Biotechnologia Acta

31-39

Authors: Gayda G. Z.1, Stasyuk N. E.1, Gonchar M. V.

Physicochemical and enzymatic methods of quantitative L-arginine estimation are described. A variety of detection procedures for L-arginine analysis have been developed. The majority of the frequently used approaches are marked by poor precision, low sensitivity and selectivity. These methods are time-consuming, expensive and require skilful labor techniques, so it needs to develop novel highly selective and sensitive ones for improvement of L-arginine monitoring in clinical diagnostics and food industry. Experimental data concerning development and testing of effective enzymatic methods of L-arginine determination, including biosensors, are presented. All proposed methods are based on human liver arginase I isolated from the recombinant yeast strain Hansenula polymorpha NCYC-495 pGAP1-HsARG1 (leu2car1 Sc:LEU2). Arginase I (EC 3.5.3.1; L-arginine amidinohydrolase), a key enzyme of the urea cycle, catalyses the conversion of L-arginine to ornithine and urea. An effectiveness of the proposed enzymatic methods for L-arginine assay, as amperometric and potentiometric biosensors so as enzymatic methods with spectrophotometric and fluorometric detection of the product, was demonstrated on the samples of commercial pharmaceuticals. These methods seem to be prospective for L-arginine analyses in food industry (juices and wines) and in medicine, for diagnostics and drug control in blood serum under treatment of cancer malignances. Hence, the proposed L-Arg selective and simple methods would be convenient and useful in the future for routine clinical analysis and food quality control.

54-59

Authors: Oliinyk O. S., Kaberniuk A. A., Kolibo D. V., Komisarenko S. V.

Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv) antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized) human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

66-74

Authors: Kucherenko I. S., Soldatkin O. O., Didukh D. Yu., Soldatkin O. P.

Analytical characteristics of a biosensor based on glucose oxidase and hexokinase and intended for ATP determination were studied. Platinum disc electrodes were used as amperometric transducers. Range of working potentials for biosensor functioning was shown. An optimal time of enzymes immobilization was determined. Optimal conditions for biosensor functioning during work with biological fluids were selected. Biosensor work in three buffer solutions (PBS, tris and HEPES) was investigated and it was shown that it was possible to obtain various operational characteristics of the biosensor depending on tasks that are assigned to it by varying the composition of sample. Reproducibility of biosensor responses to ATP and glucose during a day and of biosensor preparation was shown. The proposed biosensor can be further used for analysis of glucose and ATP content in water solutions.

80-86

Authors: Lootsik M. D., Boiko N. M., Mitina N. E., Klyuchivska O. Yu., Lutsyk M. M., Konstantinova T. E., Zaichenko A. S., Stoika R. S.

The recognition and isolation of specific mammalian cells by the biocompatible polymer coated super-paramagnetic particles with determined surface functionality were studied. The method of synthesis of nanoscaled particles on a core of iron III oxide (Fe2O3, magemit) coated with a polymer shell containing reactive oligoperoxide groups for attachment of ligands is described. By using the developed superparamagnetic particles functionalized with peanut agglutinin (PNA) we have separated the sub-populations of PNA+ and PNA– cells from ascites of murine Nemeth-Kellner lymphoma. In another type of experiment, the particles were opsonized with proteins of the fetal calf serum that improved biocompatibility of the particles and their ingestion by cultivated murine macrophages J774.2. Macrophages loaded with the particles were effeciently separated from the particles free cells by using the magnet. Thus, the developed surface functionalized superparamagnetic particles showed to be a versatile tool for cell separation independent on the mode of particles’ binding with cell surface or their engulfment by the targeted cells.

95-99

Authors: Bozhkov A. I., Menzyanova N. G., Sysenko E. I.

The effect of ethanol (final concentration is 0.3 and 0.5%) on biomass yield and composition (content of nucleic acids, proteins, lipids, and chlorophyll a and b, β-carotene, and exopolysaccharides) and morphology of the cells Dunaliella viridis in a quasi-mode of cultivation was investigated. It has been found that addition of ethanol in the culture medium to a final concentration of 0.3% increased output 2-fold compared with the quasi-continuous cultivation without alcohol and 2.5 times compared with a cumulative cultivation. Increasing the concentration of ethanol in the medium to 0.5% reduced the biomass yield of Dunaliella viridis compared with 0.3% alcohol concentration. Ethanol increased the output exopolysaccharides into culture medium and changed morphology and Dunaliella viridis motility.

110-116

Authors: Fedotov О. V., Veligodska A. K.

General content of polyphenols, carotenoids and melanin in basidiomycetes carpophorus was determined. 50 species were studied, 27 of which belong to the Polyporales form and 23 are to the Agaricales form. In order to determine the total content of phenolic substances spectrophotometric methods were used. Polyphenols were studied in alcoholic extracts through the modified Folin-Chokalteu procedure; melanin — by alkaline hydrolysis and calculated using a calibration curve (by pyrocatechol), carotenoids were studied in acetone extracts and calculated by the Vetshteyn formula. Statistical and cluster analysis of the data enabled to identify species of basidiomycetes that are perspective for biotechnology. The most promising in terms of total polyphenols, carotenoids and melanins of poliporal basidiomycetes are species Fomes fomentarius, Ganoderma applanatum, Ganoderma lucidum and Laetiporus sulphureus, and among agarikal fungi — Fistulina hepatica, Flammulina velutipes, Pleurotus ostreatus, Stropharia rugosoannulata, Agrocybe cylindracea and Tricholoma flavovirens. These species of Basidiomycetes were isolated in pure mycelia culture to find out their biosynthetic activity.

123-130

Authors: Drozdov O. L., Shulga S. M., Adab M., Glukh I. S.

The results of studies of hepatoprotective activity of biopreparations of defatted soy and sunflower lecithins under carbon tetrachloride intoxication of the experimental animals are given. Analyze of biochemical and histological data under soy and sunflower lecithin exposure changed in terms of poisoning enables to conclude that both biological products of defatted lecithin slowed the growth of mass of animal liver and transaminase activity and stimulated liver regeneration as a result of polyploidy and hypertrophy of hepatocytes, resuscitated beam and lobulation organ and created contacts between hepatocytes and venous vessels as well. It is evidenced that repair processes are activated in condition of their injection under carbon tetrachloride intoxication.