Construction and Functional Analysis of Mycolicibacterium smegmatis Recombinant Strains Carrying the Genes of Bacillary Cytochromes CYP106A1 and CYP106A2

Mycolicibacterium smegmatis mc2155 has been genetically modified to be used as a platform for the expression of foreign cytochrome P450 monooxygenases by introducing deletions in the kshB and kstD genes that encode key stages of the enzymatic destruction of the steroid nucleus. Three sets of genetic constructs have been created for heterologous expression of the genes of cytochromes P450 CYP106A1 from Bacillus megaterium DSM319 and CYP106A2 from Bacillus megaterium ATCC13368 in Mycolicibacterium smegmatis mc2155 (ΔkshBΔkstD) cells. The recombinant plasmids contained monocistronic expression cassettes of cytochrome genes (NS31 and pNS32), or tricistronic cassettes of cytochrome genes together with cDNA copies of adrenodoxin and andrenodoxin reductase genes of the bovine adrenal cortex (pNS33 and pNS34), or monocistronic gene cassettes of chimeric cytochromes fused with the DNA sequence encoding the CYP116B2 reductase domain from the soil bacterium Rhodococcus sp. NCIMB 9784 (pNS35 and pNS36). The recombinant strains of mycolicibacteria were shown to selectively monohydroxylate androstenedione (AD) under growth conditions. The product was identified as 15-hydroxyandrostenedione (15-OH-AD) by mass spectrometry and 1H and 13C NMR spectroscopy. The maximum level of 15-OH-AD production (17.3 ± 1.5 mg/L) was observed when using the recombinant M. smegmatis mc2155 (ΔkshBΔkstD) (pNS32) strain, which expresses a single cyp106A2 gene from B. megaterium ATCC13368. Host proteins of M. smegmatis mc2155 were shown to be capable of supplying electrons to heterologous cytochromes to support their hydroxylating activity. The results are of priority character, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes CYP106A1/A2 and are important for the creation of microbial strains producing valuable hydroxysteroids. cyp106A1, cyp106A2, cytochrome P450, heterologous expression, Bacillus megaterium, Mycolicibacterium smegmatis, 15β-hydroxylation, bioconversion, steroids This work was supported by the Russian Science Foundation (project No. 21-64-00024).

Co-Purification of Recombinant Modified Glucagon-Like and Glucose-Dependent Insulinotropic Peptide to Create a Two-Component Drug for the Treatment of Type 2 Diabetes Mellitus and Obesity

In addition to the previously developed recombinant modified human glucagon-like peptide 1 (rmglp1, Glypin), a recombinant modified human glucose-dependent insulinotropic peptide (RMGIP) has been obtained. A new universal reverse-phase HPLC technique has been proposed allowing quantitative analysis of rmGlp1 and rmGip separately and as part of a two-component preparation. The data show that the design of recombinant human rmGip according to the Glypine formula makes it possible to produce one-component and two-component preparations containing various rmGip and rmGlp1 protein ratios ranging from 1:0 to 20:1, using cell biomass samples mixed in predetermined proportions. Studies of human rmGip activity in a mouse model revealed reduced specific activity and signs of weak antagonistic effects. In this regard, there is a need for further study of human rmGip activity in a mouse model, including the use of alternative mouse or rat rmGip. type 2 diabetes mellitus; two-component drug, glucose-dependent insulinotropic peptide, glucagon-like peptide-1 The work was supported by the Internal Grant from National Research Center Kurchatov Institute.

Selection and Substantiation of Methods for Evaluating Cosmetic Products for Deodorant and Antiperspirant Action

The possibility of using simple and available methods for analyzing deodorants/antiperspirants has been studied. The gravimetric method was shown to have acceptable metrological characteristics under repeatability conditions when evaluating antiperspirant activity. A decrease in the number of microorganisms (CFU) on the axilla skin was observed in a rinse test experiment 4 h and 8 h after the application of deodorants/antiperspirants. The microbial population data were inversely proportional to the antiperspirant activity values of the tested compositions. The sweat secretion reducing decreases the amount of nutrients required for microbial development, which makes it possible to use the rinse test to indirectly evaluate deodorant activity in research and development of personal care products. However, due to its laboriousness and the need for volunteers, the method cannot be recommended for large-scale testing. It was shown that the disc diffusion method (DDM) used to detect Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis cannot be applied to the assessment of the intrinsic antimicrobial activity of the tested cosmetic compositions. This indicates the necessity of additional studies to select test microorganisms typical for the armpit area. In addition, DDM is useful if the deodorant effect of the composition is created by the addition of low-volatile antibacterial compounds. Therefore, microbiological methods have limited applications and are not suitable for widespread use. deodorant action; antiperspirant action, gravimetry, disc diffusion method, rinse test; deodorant; antiperspirant; cosmetic; efficiency; consumer properties, functional properties This work was supported by MUCTR (project no. K-2020-007).

Community of Hydrocarbon-Oxidizing Bacteria in Petroleum Products on the example of Ts-1 Aviation Fuel and AI-95 Gasoline

It has been shown that the studied petroleum products (kerosene and gasoline) contain microflocules of heterogeneous microbial biofilms, the cells of which are integrated into a polymer matrix containing acidic polysaccharides. Thirteen bacterial strains were microbiologically isolated from petroleum products, and their taxonomy was identified by the 16S rRNA sequence. Kerosene was characterized by a diverse bacterial composition including the following genera: Sphingobacterium, Alcaligenes, Rhodococcus and Deinococcus, while gasoline bacterial community included only two genera: Bacillus and Paenibacillus. Representatives of the Deinococcus genera capable of growing on the hydrocarbons were isolated from fuels for the first time. The strains isolated from gasoline (Bacillus safensis Bi13 and Bacillus sp. Bi14) proved to be the most effective biodegraders of all n-alkanes, isoalkanes, cycloalkanes, alkenes and aromatic hydrocarbons, whereas the kerosene strain Rhodococcus erythropolis Bi6 effectively decomposed n-alkanes and trimethylbenzene. Both types of petroleum products contained hydrocarbon-oxidizing communities, some members of which were more active in the biodegradation of hydrocarbons, while others were capable of producing biosurfactants and had either emulsifying activity (Deinococcus sp. Bi7) or cell wall hydrophobicity (Sphingobacterium sp. Bi5 from kerosene; Bacillus pumilus Bi12 from gasoline) significantly higher than the average level. The indicated properties of the studied strains make them promising for use in bioremediation. biodegradation, petroleum products, hydrocarbon-oxidizing bacteria, bio-surfactants The work was carried out within the framework of the state assignment of the Ministry of Science and Higher Education of the Russian Federation (topic no. 10.5422.2017/8.9.). Investigation of microbial potential in the use hydrocarbons was supported by the Russian Foundation for Basic Research (RFBR), contract no. 18-29-05067. Physicochemical research was performed within the framework of the state assignment to the TIPS RAS

Actinomycetes as the basis of Probiotics for Plants

The present review describes the concept of probiotics for plants and analyzes the prospects for using actinomycetes as producers of these drugs. The minimum requirements for plant probiotic microorganisms are proposed, similar to those for human probiotic microorganisms. These are utility, efficiency and safety for plants, as well as mandatory isolation from plant samples. It is noted that these requirements are usually met by endophytic and rhizosphere microorganisms that stimulate plant growth and provide them with protection from phytopathogens. Evidence is given for the possibility of attributing actinomycetes to probiotic plant bacteria, due to the close relationship of these microorganisms with plants, their wide distribution in populations of endophytic and rhizosphere microorganisms, and the presence of phytoregulatory activity. The review provides examples of genera and species of actinomycetes that are promising producers of probiotics for agronomically important crops. The most studied and commercialized of them are representatives of the Streptomyces genus. The current state, prospects and problems in commercialization of probiotics based on actinomycetes are discussed. probiotic microorganisms of plants, associative actinomycetes, endophytes, rhizosphere, biological preparations

Natural Stilbene Polyphenols as Yarrowia lipolytica Cell Cytoprotectors at Heat Stress

Using the extremophilic yeast of Yarrowia lipolytica, a new model has been proposed to study the protective properties of stilbene polyphenols, namely resveratrol and pinosylvin, under heat shock. It was shown that a short-term thermal exposure of yeast cells (55 C, 25 min) led to a 40% decrease in the colony-forming ability of the population, a fivefold decrease in the respiration rate, and a growth of cyanide resistance and catalase activity, which indicated the adaptive yeast response to heat stress. Under these conditions, natural biologically active stilbenes, resveratrol and pinosylvin, at a concentration of 10 μM each increased yeast survival by 28% and 13%, respectively. In heat shock, resveratrol additionally raised catalase activity, while pinosylvin increased the cell respiration rate and decreased cyanide resistance and catalase activity. The results obtained indicate that resveratrol acts as a mild pro-oxidant inducing antioxidant protection during the adaptive response of the yeast to heat shock. Unlike resveratrol, pinosylvin increases cell survival stabilizing mitochondrial function and preserving the ATP-generating component of respiration. Yarrowia lipolytica yeast, polyphenols, stilbenoids, resveratrol, pinosylvin, cellular respiratory activity, heat shock, superoxide dismutase, catalase

Scaling up of the Production of Recombinant Granulocyte-Macrophage Colony-Stimulating Factor and Obtaining Constructs Based on it

Abstract-A step-by-step scaling-up of the production of recombinant human granulocyte-macrophage colony-stimulating factor has been carried out. The tenfold expantion of the technology made it possible to obtain 5 mg of the target protein with a purity of more than 96% from 1 g of wet cells. The produced protein series meets the requirements for recombinant proteins of the XIV edition of the State Pharmacopoeia of the Russian Federation. Conjugates of recombinant human granulocyte-macrophage colony-stimulating factor with dextran and molecular construct of the conjugates and double-stranded RNA were obtained. The adjuvant activity of the resulting preparations was shown in different models. Key words: granulocyte-macrophage colony-stimulating factor, chromatography, molecular construct We express our gratitude to the staff of the Laboratory of Immunochemistry of the SRC VB Vector N. V. Volkova, M. B. Borgoyakova, A. A. Isaeva and S. V. Belenkaya for assessing immunogenicity in the RBD model.

Trypsinolysis of Polyurethane Particles Based on Сyclodextrin with Encapsulated Moxifloxacin

Abstract-Moxifloxacin encapsulation in polymer cyclodextrin-based particles with an average hydrodynamic diameter of 150--200 nm has led to the formation of potential delivery systems with a degree of moxifloxacin inclusion of more than 80%. Cross-linking of the moxifloxacin-cyclodextrin complexes caused a pronounced slowdown in the release of the drug molecules in acidic media to less than 10% per day. In the presence of trypsin, the drug release was accelerated by 15--20% within 90 min. It was shown by Fourier-transform infrared spectroscopy that this acceleration was due to the partial enzymatic degradation of the urethane bonds of the polymer matrix near the surface of the particles. The results obtained are important for the development of highly effective oral dosage forms of prolonged action. Key words: fluoroquinolones, cyclodextrins, FTIR spectroscopy, trypsin

Physiological and Biochemical Evaluation of the Effectiveness of a New Food Ingredient, a Source of Phytoecdysteroids and Flavonoids from Quinoa Grain

Abstract- One of the promising food sources of biologically active substances is quinoa grain, which is valued for its high content of protein, sulfur-containing amino acids, lysine, fiber, and minerals. In addition, quinoa grain can be a valuable food source of polyphenolic compounds and phytoecdysteroids. The method for production of a concentrate of flavonoids and 20-hydroxyecdysone from quinoa grains sorbed on coagulated egg protein has been developed. The in vivo evaluation of efficacy of the developed food ingredient was conducted using male Wistar rats under immobilization stress and after exhausting physical exertion. The consumption of the food ingredient prevented an increase in the level of the main stress markers, catecholamines, in animals subjected to immobilization stress. The opposite effect was observed in animals that received the food ingredient after exhausting physical exertion: their levels of catecholamines were significantly higher than in the rest comparison groups. Using the Elevated Plus Maze and Open Field tests, it was shown that the consumption of the developed concentrate neutralized the negative effect of immobilization stress and treadmill exercise on anxiety in Wistar rats. The results obtained require additional study under conditions of preventive introduction of the food ingredient into the diet of intact animals, as well as a toxicological safety assessment. Key words: quinoa, 20-hydroxyecdysone, flavonoids, stress, immobilization, exhaustive physical exercise, catecholamines, corticosterone This work was supported by the Russian Scientific Foundation, grant no. 19-16-00107.

Development of Enzyme-Linked Immunoassay Systems for Monitoring the Content of the Main Non-Meat Additives in Meat Products

Abstract-Methods for control of the content of non-meat components (connective tissue of animals, eggs, soybeans) in meat products have been developed based on competitive enzyme immunoassay of biomarker proteins: collagen, ovalbumin, and soybean trypsin inhibitor. Polyclonal rabbit antibodies against the biomarkers were produced. Screening of the following analysis conditions was carried out using the most affine preparations: the duration of the stages, the concentrations of the reagents, the composition of the reaction medium to ensure the completeness and the minimum limits of detection of analytes. It was shown that the immunochemical stage can be transferred to the kinetic mode and reduced to 20 min, while the total analysis took only 1 h. The selected conditions synchronized detection stages and provided the same signal amplitudes from all three biomarkers. An additional advantage of the method is that the analysis can be carried out at room temperature. The detection limits for collagen, ovalbumin and soybean trypsin inhibitor in the final extracts were 0.025 μg/mL, 0.012 μg/mL, and 0.001 μg/mL, respectively. Approbation of the method showed the reliability of the conclusions about the composition of the tested meat products; the relative standard deviation in the analysis of the monitored biomarkers was 8-10%. Key words: enzyme-linked immunoassay, meat products, collagen, ovalbumin, soybean trypsin inhibitor The work was financially supported by the Russian Science Foundation (project no. 19-16-00108).