increase in the plant’s resistance to stress caused by exposure to nickel and lead. However, this effect was not recorded in the experimental version with the addition of glyphosate, which confirmed its high toxicity. The results obtained indicate that the strain Pseudomonas sp. OBA 2.4.1 promoted the growth of Pisum sativum L. under stress exposure to nickel and lead, which can be used in the development of complex-action biological products intended both to protect agricultural plants from the effects of heavy metals and to reclaim contaminated soils.
In this work, oxidative damage and the level of antioxidant response in Acinetobacter calcoaceticus, Pseudomonas putida, and Rhodococcus erythropolis cells under the influence of such antibiotics as ampicillin, azithromycin, rifampicin, tetracycline, and ceftriaxone were studied. The level of protein carboxylation and lipid peroxidation (LPO), as well as the activity of superoxide dismutase (SOD), catalase, glutathione reductase (GR), and the level of glutathione 3 and 6 hours after antibiotic treatment of bacteria were assessed. It is observed that SOD induction occurs earlier and is more active than catalase induction. In A. calcoaceticus, SOD is induced together with protein carboxylation and probably protects them from oxidative damage, while catalase induction correlates with LPO. A positive correlation is also noted between catalase activity and glutathione content in R. erythropolis. Catalase activity increases insignificantly and even decreases under the studied antibiotics influence, which is associated with an insignificant level of lipid peroxidation in most prokaryotes. On the other hand, low catalase activity can contribute to genome destabilization as a result of oxidative stress and enhance the adaptive evolution of bacteria.
A significant variation in the relative representation of copies of bacterial genes of dye-decolorizing DyP peroxidases typical for the genus Shewanella and a number of other microorganisms was found in the bottom sediments of freshwater reservoirs. It was found that the specific rate of decolorization of crystal violet in a laboratory bioelectrochemical system by a mixed culture of bottom sediments, which showed the highest representation of DyP genes, depended on the method of electrical stimulation of the external circuit and the concentration of the dye. After an increase in the concentration of more than 20 microns, the maximum speed was achieved in the presence of an ionistor polarly connected to the external electrical circuit of the bioelectrochemical system and amounted to 3.23 ± 0.11 μM/h, while with the opposite polarity connection, a minimum value of 2.07 ± 0.08 μM/h was observed. In the case of an open circuit and a resistor, similar indicators occurred with 2.88 ± 0.09 and 2.67 ± 0.12 μM/h, respectively. When analyzing the decolorization products, a consistent decrease in the maxima of the absorption bands of the dye was noted, indicating its more complete degradation by the mixed culture. The results may be of interest for the development of methods to improve the efficiency of bioelectrochemical methods of environmental biotechnology by electrostimulation of the external circuit.
Methionine biosynthesis in most microorganisms proceeds in two alternative ways. Each pathway is catalyzed by independent enzymes and is tightly regulated by methionine. The transulfurylation pathway involves the formation of a cystathionine, and cysteine acts as a source of sulfur. The enzymes of this metabolic pathway are characterized in detail. The direct sulfhydrylation pathway involves the synthesis of homocysteine with the participation of an inorganic sulfur source directly from O-acetylhomoserine and is predominant in most classes of bacteria. The subject of this review is the properties and functioning of one of the least studied enzymes of the direct sulfhydrylation pathway-O-acetylhomoserine sulfhydrylase. A deep understanding of the mechanisms controlling the substrate and reaction specificity of O-acetylhomoserine sulfhydrylase is a necessary step in the rational redesign of the enzyme in order to create a promising catalyst for the synthesis of methionine and its derivatives, as well as, in combination with crystallographic data, for the development of new antimicrobial compounds based on effective enzyme inhibitors.
The influence of organic solvents such as methanol and acetonitrile on the results of butachlor immunoassay in samples of rice and rice products was studied. The schemes of enzyme immunoassay using: (a) native antiserum containing specific antibodies to butachlor and antispecies antibodies labeled with horseradish peroxidase, and (b) biotinylated specific antibodies and streptavidin labeled with peroxidase are considered. The close values of IC10 (0.77 and 0.68 ng/mL, respectively) and working range (2.6–165 and 2.4–192 ng/mL, respectively) were established for the two schemes, when analyzing in a mixture of phosphate buffer and methanol in a percentage ratio of 85 : 15, respectively. For the second scheme, the detection of butachlor in butachlor in rice-containing products is shown at 80-132
CRM197 (Cross Reacting Material 197) is an inactive form of the C. diphtheriae exotoxin used as a carrier protein for the development and production of conjugated polysaccharide vaccines and immunotherapeutic drugs. However, the development of these research areas is not possible without an efficient and cost-effective technology to produce CRM197 of the proper quality. In this study, we developed a highly efficient method to produce recombinant CRM197 as a fusion with SUMO protein, yielding more than three grams per liter in the form of inclusion bodies. We examined the significant effect of the type of expression vector, the heterologous gene expression conditions, and cultivation on its solubility. Using a combination of reduced cultivation temperature and the promoter of the gene encoding the heat shock protein CspA, we achieved an increase in the solubility level of SUMO-CRM197 of more than 30
Grafted copolymers of chitosan–vinylpyrrolidone, water-soluble at a pH of 6.8–7.5, were obtained. A technique has been developed for obtaining an aggregatively stable system of platinum nanoparticles in copolymer solutions with an average size of 4 nm. The thermophysical and structural characteristics of the powdered composition of a platinum nanoparticle-copolymer are investigated. An in vitro comparison of the antitumor activity of solutions of the developed composition and cisplatin at the same platinum concentration was performed. It was found that with respect to the culture of HeLa Kyoto and A431 cancer cells, the composition is five and two times less effective than cisplatin, respectively. Along with this, the biocompatibility of the composition is 17 times higher than that of cisplatin, which allows its use at elevated concentrations and the development of an antitumor agent with platinum nanoparticles commensurate in effectiveness with cisplatin.
The role of surface antigens of Yersinia pestis in reception of the phage L-413C was evaluated experimentally. Based on the methods of phage inactivation after its co-incubation with soluble or bead-bounded antigens, the importance of the plague microbe LPS in the phage reception was confirmed, and the inability to bind the capsular antigen F1, Ail protein, and two autotransporters YapF and YapM was shown. The native and recombinant PsaA, being solved, significantly inhibited the lytic activity of the phage in contrast to the bead-bound antigens. The knockout EV cells (ΔpsaA) are able to bind the phage particles as well as the wild strain. The use of three methods to evaluate the role of the PsaA antigen in phage L-413C reception gave contradictory results. On the one hand, the reactive domains of PsaA are able to interact with phage particles in solution. At the same time, these domains appear to determine nonspecific binding of the PsaA protein to the underlying bacterial cell structures or polystyrene microsphere, preventing phage adhesion.
The chaperone system of the cell is the first line of defense in plants under stress. In the present work, the effect of heat stress on the levels of cytoplasmic HSP70 and chloroplast HSP70B chaperones in three Cucurbita species (C. maxima Duchesne, C. pepo L., and C. moschata, Duchesne) differing in their stress resistance was studied. A correlation between the levels of cytoplasmic HSP70 and chloroplast HSP70B chaperones and pumpkin species under heat stress was demonstrated. Under stress, a significant increase in the chaperone levels was observed in the cells of the C. maxima pumpkin, namely, the level of the cytoplasmic HSP70 increased by 3.6 times, and the level of chloroplast HSP70B increased by two times. Heat stress caused a 1.7-fold increase in the level of the cytoplasmic chaperone HSP70 in the cells of the C. pepo pumpkin, while no significant change in the level of the HSP70B protein was observed. However, heat stress led to a decrease in the levels of both HSP70 and HSP70B compared to untreated plants in the C. moschata pumpkin. The dynamics of changes in the levels of cytoplasmic and chloroplast chaperones under heat stress is similar. It should be noted that the constitutive levels of HSP70 and HSP70B under normal conditions are higher in C. moschata and C. pepo compared to C. maxima. The analysis of the obtained data revealed an interesting pattern: high constitutive levels of HSPs result in an insignificant induction of HSPs, and, vice versa, low constitutive levels of these proteins correlate with the high induction rate of these proteins after exposure to heat stress. These findings are important in understanding the mechanisms of plant stress resistance and may be useful for selection and development of highly resistant productive varieties of agriculturally important plants.
Previously it was shown that endophytic bacteria had the ability to move out of the roots of pea plant seedlings (Pisum sativum L.) into the rhizosphere. In this study, six distinct bacterial strains were isolated from the root growth medium during the cultivation of seedlings in an aqueous medium. By analyzing the nucleotide sequence of 16S rRNA genes, the taxonomic position of these strains was established. Their morphological and cultural parameters were assessed, and the activity of hydrolytic enzymes (pectinase, cellulase, protease) and the IAA-producing capability were examined. It has been observed that the quantity of endophytic bacteria that appears on the root surface during the growth of pea seedlings significantly surpasses the quantity present in the root tissues. It is assumed that hydrolytic enzymes such as pectinase and cellulase are involved in the release of bacteria into the external environment, causing the destruction of carbohydrate structures in plant cell walls. The metabolic parameters established in the studied strains, and the significance of these endophytic bacteria for the host plant after their exit from the roots into the rhizosphere are under discussion.
In this work, oxidative damage and the level of antioxidant response in Acinetobacter calcoaceticus, Pseudomonas putida, and Rhodococcus erythropolis cells under the influence of such antibiotics as ampicillin, azithromycin, rifampicin, tetracycline, and ceftriaxone were studied. The level of protein carboxylation and lipid peroxidation (LPO), as well as the activity of superoxide dismutase (SOD), catalase, glutathione reductase (GR), and the level of glutathione 3 and 6 hours after antibiotic treatment of bacteria were assessed. It is observed that SOD induction occurs earlier and is more active than catalase induction. In A. calcoaceticus, SOD is induced together with protein carboxylation and probably protects them from oxidative damage, while catalase induction correlates with LPO. A positive correlation is also noted between catalase activity and glutathione content in R. erythropolis. Catalase activity increases insignificantly and even decreases under the studied antibiotics influence, which is associated with an insignificant level of lipid peroxidation in most prokaryotes. On the other hand, low catalase activity can contribute to genome destabilization as a result of oxidative stress and enhance the adaptive evolution of bacteria.
The reactivity during enzymatic hydrolysis of eight industrially produced samples of pulps and semichemical pulps by enzyme preparations of glycosyl hydrolases B151 and F10 produced by a strain of the ascomycete fungus Penicillium verruculosum has been determined. It is shown for the first time that among fibrous pulps available on the market of pulp and paper industry in Russia, the highest level of yield of glucose from the initial wood during biocatalysis using cellulases and hemicellulases is characteristic of semichemical pulps obtained after green liquor cooking of hardwood. A high degree of enzymatic conversion of softwood bleached kraft pulp has been established, which in combination with the possibility of obtaining modified polysaccharide materials from non-hydrolysable residue makes this cellulosic substrate the most promising for the development of biological processes at pulp and paper industries. It is shown that drying of pulp negatively affects the efficiency of cellulose hydrolysis, while mechanical refining improves the performance of the enzymatic saccharification process.
The current information about hydrophobins, low molecular weight proteins synthesized by filamentous fungi, which are one of the strongest cellular biosurfactants, is summarized. The mechanism of biosynthesis of hydrophobins, the chemical structures and spectrum of its natural and synthetic isoforms, biological activity, and role in the regulation of vital processes of producers are presented. The potential for using hydrophobins in biotechnology has been demonstrated.
The results of studies of an intensive culture of a new species of bentoplanktonic diatom N. shiloi (Lee, Reimer et McEnery) Round, Hallsteinsen et Paasche 1999 isolated from the Black Sea are presented. Detailed descriptions are provided of the methods used to isolate the species into an algologically pure culture and its morphological and taxonomic features under light and electron scanning microscopy. The biochemical and production characteristics were also studied including the ability of the strain to accumulate fucoxanthin (Fx) and polyunsaturated fatty acids (PUFA) in laboratory conditions. During the exponential growth phase, the specific culture growth rate was µ = 0.8 1/day, and the maximum productivity P = 0.46 g dry weight/(L day). The accumulation of PUFAs in the biomass of N. shiloi reached 67.39 mg/g dry weight of algae. The Fx concentration in the biomass at the beginning of the stationary growth phase was 10 mg/g dry weight. The fairly high rate of Fx biosynthesis in microalgae cells, as well as the composition of fatty acids of the Black Sea strain, makes it possible to classify N. shiloi as a promising object in biotechnology.
In this article, strong promoters of thermotolerant methylotrophic yeast Ogataea haglerorum have been characterized. Promoters play a key role in the regulation of gene expression; therefore, they are the important element of expression vectors. Strong and strictly regulated promoters are a powerful tool for creating highly productive strains — producers of recombinant proteins. To expand the potential of the O. haglerorum expression system natural methanol-induced promoters of the OhMOX and OhFMD genes and the constitutive promoter of the OhGAP gene were studied in comparison with the promoter of the MOX gene from O. polymorpha yeast. A gene encoding recombinant β-mannanase was used as a reporter gene. It has been shown that in O. haglerorum yeast cells, the expression level (strength) of the pOhMOX promoter is about 1.4–1.9 times higher relative to the pOpMOX promoter from O. polymorpha yeast. The obtained data on the strength of promoters from yeast O. haglerorum can be useful in designing producers of recombinant proteins and optimizing metabolic pathways in methylotrophic yeast O. haglerorum.
