Biotechnologia Acta

5-28

Authors: O. M. Klyuchko

The purpose of the present work was to alalyse the prototypes of information expert systems, their structure, functions and practical application, and to develop a new one for solving practical problems in biotechnology, laboratory practice and environmental protection. The observed prototypes were developed for the use in genetic studies, agricultural production, nature protection from pests and environmental pollutants, for works in medicine, and etc. During the work, following methods were used such as methods of comparative research of the samples of technical devices, imitation and program modeling, which were based on numerical results obtained in experiments with the recording of chemosensitive transmembrane electrical currents in neurons in voltage clamp mode. As a result, an original expert system was developed. It was coupled with a detector group, databases and interface. The developed expert system was able to distinguish automatically the certain types of chemicals at the input, to display their identification data and, if necessary, the reports about their harmfulness. Conclusions were done about the practical value of these data for the elaboration of new electronic expert systems for monitoring the presence of harmful substances in the environment. It was also discussed the possibility of developed expert system application for new methods of qualitative and quantitative analysis of some organic compounds.

39-46

Authors: A. V. Kotinskyi, A. I. Salyuk, S. A. Zhadan

The aim of the work was to study the impact of glycine on Spirulina growth and photosynthetic processes intensity. For research the culture of Spirulina platensis (Gom.) Geitl cyanobacterium strain LGU — 603 from collection of Kholodny Institute of Botany of the National Academy of Sciences of Ukraine was used. The cultivation process was carried out in Zaruk nutrient medium in a vertical tubular apparatus of 8 cm in diameter and of volume 2 dm3 in a constant mixing of the culture medium with air. Introduction of glycine in a cultural environment resulted in the increase of the Spirulina productivity, maintenance of basic photosynthetic pigments and protein. Glycine introduction allowed to increase the growth rate of spirulina and to content achieve the high density of the culture due to the increased length of log-phase. Intensification of Spirulina platensis growth and its productivity increase was due to the fragmentation of cyanobacteria trichomes, so the increase of trichomes number with a small number of cells, which grow quickly. The degree of fragmentation depended on the concentration of introduced glycine and on the culture development stadium and also on introduced glycine concentration.

55-66

Authors: I. A. Blaydа, N. Yu. Vasylieva, T. V. Vasylieva, L. I. Sliusarenko, O. I. Dzhambek

The aim of the work was to optimize the process of bacterial desulfurization of energy coal, namely, to determine the influence of the component composition of the nutrient medium and the conditions of the process, which ensure the maximal development and activity of the aboriginal association of acidophilic chemolithotrophic bacteria and, as a consequence, the maximal index of sulfur decrease in coal in minimal time. We used the method of mathematical planning of the experiment adapted to the plan in Greek-Latin squares. The calculations in this approach are based on the analysis of variance (ANOVA). The formal planning of experiments has been carried out with four operating factors (nutrient medium components) at four levels (concentrations). The calculations were performed in Excel. The selection of operating factors and their combinations was made with the usage of unifactor ANOVA, correlation analysis and the method of principal components PCA. Researches were carried out in R 3.4.0 program and were founded on data of the preliminary evaluating experiments. Acidithiobacillus ferrooxidans Coal 17 aboriginal strain was used to obtain the most significant desulfurization effect. This strain was isolated from the investigated coal, studied and identified. The significance of the factor level for each nutrient medium component was analyzed using the Duncan’s multiple range test, the uniformity of the variances was examined with the Cochran’s test, and the significance of the factors was tested by the Fisher’s criterion. As a result, for the optimal nutrient medium the next combination of factors and their levels, which corresponds to the composition, g/dm3, was recommended: (NH4)2SO4 — 0.15; K2HPO4 — 0.50; FeSO4.7H2O — 44.0; KCl — 0.10; MgSO4·7H2O — 0.10; Ca(NO3)2 — 0.10; yeast extract — 0.025% (vol.); strain A. ferrooxidans Coal 17 (titre 1·108 CFU/ml) — 1.60% (vol.). This makes it possible to reduce the sulfur content in coal by 66.31% in a short period (seven days). This result could not be got before.

73-81

Authors: O. B. Tashyrev, I. B. Sioma, G. O. Tashyreva, V. M. Hovorukha

The aim of the research was to develop theoretic principles of an efficient biotechnology for microbial purification of concentrated sewage filtrate from a wide range of organic and non-organic compounds, and experimentally confirmed these principles. The experiment combined conventional microbiological, physical and chemical methods in which ten different types of solid carriers, both natural and artificial, were used. A comparative analysis of these types of solid carriers used in the flow system and a structured evaluation of treatment parameters was provided. The obtaining results demonstrated the effectiveness of the use of approach sewage microbial purification in a flow system. Developed principles can be used as a basis for new efficient biotechnologies for sewage microbial purification.

5-25

Authors: O. M. Klyuchko

The purpose of the work was to analyze existing information systems (IS) of biological objects and to propose the methods for development of such IS for insects on the example of Noctuidae (Lepidoptera). A detailed analysis of technical information concerning the distributed networked systems, access to computer systems to the common data in electronic IS and the organization of biomedical databases in the Internet was done. The peculiarities of IS’ prototypes development for environmental monitoring of the fauna have been discussed, in particular changes in the number of butterflies’ populations throughout France (including western and southern departments), the Noctuidae (Lepidoptera) in the steppe zone of Ukraine (Striltzivskyi Steppe), and the development of such IS for all territory of Ukraine. The results could be used to develop electronic IS for other biological organisms.

35-41

Authors: N.V. Borzova, O. V., Gudzenko, L. D. Varbanets

The aim of the work was to investigate the possibility of practical use substrate specificity of α-Lrhamnosidase Cryptococcus albidus. p-Nitrophenyl derivatives of monosaccharides were used to determine the activity and specificity of the enzyme. The ability to hydrolyze of natural substrates was evaluated by Davis and high-performance liquid chromatography methods. It was shown that the enzyme exhibits narrow specificity towards the glycon of synthetic substrates and hydrolyzes only p-nitrophenyl-α-L-rhamnopyranoside (Km 4.5 mM) and p-nitrophenyl-α-D-glucopyranoside (Km 10.0 mM). C. albidus α-L-rhamnosidase the most active degrades naringin (Km 0.77 mM), releasing prunin and naringenin. Km for neohesperidin was 3.3 mM. The efficacy of the naringin hydrolysis in grapefruit and pomelo juice was 98 and 94% in 60 min (40 оC, 2 U/ml). As the result of treatment by α -L-rhamnosidase of green tea and orange juice, there was a decrease in the content of rutin, narirutin and hesperidin, indicating that the α -1,2- and α -1,6-linked rhamnose can be cleaved from natural flavonoids. Thus, the study shows the efficiency of treating citrus juices and green tea with C. albidus α -L-rhamnosidase for the purpose of improving their taste qualities and obtaining bioavailable flavonoids glucosides.

49-53

Authors: O. V. Kravchenko, O. S. Panchenko

The aim of the research was the isolation from drinking water the pure cultures of iron- and manganese-oxidizing microorganisms with further assessment of their efficacy to remove these contaminants on rapid filters. To assess the effectiveness selected strains were grown on the solid nutrient medium; the suspension was prepared and was treated to zeolite loading. Ten pure cultures of iron- and manganese-oxidizing bacteria were isolated and identified as 6 genuses: Siderocapsa, Leptothrix, Sphaerotillus, Galionella, Metallogenium, Hyphomicrobium. Comparison the efficiency of genuses Leptothrix, Sphaerotillus, Metallogenium has shown that under conditions of these experiments Leptothrix more effectively removed iron and manganese at low concentrations in model solution.

65-74

Authors: L. Oriabinska, O. Dziuba, O. Dugan

The aim of the work was to find strains of lactic acid bacteria capable to synthesize extracellular tannase enzyme − the key enzymehe hydrolyzing tannins which are plant food constituent. One of the main product of tannins hydrolyzis is gallic acid- the compound with proven antioxidant and onco-protective properties. As a result of lactobacteria screening, two biocompatible strains of lactic acid, namely L. rhamnosus LB3 and L. delbrueckii subsp. delbrueckii with a high level of enzyme productivity, were selected. The maximum accumulation of tannase, corresponding to 0.031 ± 0.002 U/ml for L. rhamnosus LB3 and 0.03 ± 0.002 U/ml for L. delbrueckii subsp. delbrueckii, was observed after 48 h of cultivation. Both strains showed rapid growth and performance of tannase in MRS medium in the presence of glucose or lactose as a carbon source. It was shown that gallic acid, which was a necessary component of the medium as a target enzyme inducer, did not affect the accumulation of lactobacilli biomass. The selected strains are of interest as producers of a bicomponent probiotic with antioxidant properties and require further investigation.

5-27

Authors: T. P. PIROG, A. A. VORONENKO, M. O. IVAKHNIUK

Data on exopolysaccharides synthesis by psychrophilic fungi and bacteriae, halo- and thermophilic archaea and bacteriae, including those isolated from deep-sea hydrothermal vents — sources — were provided. Physiologic significance, physico-chemical properties and possible practical applications of exopolysaccharides from unusual sources were analyzed. Most of them have immunomodulating, antiviral, anticoagulant, antitumor, antioxidant activities promising for medical and pharmaceutical applications. Meanwhile, based on the literature date, the conclusion follows about the urgent necessity to develop efficient technologies for synthesis of these exopolysaccharides by nontraditional producers, which currently lags far behind common techniques.

50-56

Authors: O. V. Shtapenko, I. I. Gevkan, Yu. I. Slyvchyk, Ye. O. Dzen, V. Ya. Syrvatka, N. M. Matvienko

The purpose of this study was to investigate the effect of supplementation with organic zinc, manganese and chromium in the form of liposomal complex on the fertilizing ability and the level of antioxidant responses of female rabbits. Feeding of female rabbits with supplementation of organic forms of trace elements prior to insemination resulted in increase the numbers of corpora lutea, implantation and living fetuses compared to the control group. Moreover, there were the 4.4% and 1.7% decrease in pre- and post-implantation losses in animals receiving the organic microelements prior to insemination, respectively. The level of thiobarbituric-acid-reacting substances in ovary of experimental group was significantly higher (P ≤ 0.05) compare to the control group, while the level of lipid hydroperoxides in experimental group was decreased. In the uterus of rabbits after addition organic compound of trace elements significantly decreasing the thiobarbituric-acid-reacting substances level was by compare to the control animals (P ≤ 0.001). The level of the superoxide dismutase activity in uterus and ovary of female rabbits in the experimental group were significantly higher than in the control group (P ≤ 0,01). Our studies indicated that supplementation organic microelements in liposomal form to the basal diet for 2 weeks before insemination had a beneficial effect on the metabolism intensity and maintaining antioxidant-prooxidant balance in reproductive organs that improve fertilization and mbryo implantation.

68-72

Authors: O. B. Zlenko, A. P. Gerilovych

The aim of the work was to construct and test the recombinant positive control for F. tularensis detection by a real-time polymerase chain reaction (qPCR). The molecular TA-cloning of pTZ57_F/R plasmid ligated with tul4 gene PCR product into DH5α E. coli. The minimal detection level in qPCR was one copy number per reaction. The obtained positive control was highly sensitive, specific and safe qPCR in the laboratory tularemia diagnostics.

84-90

Authors: Y. Hurmach, М. Rudyk, V. Svyatetskaya, L. Skivka

The aim of the work was to investigate the effect of the intracranial catheter placement on the metabolic profile of rat microglia. Microglial cells were isolated by the centrifugation in Percoll gradient. Oxidative metabolism and phagocytic activity were investigated by flow cytometry. Arginase activity was examined by colorimetric method. Nitrite level was assayed in Griess reaction. It was found that intracranial catheter placement caused down-regulation of nitrite synthesis by 3 times, augmentation of the reactive oxygen species generation by 1.5 times, and slightly decreased microglia phagocytic activity. Thus, intracranial catheter placement causes long term anti-inflammatory shift of microglia metabolism in rats.

5-26

Authors: O. M. Klyuchko

The aim of the work was to generalize and analyze the use of electronic information systems in biotechnology in order to create new versions of these systems. The publications concerning the systems of different types for solving the problems in biotechnology were studied. Similar systems which traditionally belong to biology and medicine were classified. The prospects of their application for development of more advanced electronic systems were considered.

47-55

Authors: Ogbuagu Dike Henry, Nwachukwu Ikenna Ndubuisi, Ejike Onyinye Joy

The aim of the research was to utilize biofilms as a model in ecotoxicology to remove selected heavy metals (Cd, Cu, Cr, Zn and Pb) from wastewater in a static condition. Biofilms were grown in three graded concentrations of the metal leachates (0.625, 0.417 and 0.250 %), harvested after 1, 2 and 3 weeks and analyzed for heavy metals. Mean accumulations peaked on Day 21, and of Cd ranged from 0.000 to 0.040 (mean = 0.00837 ± 0.002), Cu from 0.000 to 0.212 (meam = 0.03929 ± 0.012), Cr from 0.000 to 0.500 (mean = 0.05821 ± 0.021), Zn from 0.000 to 1.456 (mean = 0.31833 ± 0.109) and Pb from 0.000 to 0.099 (mean = 0.02129 ± 0.006) mg/g in resultant biofilm formations. Accumulation of the metals increased significantly with time [F(205.59) > Fcrit(3.95)] at the 95% confidence interval. Those of Pb was significantly higher in the 0.625% leachate mixture than control (Sig F = 0.034) at P < 0.05, even as those of Cd and Cu were slightly higher in the concentrations than control. Biofilm model removed small amounts of metals from wastewater stream in static condition.

69-77

Authors: V. Raks, M. Öztürk, O. Vasylchenko, M. Raks

Research aimed to isolate biologically active compounds from mushrooms fruiting bodies of Ganoderma lucidum, Ganoderma adspersum and Ganoderma applanatum and estimate their antioxidant activities. Variety of sample preparation techniques were used to isolate biologically active compounds: solid-liquid, ultrasonic extraction, Soxhlet extractor extractions. Antioxidant properties were estimated with spectrophotometricaly measuring free radical scavenging activity. High performance liquid chromatography was applied to get fingerprints of isolated extracts. In β-carotene-linoleic acid assay, methanol extracts demonstrated mainly the best antioxidant activities: optimum values of half maximal inhibitory concentration (IC50) − for G. applanatum and G. adspersum were 8.25 ± 0.88 µg/ml and 1.70 ± 1.13 µg/ml respectively. However, petroleum ether and chloroform extracts of G. lucidum demonstrated the highler antioxidant activity with an IC50 about 33.66 ± 3.69 µg/ml. Chromatograms of dry components of acetone and methanol extracts of G. lucidum were recorded. The main outcome of such chromatograms is the possibility to compare presence active components in various mushrooms species without usage of expensive standards.

83-88

Authors: Makogonenko S. Yu., Baranov V. I., Ponomarenko S. P.

The aim of study was to investigate the effect of new growth regulators “Stimpo” and “Regoplant”, produced by State Enterprise Interdepartmental Science and Technology Center − ISTC "Agrobiotech", on the proline and free amino acids content, and lipid peroxidation reactions intensity in comperison to the action of gibberellic acid and “Treptolem” (a second-generation growth regulator) in 14 day sunflower sprouts. The plants were cultured on soil substrates made from the coal mine rock waste of the Central Concentrating Factory in the Chervonohrad mining region. It was shown that the free amino acids and proline content increased, and the intensively of lipid peroxidation reactions (evaluated by the content of malonic dialdehyde) decreased. This indicated to the increase of plants resistance to unfavorable conditions of the coal mine rock under the action of the growth regulators studied. One could assump that “Stimpo” and “Regoplant” are promising agents in phytochemical treatment of coal mine rock dumps.

5-22

Authors: O. M. KLYUCHKO

The aim of the work was to generalize and analyze the use of electronic information systems in biotechnology in order to create new versions of these systems. The publications concerning the systems of different types for solving the problems in biotechnology were studied. Similar systems which traditionally belong to biology and medicine were classified. The prospects of their application for development of more advanced electronic systems were considered.

30-39

Authors: A. A. Siromolot, O. S. Oliinyk, D. V. Kolybo

The goal of this study was to characterize serum immunoglobulin G (IgG) antibody responses during experimental immunization of laboratory mice by purified recombinant proteins MPT63, MPT83 of M. tuberculosis and artificial fusion protein MPT83-MPT63 and obtain the recombinant single chain variable fragments of antibodies (scFv) against these antigens. This study demonstrates that the humoral immune response to MPT63, MPT83, MPT83-MPT63 fusion protein and equimolar set of MPT63 and MPT83 was highly different. For each antigen, serum antibody levels were evaluated by a cutoff value based on optical density index. A crucial role of MPT83 for immunogenicity of chimeric protein and/or cocktail of individual antigens under conditions of immunization of laboratory animals. We obtained also specific scFv antibodies against MPT63 and MPT83. These antibodies can be used for the development of the system for quantitative determination of antigens as well as for their biological properties investigation. Thereby, based on the results of the immune response and mycobacterial proteins antigenicity we showed highly immunogenicity properties of N-terminal part of MPT83 antigen for enhencement of ELISA sensitivity. We suggest MPT83-MPT63 fusion protein as a potential candidate on the role of antigenic substance for the serological diagnosis of tuberculosis.

47-56

Authors: Lakhneko O. R., Stepanenko A. I., Kuzminskiy Ye. V., Morgun B. V.

The aim of the research was to study polymorphism of preselected gene loci of three transcription factors (TaNAC2a, TaWRKY2, and TaWRKY19) and the Late Embryogenesis Abundant (LEA) proteins dehydrin (Td29b) related to wheat drought tolerance. The genes structure and chromosome location were established via bioinformatics tools. It is stated that TaWRKY2 and TaWRKY19 genes were comprised of 4 exons and 3 introns located on 2BS and 1DS chromosome arms, respectively; TaNAC2a — 2 exons and 1 intron 7AS; Td29b — single exon gene 3AS. Using polymerase chain reaction, no polymorphism was observed. Polymorphic bands were detected for TaWRKY2 locus. The screening of the distribution of the revealed polymorphic loci was carried out for a set of wheat and rye varieties, old landraces and interspecific hybrids. The polymorphism of TaWRKY2 locus indicated the presence of some other possible alleles of the gene. The obtained data are important for further investigations of wheat drought tolerance.

64-71

Authors: K. Y. Manoilov, O. I. Krynina, A. Ju. Labyntsev, S. I. Romaniuk, D. V. Kolybo

The aim of the work was to evaluate in vitro the cytostatic effect of recombinant fragments of the non-toxic point mutant of diphtheria toxin — CRM197, which was suggested as a potent medication for treatment of triple negative breast cancer. For this purpose, non-toxic recombinant derivatives of diphtheria toxin — CRM197, subunit B SbB and receptor domain Rd had been isolated by Ni-NTA agarose affinity chromatography and their effect on the growth of individual colonies of triple negative breast cancer MDA–MB–231 cells were characterized by such parameters as average colony area, perimeter and circularity index. According to the obtained results, CRM197 and SbB, whose molecules contain the translocation domain Td, exhibited the same cytostatic effect against MDA–MB–231 cells, reducing the area and perimeter of individual colonies. Rd protein did not affect the last two parameters that characterize the size of the colonies, but changed the form of the margin of colonies, as evidenced by an increase in the circularity index. It is supposed that Td may be involved in the implementation of cytostatic action due to its inherent pore-forming activity in relation to lipid membranes. It is concluded that Rd and Td, unlike the catalytic domain of diphtheria toxin, play important roles in the implementation of the cytotoxic properties of CRM197, while SbB consisting of Rd and Td is the structural DT fragment of smallest molecular weight that can be used as the analog of CRM197.