Biodegradation of Guar Gum in Hydraulic Fracturing Fluid under the Action of Enzyme Preparations of Basidiomycetes

Guar gum is a polymer that is widely used as a gelling agent for technological liquids in the petroleum industry. In this paper, we have studied the potential for the environmentally friendly biodegradation of guar gum by enzymes of basidiomycetes for efficient disposal of oil industry wastes. For the first time, we compared the enzymatic activity towards guar gum of seven basidiomycete strains, namely Trametes hirsuta MT-24.24, Lactarius necator, Trametes hirsuta MT-17.24, Schizophyllum commune MT-33.01, Fomes fomentarius MT-4.05, Fomitopsis pinicola MT-5.21, and Trametes versicolor It-1. This comparison showed that the preparation based on Fomitopsis pinicola MT-5.21 fungal mycelium at a concentration of 0.05% provides the most efficient decomposition of a frac fluid containing guar gum. By varying the enzyme concentration in this fluid it is possible to control the decrease in its viscosity over time. The developed enzyme preparation is an efficient and environmentally friendly guar gum biodegradant and can be used to process waste fracturing fluids based on polysaccharides in order to reuse water resources. Key words: biodegradants, basidiomycetes, guar gum, enzymatic hydrolysis, enzyme destructors, fracturing fluids. Funding - The work was financially supported by the National University of Oil and Gas "Gubkin University" (Internal grant no. 120720 "Development of New Biotechnological Methods and Materials for Environmental Protection and Biomedicine").

Reconstruction of Industrial Processes for Cultivating CHO Cells in a Scale-Down Bioreactor Model

According to generally accepted international standards, the characterization and confirmation of the quality of biological pharmaceutical substances requires the use of not only a combination of physicochemical and biological tests, but also an in-depth knowledge of the production process and its control. The study of such an industrial process involves considerable expenditure of time and material resources. Gaining a deep knowledge of the production process is greatly facilitated by using laboratory models of industrial installations that effectively reproduce large-scale processes in a research laboratory. On the basis of a laboratory bioreactor for the cultivation of eukaryotic cells with a working volume of 2 L, a scale-down model of a pilot bioreactor has been developed, linearly scalable to a production volume of 1000 L. The laboratory model reproduces the key parameters of cultivation on a pilot scale: the ratio of the geometric dimensions of the reactor, the peripheral speed of the mixer, the specific power input, the coefficient of oxygen mass transfer, the type and intensity of aeration, strategies for defoaming, feeding and maintaining the pH level. Experiments using the developed model showed high similarity in the kinetics of cell growth, productivity and quality of the expressed monoclonal antibodies in laboratory and pilot bioreactors. Key words: cultivation, CHO cells, monoclonal antibodies, scale-down model

Recombinant strains producing thermostable lipase from Thermomyces lanuginosus and their use in heterogeneous biocatalysis, including in the processes of low-temperature synthesis of esters

Abstract-This work was devoted to the construction of recombinant strains Escherichia coli BL21 (DE3) and Pichia pastoris X33, producing a 1,3-specific thermostable lipase from Thermomyces lanuginosus. The sequences of two lipase genes were optimized for expression in bacteria and methylotrophic yeasts, then synthesized and cloned into the corresponding expression vectors. As a result of genetic engineering manipulations, E. coli and P. pastoris strains were constructed that efficiently produced recombinant lipase from T. lanuginosus, which accumulated in the cytoplasm in an amount of 30-40% of the total cellular protein. Recombinant P. pastoris clones secreted lipase into the nutrient medium at a concentration of at least 1 g/L. Lipases produced by the recombinant clones, designated as rE.coli/lip and rPichia/lip, respectively, contained a six-histidine sequence (-His6) in the C-terminal region. The resulting lipases were immobilized on/in solid inorganic supports in order to develop heterogeneous biocatalysts (HB) for the enzymatic conversion of triglycerides and fatty acids. The rPichia/lip enzyme was adsorbed on mesoporous silica and macroporous carbon aerogel. The properties of the prepared HB, their enzymatic activity, substrate specificity and operational stability were studied in the reaction of esterification of fatty acids with aliphatic alcohols in organic solvents at 20 ± 2°C. It was found that immobilized lipases had a relatively wide substrate specificity, as well as high operational stability, and the prepared HB almost completely retained their high esterifying activity for several tens of reaction cycles. Key words: Escherichia coli, Pichia pastoris, recombinant strains-producers, Thermomyces lanuginosus lipase gene, immobilization, biocatalysts, esterification The authors are grateful to V. L. Kuznetsov for the provided samples of carbon aerogel and A. V. Ryabchenko for gene-engineering manipulation aimed at obtaining the recombinant rE. coli strain, a producer of the rE.coli/lip enzyme. The work was carried out under the Project on Fundamental Research within the framework of a state assignment to the Institute for Catalysis "Catalysts and Processes of Renewable Raw Material Conversion" (no. 0239-2021-0005).

The Expression Potential of New Yeast Strains of the Komagataella Genus

The expression potential of various strains from the Collection of the National Bio-Resource Center (BRC VKPM) collection belonging to the species Komagataella kurtzmanii, K. phaffii, K. mondaviorum has been assessed by the level of production of the heterologous enzyme Citrobacter freundii phytase. Heterologous expression in the K. mondaviorum strains was observed for the first time. The strains of K. phaffii Y-4288, K. mondaviorum Y-4331 and K. phaffii Y-4287 were identified with a high level production of the heterologous enzyme, a high growth rate, the ability to accumulate a large amount of biomass and moderate thermotolerance. It was shown that the average productivity of the transformants based on K. phaffii Y-4288, K. mondaviorum Y-4331, and K. phaffii Y-4287 strains exceeds that of the commercial industrial recipient strain K. phaffii GS115 Y-2837 by more than 3, 5 and 6 times, respectively. The K. phaffii Y-4287 and K. mondaviorum Y-4331 strains exhibited moderate thermotolerance and the ability to accumulate a heterologous product at 37° C. The high expression potential of the identified strains opens up the possibility of creating recipient strains on their basis for high-level production of heterologous proteins. Key words: Komagataella, Pichia pastoris, thermotolerance, expression of heterologous proteins Funding - This work was supported by the Ministry of Science and Higher Education of the Russian Federation (grant no. 075-15-2019-1658 dated October 31, 2019) and was carried out using the resources of the Unique Scientific Facility of the "All-Russian Collection of Industrial Microorganisms" National Bio-Resource Center, NRC «Kurchatov Institute»---GOSNIIGENETIKA.

Influence of Amino Acid Substitutions in a Recombinant Cow Chymosin Molecule on the Dependence of its Coagulation Activity on the Concentration of Calcium Chloride in Milk

The influence of substitutions of amino acids involved in the formation of salt bridges on the cow chymosin technological properties has been studied. It was shown that point amino acid mutations Asp198→Lys, Asp156→Val and their combination decrease the sensitivity of coagulation activity of engineering recombinant chymosins to change in the concentration of calcium chloride in milk in the range of 1-5 mM. The found effect is a positive technological evidence in terms of cheese making, since it allows varying the content of calcium chloride added to the milk mixture without a threat of significant changes in the undesired proteolytic activity of used milk-clotting enzyme. recombinant chymosin, technological properties, amino acid substitutions, milk-clotting activity, concentration of calcium chloride. The work was performed within the framework of the state task of the Ministry of science and higher education of the Russian Federation (topic number fzmw-2020-0002, "Development of recombinant enzyme producers for cheese making").

Study on the Toxicity of Calix[4]- and [6]-Arenes and their Sulfated Derivatives in Two- and Three-Dimensional Cell Cultures of Colorectal Adenocarcinoma

A comparative study of the toxicity of two unsubstituted calixarenes consisting of 4 and 6 phenolic fragments, as well as their p-sulfated derivatives, was carried out on the HT-29 colorectal adenocarcinoma cells cultured in two-dimensional (2D) and three-dimensional (3D) formats. It was shown that both unsubstituted calixarenes decrease the viability of tumor cells; calix[4]arene and calix[6]arene exhibited a cytostatic and a cytotoxic effect, respectively. Sulfated derivatives of calixarenes did not have a pronounced toxic effect on HT-29 cells. However, due to their high hydrophilicity and the ability to form adducts with various therapeutic molecules, they can be used for delivery of anticancer drugs. calixarenes, cytotoxicity, HT-29 cells, 2D cell culture, 3D cell culture The work was financially supported by the Russian Science Foundation (project no. 19-15-00397).

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