Biotechnologia Acta

9-15

Authors: D. O. Tsymbal, О. H. Minchenko

To investigate IRE1-dependent branch of endoplasmic reticulum stress pathway in various cancer cells we created cDNA-constructs for expression of dominant-negative inositol-requiring enzyme – 1 IRE1 and cytosolic domain of IRE1 fused on a C-terminus with c-Myc and 6xHis tags. The non-small-cell lung carcinoma cells H1299-shE6AP were transfected with these constructs. Using anti-c-Myc antibodies we demonstrated effective, dose-dependent expression of dominant-negative and cytosolic IRE1 proteins. In order to investigate IRE1-mediated, heat shock element-dependent transcription, the cells were further transfected with a reporter construct containing heat shock element. We observed that overexpression of dnIRE1 in H1299-shE6AP cells led to significant induction of heat shock element-dependent transcription. This observation may reflect the induction of heat shock genes, which contribute to cellular adaptation to inhibition of native IRE1, a key sensory-signaling enzyme of endoplasmic reticulum stress pathway, which suppresses cancer cell proliferative capacities and alternates the expression of numerous genes, including many transcription factors.

23-31

Authors: T. P. Pirog, T. A. Shevchuk, K. A. Beregova, N. B. Kudriya

The purpose of this study was to establish optimal molar glycerol to glucose ratio for enhanced biosurfactant synthesis by the cultivation of Nocardia vacсinii MV В-7405 on a mixture of energetically nonequivalent substrates (glucose and glycerol). Based on theoretical calculations of the energy requirements for biomass production and the synthesis of surface-active trehalose monomycolates on the energy-deficient substrate (glycerol), the concentration of the energy-excessive substrate (glucose), which increased the efficiency of the substrate carbon conversion to biosurfactant, was determined. It was found that the synthesis of extracellular biosurfactant on a mixture of glucose and glycerol at molar ratio of 1.0:2.5 increased 2.0–2.3-fold ompared to that on corresponding monosubstrates. The increased level of biosurfactant on the mixed substrate is determined by intensification of surface-active trehalose monomycolate synthesis, confirmed by 1.2-5.7–fold increase in activity of the enzymes involved in their biosynthesis (trehalose phosphate synthase, phosphoenolpyruvatecarboxykinase, phosphoenolpyruvate-synthetase) compared to cultivation on monosubstrates glucose and glycerol. The results indicate the feasibility of using a mixture of energetically nonequivalent substrates for enhancing the synthesis of secondary metabolites, as well as possibility of achieving the high efficiency of these mixed substrates if they are properly chosen and the molar ratio of their concentrations are correctly determined.

41-47

Authors: S. S. Dekina, I. I. Romanovska, A. M. Ovsepyan, M. G. Bodyul, V. A. Toptikov

The aim of the research was the development of the method of lysozyme isolation from hen egg proteins. Lysozyme was isolated by differential heat denaturation of proteins with changing of the medium pH value, followed by neutralization, dialysis and additional purification by gel chromatography on Sephadex G-50. Activity was determined by bacteriolytic method (with Micrococcus lysodeikticus 4698 as a substrate). The enzyme purity and molecular mass were determined using SDS-electrophoresis and massspectrometry. The method of lysozyme isolation from hen egg proteins with the enzyme yield of 3.2 ± 0.2% and bacteriolytic activity of 22 025 ± 1 500 U/mg is modified. According to electrophoresis data, the isolated enzyme is characterized by high degree of purity (~95–98%) and is comparable with lysozyme of AppliChem company by main physical and chemical characteristics. The obtaining product is stored in a crystalline form at low temperature (–24 оC) for 9 months. The proposed method allows obtaining active and stable lysozyme with high purity from hen egg protein in laboratory conditions for the usage in biotechnology.

56-62

Authors: I. A. Blayda, T. V. Vasyleva, V. I. Baranov, K. I. Semenov, L. I. Slysarenko, I. M. Barba

The purpose of the research was determination of strains Acidithiobacillus ferrooxidans MFLv37 and Acidithiobacillus ferrooxidans MFLad27, isolated from aboriginal consortium of coal beneficiation dumps and fly ash from coal combustion, resistance to heavy metals, forming part of these waste, as well as adaption ability of the strains to new substrates. New strains increased resistance to heavy metal ions as compared to A. ferrooxidans standard and collection strains is found; minimal inhibitory concentrations of heavy and toxic metals are determined; a number of metals that have negative impact on growth of isolated cultures are identified. It is shown that the minimal metals concentrations, at which strains growth still happens, are several times higher than their concentrations in technogenic waste. It has been established that isolated strains differed in their ability to adapt, as well as in growth rate and substrates oxidation. This is due to the specific conditions of microbiocenoses formation in making and further storage of rock dumps and fly ash, whereof the appropriate strains are isolated. The investigations indicate the necessity in directional selection of strains that are resistant to the toxic compounds and are able to oxidize various mineral substrates, as well as in their adaptation to new substrates for the extraction of heavy metals.

71-76

Authors: K. V. Liapina, P. G. Dulnev, A. I. Marinin, N. N. Pushanko, V. V. Olishevskiy

Aim of the work was to determine the efficiency of combined application of lime and high-viscous suspensions, containing the aluminium nanoparticles as a precursor in treatment of sugar-containing solutions. At the first stage the aluminium nanopowder, encapsulated into a salt matrix, was produced by the combined precipitation from a gas phase of metal and halogenide of alkali metal (NaCl). For the long-term stabilization of aluminum nanoparticles the method, developed by the authors, for dispersing these powders in the composition of polyethylene glycols was used, providing the colloidal solution of high viscosity (gel). At the second stage, as an object of investigation a juice of sugar beet, produced in the laboratory conditions by water extracting from the beet chips, was applied. In the produced juice the main characteristics of its quality were determined: the content of solids, sucrose, its purity was calculated (ratio of sucrose to solids content, in%). The content of protein and pectin components was also determined (as the main components of the colloidal fraction of the diffusion juice). Conventionally, as a basic reagent for the process of a lime pretreatment a lime milk of 1.18 g/cm3 density, prepared by liming the burned lime using hot water, was used. During the experiments the effectiveness of reagents, containing aluminum in nanoform, on the degree of removal of the colloidal dispersion substances in the process of juice purification in sugar beet production and improvement of its quality, is shown. However, the obtained results show that, depending on the method of producing, the additional reagents with aluminium nanoparticles have different effect on change of diffusion juice purity in the process of its treatment by the lime milk.

87-99

Authors: T. V. Krupska, S. V. Pakrishen, O. V. Serov, O. T. Volik, V. V. Turov

By the method of low-temperature 1Н NMR spectroscopy the structure of the hydrate layers of water associated with brain cells, the changes of these parameters during necrotic lesions (stroke) and in the presence of trifluoroacetic acid, which allows differentiating intracellular water clusters according to their ability to dissolve the acid, were studied. Also the impact of silica TS-100 nanoparticles on the state of water in brain tissue, namely on the water binding parameters in the air and in the presence of a weakly polar solvent was considered. The distributions by the radii and change of Gibbs free energy for clusters of strongly bound interfacial water were obtained. It was shown that the hydration properties of the native brain tissue differ from the hydration properties of necrotic damaged tissue by the structure of weakly bound water clusters. In intact tissue all the water is associated and is a part of clusters and domains, most of which have a radii R = 2 and 20 nm. The media with chloroform stabilizes water polyassociates with the radius up to R = 100 nm and trifluoroacetic acid stabilizes water polyassociates with radii R = 7–20 nm. It was found that the partial dehydration of the investigated tissue samples is accompanied by decreasing of weakly bound water amount and some increasing of strongly bound water that indicates a change of molecular interactions between the components of cells-nanoparticles composite system. The ischemic necrosis area presence leads to a decrease of water binding due to the average size water polyassociates increasing. This effect is observed both in air and in a weakly polar organic solvent medium (deuterochloroform).