Biotechnologia Acta

19-26

Authors: А. V. Bazalii, A. V. Vorotnikov, P. A. Тiurin-Кuzmin, V. А. Тkachuk, S. V. Коmisarenko, L. B. Drobot

The aim of this study was to design the expression vector encoding fluorescent sensor of hydrogen peroxide HyPer fused with adaptor protein Ruk/CIN85 as well as to check its subcellular distribution and ability to sense hydrogen peroxide. It was demonstrated that in transiently transfected HEK293 and MCF-7 cells Ruk/CIN85-HyPer is concentrated in dot-like vesicular structures of different size while HyPer is diffusely distributed throughout the cell. Using live cell fluorescence microscopy we observed gradual increase in hydrogen peroxide concentration in representative vesicular structures during the time of experiment. Thus, the developed genetic construction encoding the chimeric Ruk/CIN85-HyPer fluorescent protein represents a new tool to study localized H2O2 production in living cells.

39-48

Authors: A.A.Peterson, M. Yu. Vasylenko, N. A. Matvieieva, M. V. Kuchuk

Determination of the presence of the recombinant fusion protein (ESAT6-Ag85B(ΔTMD)-6His) and its accumulation level in duckweed plants (Lemna minor L.) was the aim of the research. ESAT6 and Ag85B are secretory proteins of Mycobacterium tuberculosis and are considered as potential candidates for development of new vaccine against tuberculosis (TB). Transgenic duckweed plants were obtained previously by Agrobacterium rhizogenes-mediated transformation and possessed fusion gene sequence esxA-fbpBΔTMD. Specific polyclonal antibodies were produced in immunized mice to identify levels of accumulation of TB antigens in plants. Recombinant antigen used for mice immunization was obtained in our laboratory by expression in E. coli. Western blot analysis revealed the recombinant tuberculosis antigen ESAT6-Ag85B(ΔTMD)-6His in extracts from transgenic L. minor plants. The level of accumulation of the protein corresponds to 0.4-0.5 µg protein per 1 g of fresh weight of plant. Additionally, the accumulation of recombinant protein was investigated in lyophilized transgenic plants after 1.5 year storage. Duckweed plants accumulating a recombinant analogue of M. tuberculosis secretory proteins can be used for development of plant-based edible vaccines.

55-63

Authors: L. A. Kotyuk

The aim of the paper is to research biochemical composition of Hyssopus officinalis L. (Lamiaceae) in relation to plant age and phenological growth stage under conditions of Ukrainian Polissya, bin order to determine the optimal harvest dates of the herbal material and its application spheres. The raw material samples under analysis were cut at various growth stages: the vegetative, budding, blooming, ripening stages. To study the hyssop oil composition, areal parts of H. officinalis were used. The composition analysis was aimed at determining absolute dry matter (by drying samples at 105 °C up to the constant mass), “crude” cellulose, amounts of protein, fats, calcium, potassium, phosphorus, ascorbic acid, carotene, discernible sugars and tannins and essential oil. The present study has proved that in the plant ontogenesis the amount of essential oil, obtained from H. officinalis areal parts, does not markedly decrease: volatile oil yield in plants of the first, second and third years of life amounted to 1.007%, 0.75% and 0.71% respectively. The composition of volatile oil in the plants of the first year of life reveals 46 components, of which pinocampone (53.73%), isopinocampone (4.66%) myrtenol (9.35%) and camphor (3.86%) prevailed. In H. officinalis volatile oil of the third year 30 components were identified, the prevailing of which were isopinocampone (44.43%), pinocampone (35.49%), myrtenol (5.26%), germacrene D (3.15%), pulegone (2.93%) and bicyclogermacrene (1.35%). We could observe the change in the quantitative and qualitative composition of H. officinalis volatile oil throughout the entire vegetation period. Thus, in the phase of vegetative growth one can identify 25 compounds, the most predominant being elemol (33.25%), germacren D (21.59%) and bicyclogermacrene (15.78%). In the phase of blossoming 30 components can be identified, a high amount of isopinocampone and pinocampone (44.43% and 35.49%) and somewhat lover amount of myrtenol (5.26%), pulegone (2.93%) and bicyclogermacrene (1.35%) can be noted. During the fruit-bearing period 21 compounds with the prevalence of elemol (44.46%), bicyclogermarcren (10.30%), germacren D (5.86%), spatulenol (4.36%), β-eudesmol (4.34%), α-eudesmol (4.04%) and γ-eudesmol (3.92%) can be identified. H. officinalis raw material from one-year-old plants is to be used in food industry, whereas plants collected at the blooming stage are preferable for cosmetics and perfumery.

71-77

Authors: A. R. Banya, O. Y. Karpenko, V. I. Lubenets, V. I. Baranov, V. P. Novikov, O. V. Karpenko

The aim of the work was to study the influence of rhamnolipid biocomplex and ethylthiosulfanilate on field pea and sorghum plants when growing in petroleum contaminated soil. Plant seeds were treated with solutions of rhamnolipid biocomplex or ethylthiosulfanilate (0.01 g/l) before planting and grown in containers with soil artificially contaminated with petroleum (5, 8 and 10%). Effect of rhamnolipid biocomplex and ethylthiosulfanilate was determined by the determination of growth (weight, length plants) and biochemical parameters (content of photosynthetic pigments, hydrogen peroxide, malondialdehyde). The stimulating effect of rhamnolipid biocomplex and ethylthiosulfanilate on growth parameters of field pea was shown: the shoot mass has significantly increased on 39%, root mass – on 26% if compared with the control. For sorghum somewhat smaller increase of growth parameters was observed. Under the influence of rhamnolipid biocomplex and ethylthiosulfanilate the content of photosynthetic pigments in field pea and sorghum has also increased. It was determined that the action rhamnolipid biocomplex and ethylthiosulfanilate promoted the decrease of indicators of plant oxidative reactions if compared with control: the content of hydrogen peroxide – in average on 15% and 16%, malondialdehyde – on 13.5% and 16% respectively. The results of the study testify to the effectiveness of rhamnolipid biocomplex and ethylthiosulfanilate as growth stimulators for field pea and sorghum, as well as improvement of adaptive capability of plants to unfavorable conditions. It creates the prospects of their application as effective and ecologically safe substances for the intensification of contaminated soil phytoremediation.

86-92

Authors: I. A. Blayda, T. V. Vasilyeva, L. I. Slyusarenko, V. F. Khytrych

The study's aims were to isolate and establish pure cultures of prevailing microorganisms from the aboriginal consortium in coal fly ash (FAAC), describe their physiology, biochemistry and practically-useful properties, and compare the efficiency of bioleaching metals from fly ash using pure cultures and the consortium. Through enrichment cultures on standard media we isolated pure cultures of the microorganisms which were then preliminarily identified using standard techniques. This allowed us to isolate from FAAC pure cultures of three prevailing strains of mesophilic and moderately thermophilic acidophilic chemolithotrophic bacteriae, belonging to Acidithiobacillus, such as Acidithiobacillus ferrooxidans, and Sulfobacillus. The strains exhibited high oxidative activity in leaching the rare metals Gallium and Germanium, as well as some heavy metals, from fly ash substrate. A comparison of oxidative activity of the isolated strains and the aboriginal consortium under mesophilic conditions led to the conclusion about advantage of consortium, because it had arisen from syntrophy of microbes in the community. This should be taken into account at the developing of bacterial preparations that are optimal for the technogenic substrate.

103-11

Authors: Govorukha V.M., Tashyrev O.B.

The aim of the study was regulation of metabolism of Citrobacter freundii Ml-31.1/1 on the basis of thermodynamic calculations. The essence of metabolism regulation was in ability to perform two opposite processes, namely mobilization and immobilization of iron compounds, depending on the culture conditions by single strain C. freundii Ml-31.1/1. The following methods were used: potentiometric measuring of pH and Eh values, colorimetric determination of microbial biomass increase and changes of iron compounds concentration, gas chromatography. The possibility of theoretically grounded regulation of iron transformation pathways was experimentally confirmed. Single culture (C. freundii Ml-31.1/1) provided both immobilization and mobilization of iron compounds due to specific conditions for microbial growth in nutrient medium.. Obtained regularities are the basis for assessing the role of microorganisms in iron biogeochemical cycles. This data could be used as a basis of effective biotechnologies of iron extraction from depleted deposits and water purification from iron compounds.