Biotechnologia Acta

25-37

Authors: Gulevskyy O. K., Akhatova Yu. S.

This paper was aimed to review the literature data from native and foreign sources accumulated for 40-years period of research of the features of the molecular structure, functions, production and application of human alpha-fetoprotein (AFP), which is known as one of the most studied and increasingly demanded proteins. Results of fundamental studies performed with the use of modern methods, including various types of electrophoresis, chromatography, electron microscopy and immunoassay, in order to characterize the principal physicochemical capacities and localization of free and bound forms of AFP, as well as polypeptide structure, heterogeneity and topography of AFP receptors are highlighted here. The data on the mechanisms of AFP synthesis, its conformational features, binding sites and intracellular metabolism are also presented. The concepts of physiological functions and mechanisms of AFP transport in an organism are presented. Data on AFP isolation from the natural primary products and its production by means of recombinant and synthetic methods are shown. This review also summarizes information on the current possibilities of clinical application of AFP and the prospects for its usage in anticancer therapy for targeted delivery of chemotherapy drugs, with emphasis on the description of the recent progress in this field.

46-56

Authors: Bakalchuk M. M., Besarab O. B.

Fecal microbiota transplantation (FMT) is a treatment method based on donor's fecal solution injection into the patient's gastrointestinal tract. FMT is effectively used in the treatment of recurrent Clostridium difficile infection. There is also growing interest in the therapeutic application of the method to treat metabolic, autoimmune and other disorders that was not previously associated with intestinal microbiota. Despite the promising results of FMT use, the organizational and legal matters and that of the safety FMT application have not yet been resolved in the European and Ukrainian medical community. The purpose of this review was to summarize information on the FMT application and the regulatory aspects of its use. The analysis of the practical instructions provisions of for FMT applying in clinical practice was carried out, and the bioethical problems associated with the FMT use were investigated.

69-80

Authors: O. А. Havryliuk, V. М. Hovorukha, А. V. Sachko, G. V. Gladka, О. B. Tashyrev

Copper is a highly toxic metal common in both natural and man-made ecosystems. The goal of the work was to determine the level of resistance of microorganisms of natural ecosystems to cationic form and organometallic complex of Cu2+. Microorganisms of 9 natural ecosystems of five geographic zones (the Antarctic, the Arctic, the Dead Sea (Israel), middle latitude (Ukraine) and the equatorial zone of South America (Ecuador) were investigated. Resistance of microorganisms was determined by cultivation in the medium with concentration gradient of Сu2+. The amount of Cu2+-resistant microorganisms in natural ecosystems was determined by colony counting on nutrient agar with Сu2+ citrate and Cu2+ cation. The Cu(II) concentration in soil and clay samples was analyzed by atomic absorption spectroscopy method. We have confirmed the hypothesis that microorganisms resistant to toxic Cu2+ compounds in high concentrations exist in any natural ecosystem. The resistance to Cu2+ cation was 8 – 31 and 14 –140 times less than to Cu2+ citrate in nutrient and mineral agar media respectively. The amount of Cu2+-resistant microorganisms in natural ecosystems reached hundreds and thousands at the presence of 175…15 500 ppm Cu2+. Thus, the soils, clays and sands of natural ecosystems are a “genetic resource” of copper-resistant microorganisms that are promising for development of novel biotechnology of purification of copper-containing wastewater and soil bioremediation.

13-23

Authors: O. Obodovych, V. Sydorenko

The use of plant biomass as a primary source of energy is currently unacceptable both from an economic and environmental point of view. The experience of a number of industries, in particular hydrolysis production, enables to solve the problem of profitability of organic biomass treatment by its deep complex processing with the resulting components whose cost exceeds the cost of organic raw materials as fuel. Currently, the main results of complex processing of organic raw materials are still energy-intensive products ̶ bioethanol and hydrolyzed lignin, which energy characteristics are commensurate with fossil fuels. Bioethanol production from starch-containing, sugar-containing or lignocellulosic raw materials requires the use of different technological stages and, accordingly, the cost of bioethanol for each type of raw material is different. Compared to bioethanol produced from sugar and starch raw materials, bioethanol manufactured from lignocellulosic raw materials is more expensive. Bioethanol obtained from lignocellulosic raw materials is more expensive compared to bioethanol from sugar and starch raw materials. The most energy-intensive in the technology of bioethanol obtaining from lignocellulosic raw materials is the stage of pretreatment of raw materials for hydrolysis, because the process of preliminary preparation and hydrolysis with dilute acids occurs at high temperatures and pressures. During enzymatic hydrolysis, the process temperature is maintained for a long time (up to several days). To ensure deep integrated processing of plant raw materials, as well as to reduce overall costs, it was proposed to improve the technology and equipment, which allow increasing the degree of conversion of raw materials into basic and by-products.

30-40

Authors: E. G. Fomina, E. E. Grigorieva, V. V. Zverko, A. S. Vladyko

A heterologous host has got a unique expression ability of each gene. Differences between the synonymous sequences play an important role in regulation of protein expression in organisms from Escherichia coli to human, and many details of this process remain unclear. The work was aimed to study the composition of codons, its distribution over the sequence and the effect of rare codons on the expression of viral nucleocapsid proteins and their fragments in the heterologous system of E.coli. The plasmid vector pJC 40 and the BL 21 (DE 3) E. coli strain were used for protein expression. The codon composition analysis was performed using the online resource (www.biologicscorp.com). 10 recombinant polypeptides were obtained encoding the complete nucleotide sequence of nucleocapsid proteins (West Nile and hepatitis C viruses) and the fragments including antigenic determinants (Lassa virus, Marburg, Ebola, Crimean-Congo hemorrhagic fever (CCHF), Puumaravala, Hantaan, and lymphocytic choriomeningitis (LHM)). Hybrid plasmid DNAs provide efficient production of these proteins in the prokaryotic system with the recombinant protein yield varying by a factor of 8: from 5 to 40 mg per 1 liter of bacterial culture. No correlation was found between the level of protein expression and the frequency of occurrence of rare codons in the cloned sequence: the maximum frequency of occurrence of rare codons per cloned sequence was observed for the West Nile virus (14.6%), the minimum was for the CCHF virus (6.6%), whereas the expression level for these proteins was 30 and 5 mg/L culture, respectively. The codon adaptation index (CAI) values, calculated on the basis of the codon composition in E. coli, for the cloned viral sequences were in the range from 0.50 to 0.58, which corresponded to the average expressed proteins. The analysis of the distribution profiles of CAI in the cloned sequences indicated the absence of clusters of rare codons that could create difficulties in translation. A statistically significant difference between the frequencies of the distribution of amino acids in the cloned sequences and their content in E. coli was observed for the nucleocapsid proteins of the Marburg, Ebola, West Nile, and hepatitis C viruses.

50-57

Authors: T. A. Yatsenko, S. M. Kharchenko

The plasminogen/plasmin system plays a crucial role in fibrinolysis and regulation of cell functions in a wide range of normal and pathological processes. Investigation of plasminogen/plasmin functions requires the availability of well-characterized and effective molecular tools, such as antibodies. In the present work, the isolation and characterization of rabbit polyclonal antibodies against human plasminogen are described and approaches for the identification of plasminogen and its fragments using the purified antibodies are demonstrated. For the antibodies isolation, standard animal immunization and blood collection procedures, serum isolation, protein salting out and affinity chromatography were performed. For the antibodies characterization and application, the following methods were used: enzyme linked immunoassay (ELISA), Western blotting, FITC-protein conjugation, flow cytometry and spectrofluorometry. The obtained polyclonal rabbit anti-human plasminogen antibodies interacted with human Glu- and Lys-plasminogen, kringles 1-3 and 1-4 of plasminogen, mini-plasminogen, the heavy and light chain of plasmin. We propose the application of anti-plasminogen antibodies for the direct ELISA, Western blot analysis, and for flow cytometry and spectrofluorometric analysis of plasminogen binding with cells. The obtained anti-plasminogen antibodies are promising tools for the investigation of plasminogen/plasmin system functions, either fibrinolytic or signaling.

19-31

Authors: O. KUZNETSOVA, E. VLASENKO

The paper is aimed to analyze the current data on the influence of exogenous growth stimulators on growth and development of higher Basidiomycetes. The historical aspects about discovery and role of phytohormones in fungi physiology are reviewed. The taxa of the basidiomycetes, which are capable of synthesizing phytohormones of all known types, including Aphyllophorales, Boletales, Agaricales, Sclerodermales, Hymenogastrales, Uredinales, Ustilaginales are described. The data from different sources describing the effects of natural and synthetic growth stimulators on the development of basidiomycetes are summarized and compared. It is noted that various concentrations of phytohormones (auxins, gibberellins, cytokinins) from 0.01 to 400 mg/ml were used in the experiments to study their effects on mycelial growth. Possible causes of conflicting results obtained by different authors, such as application of diverse methods of mushroom cultivation, different media and methods of substrate sterilization, are described.

42-61

Authors: A. K. Gulevsky, I. I. Shcheniavsky

This review presents the current scientific literature data about structure, properties, and functions of collagen, which is known as one of the most abundant human and animal proteins. The building of collagen molecule from the primary structure to submolecular formations, the main stages of its synthesis and biodegradation are briefly described. The information about collagen diversity, its features and metabolic ways in various tissues, including skin, tendons, bones, etc. is presented. The problems of pathologies caused by collagen synthesis and breakdown disorders as well as age-related changes in collagen properties and their causes are discussed. A comparative analysis of the advantages and disadvantages of collagen and its derivatives obtaining from various sources (animals, marine, and recombinant) is given. The most productive methods for collagen extraction from various tissues are shown. The concept of collagen hydrolysis conditions influence on the physicochemical properties and biological activity of the obtained products is described. The applications of collagen and its products in various fields of industrial activity, such as pharmaceutical, cosmetic industry and medicine, are discussed. Further prospective directions of fundamental and applied investigations in this area of research are outlined.

73-86

Authors: V. M. Hovorukha, O. A. Havryliuk, G. V. Gladka, I. O. Bida, O. B. Tashyrev

Increasing pollution of environment by toxic metals is the urgent problem requiring effective solution worldwide. The goal of the work was to study the dynamics of the interaction of Cr(VI), Mo(VI), W(VI) compounds with obligate anaerobic microorganisms Clostridium butyricum GMP1, which ferment organic compounds with the synthesis of hydrogen. The standard methods were used to determine рН and redox potential (Eh), the gas composition, and the concentration of metals. The application Clostridium butyricum GMP1 was showed to be useful to investigate its interaction with toxic metals. The higher redox potential of metal provided the opportunity for its faster and more effective reduction. The patterns of the reduction of toxic metals Cr(VI), Mo(VI) and W(VI) by obligate anaerobic strain Clostridium butyricum GMP1 were obtained. The experimental data confirmed the thermodynamically calculated correlation between the redox potential of the metal reduction to insoluble form and effectiveness of its removal. Obtained results can serve as the basis for further optimization and development of environmental biotechnologies for wastewater treatment with the simultaneous destruction of solid organic waste and hydrogen synthesis.

14-25

Authors: І. V. Kosakivska, M. M. Shcherbatiuk, L. V. Voytenko

The review analyzes and summarizes the latest literature on the history of development and the current state of methodological approaches to the identification and quantification of phytohormones in plant tissues. Phytohormones play a key role in the regulation of physiological processes throughout the life cycle of plants – from seed germination to aging. In tissues of plants, their concentrations are very low (from 10-9 M to 10-6 M), so the development of fast, highly efficient, comprehensive, and at the same time reliable approaches to the determination of phytohormones is extremely urgent. The article gives a brief description of the main classes of phytohormones, describes their functional activity. The substantiated and checked sequence of procedures on the extraction of plant hormones, their separation from interfering substances, quantification methodology for indol-3-acetic, abscisic, gibberellic (GA3), salicylic acids and five cytokinins that combines a high-performance liquid chromatography with mass spectrometry are presented. The article describes four chromatographic methods of profiling, gives the conditions for detecting phytohormones of different classes and ionization of these substances in a mass spectrometer. The importance of phytohormones profiling in plant tissues for further applied in biotechnological approaches, in particular in metabolic engineering, for the formation of stress resistance in crops is discussed.

39-48

Authors: M. Gahramanova, A. Ostapchuk, O. Molozhava, V. Svyatetska, M. Rudyk, Y. Hurmach, R. Dovhyi, L. Skivka

The aim of the work was to explore fatty acid composition of purslane seed water extract and its effect on the metabolic profile of murine peritoneal macrophages. Fatty acid composition was evaluated by gas chromatography–mass spectrometry. Collection of murine macrophages from the peritoneal cavity was done without preliminary sensitization. Reactive oxygen species generation was assayed by flow cytometry and nitroblue tetrazolium test. Phagocytic activity was evaluated by flow cytometry. Nitric oxide production was analyzed in cell supernatants by Griess reaction. Arginase activity was measured in cell lysates by standard colorimetric assay. Reactive oxygen species and nitric oxide production were significantly lower in murine macrophages simultaneously treated with purslane seed water extract and lipopolysaccharide in comparison to macrophages treated with lipopolysaccharide only. Also, the studied extract caused statistically significant increase in arginase activity of unsensitized peritoneal macrophages. That is consistent with the fatty acid content of this extract, since it contained comparatively higher proportion of unsaturated fatty acids exhibiting anti-inflammatory properties, than saturated fatty acids known for their pro-inflammatory effects.

60-70

Authors: G. O Iutynska, M.Ya Vortman, D. R Abdulina, Zh. P Kopteva, A. Ye Kopteva, A. V Rudenko, V. V Tretyak, V. N Lemeshko, V. V Shevchenko

Biodegradation of various materials in the environment is important because many synthetic materials are stable for a long time. At the end of the service life, the materials must be involved in the natural cycle and decompose. In modern polymer chemistry, one of the promising areas is to obtain and study the properties of hydrogel systems, among which a significant place is occupied by polyethylene oxide hydrogels. The aim of the study was to determine the biodegradation of guanidine-containing polyethylene oxide hydrogel under the action of bacteria and to study its antimicrobial properties. The antimicrobial activity of a guanidine-containing oligomer and its newly synthesized polyethylene oxide hydrogel was studied by a disco-diffusion method. The enzymatic activities of bacteria were determined by spectrophotometry. To study the resistance of guanidine-containing polyethylene oxide hydrogel to microbial destruction, bacteria were grown in Tauson's liquid medium with the addition of meat peptone broth at a temperature of 28±2 °C. Changes in the chemical composition of the studied materials were analyzed by infrared Fourier spectroscopy of the Tensor 37 (Bruker) and 1H NMR spectroscopy. The antimicrobial properties and biodegradation of guanidine-containing polyethylene oxide hydrogels were determined, which were obtained by reacting oligooxyethylene glycol MW 6000 with toluene diisocyanate and guanidine-containing oligomer, which acted as a crosslinking, ion-containing and antibacterial-agent. The synthesized hydrogel showed antimicrobial activity against gram-positive and gram-negative bacteria. The biodegradation of hydrogels under the action of various bacterial strains and enzymes that they synthesize was studied. The presence of the tested materials lead to a decrease in the enzymatic activity of bacteria in 1.4 – 2.5 times compared with the control. Synthesized guanidine-containing polyethylene oxide hydrogel showed antimicrobial activity against the studied test cultures. The introduction of the studied materials into Tauson's environment as additional sources of carbon and energy helped to reduce the catalase and lipase activities of hydrocarbon-oxidizing bacteria. Under the influence of the studied bacteria, the hydrogels were destroyed with 88.4% from the initial value.

30-39

Authors: N. I. Aralova, O. M. Klyuchko, V. I. Mashkin, I. V. Mashkina, T. A. Semchyk

The aim of the work was to create a complex mathematical model simulating the course of the disease caused by the SARS-CoV-2 virus on the level of interaction between functional systems of organism and pharmacological correction of organism hypoxic states arising in the complicated course of the disease. In the present work the methods of mathematical modeling and theory of optimal control of moving objects were used. The proposed integrated mathematical model consisted on the mathematical models of functional systems of respiration and blood circulation, thermoregulation, immune response, erythropoesis, and pharmacological correction. Individual patient data were taken for this model, and the disturbing effect in the form of viral disease was simulated. The reactions of functional respiratory and blood circulatory systems were predicted. Partial pressures of respiratory gases in alveolar spaces and their tensions in lung capillaries blood, arterial and mixed venous blood, and tissue fluid were calculated. Further the intravenous injection of antihypoxant was simulated and the values of the same parameters were calculated. In such a way it was possible to choose the most optimal way of hypoxic state correction for any individual. This model is theoretical only for today because the models of respiratory and blood circulation systems were designed for the average person and it does not suppose peculiarities of individual persons infected with SARS-CoV-2. In particular, this concerns the peculiarities of gas exchange in the alveolar space and characteristics of respiratory gases diffusion through the alveolar-capillary and capillary-tissue membranes. However, it is one of possible directions for solving the complex tasks related to treatment of the disease caused by SARS-CoV-2 virus. In the result of the work the complex of information support for the imitation of viral disease course was developed at the level of interaction of organism functional systems, as well as pharmacological correction of caused by it hypoxic states.

64-72

Authors: M. Kryvtsova, M. Hrytsyna, I. Salamon

Essential oils are widely used in beauty therapy, medicine and food industry, and they are considered to be a valuable consumer product. At the same time, the biochemical composition and properties of essential oils, including their antimicrobial activity, varies depending on the habitat, climatic conditions and plant chemotype. The purpose of our work was to study the qualitative and quantitative composition of essential oils and their antimicrobial properties, from Origanum vulgare plants harvested in eastern Slovakia and Lviv region, Ukraine. In the wild, O. vulgare L. were gathered in close vicinity of the village of Trostianets, Lviv region, Ukraine, in 2019. In Slovakia, the plants were grown by Agrokarpaty Company, Plavnica. Essential oils were extracted by hydrodistillation (2 hours) in a Clevenger apparatus, according to the European pharmacopoeia. The analysis of the biochemical properties of essential oils extracted from plant populations from Lviv region, showed that the contents of essential oils were within 0.35 ± 0.05%. The composition of essential oils shows that Origano vulgare L. plants from the natural population grown in Lviv region, belongs to the monoterpene chemotype. Monoterpene hydrocarbons α-terpinene and α-terpineol together accounted for 29‒33%, acyclic monoterpenes – β-myrcene – 7%, linalool – 4%, while the polyphenol compound p-cymene accounted for only 15%. The O. vulgare plants from Slovakia were characterised by the essential oil content of 0.15 to 0.50%, and the composition which allowed us to refer them to the carvacrol chemotype, with phenols as its main ingredients – carvacrol and thymol (together 71%), and isopropyltoluene (4.0%). Monoterpene hydrocarbons terpinene (5.0%) and terpineol alcohol (6.0%) jointly accounted for 11%; acyclic monoterpene mycrene – 3%; and sesquiterpene β-caryophyllene – 4,5%. Essntial oil from O. vulgare harvested in Slovakia demonstrated high antimicrobial activity against reference and clinical isolates of opportunistic microorganisms. Essential oil from the samples gathered in Lviv region, showed low antimicrobial activity. Thus, it has been shown that the reviewed plants referred to different chemotypes, which calls forth the prospect of the use of essential oils extracted from different plant chemotypes for different purposes, depending upon their biochemical composition and properties.

81-88

Authors: L. Sabliy, V. Zhukova

The work was aimed to study the processes of industrial wastewater purification of a pharmaceutical company treatment and to recommend an effective technology for local treatment of such wastewater in accordance with the requirements of normative documents on the treated wastewater disposal into the urban sewerage system. Model solutions of cefuroxime, a cephalosporin antibiotic, in distilled water with a concentration of 25 and 35 mg/dm3 were used for the study. COD model solutions, which is, respectively, 90 and 120 mg/dm3. Chemical oxygen demand was etermined by the dichromate method (according to the guiding normative document (GND) 211.1.4.021-95. “Methods for Chemical Oxygen Demand (COD) Determining in Surface and Wastewater”). The proposed technology included the following successive processes of physical and chemical treatment of wastewater: aeration – equalization of quantitative and qualitative composition of wastewater and oxidation up to 30% of organic matter; coagulation – removing of antibiotics from wastewater by their adsorption on a highly developed surface of mineral coagulants flakes; sedimentation – separating of the formed flakes; oxidation of antibiotics destruction products by hydrogen peroxide; filtration – removal of fine impurities.Effect of COD reduction rate in the coagulation and setteling of wastewater in the case of ferrous sulphate III was 79.2% and 75%, which is higher by 4,2-6,7% than when using aluminum sulphate. The COD index changing of the pharmaceutical enterprise wastewater by the stages of its purification was found as follows: “aeration — coagulation with iron sulfate III — sedimentation — oxidation — filtration”. After filtering effect of reducing COD index was 95.8-100% at the initial value of 120 and 90 mg/dm3 respectively. The technology of local wastewater treatment from antibiotics was developed, which was based on the consistent use of physico-chemical methods of treatment and enabled to remove antibiotics and related substances from wastewater in accordance with the requirements of normative documents and disposal of treated wastewater into the urban wastewater system. Application of the developed technology of local wastewater treatment from antibiotics and related substances by the pharmaceutical companies would lead to meet the requirements of industrial wastewater disposal into the city drainage system, to a significant risk reduction of the antibiotics influence on the microorganisms of active sludge of the urban wastewater biological treatment facilities, to operating costs reduction to achieve maximum allowable discharges (MAD) of wastewater into a natural reservoir.