4_2020(en)

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ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)

Biotechnologia Acta V. 13, No 4, 2020
Р. 71-80, Bibliography 30, English
Universal Decimal Classification: 579.841: 577.114
https://doi.org/10.15407/biotech13.04.071

PRODUCTION OF EXOPOLYSACCHARIDE ETHAPOLAN UNDER Acinetobacter sp. IMV B-7005 CULTIVATION ON THE MIXTURE OF ACETATE AND SUNFLOWER OIL

Pirog Т. P., Voronenko А. А., Yarosh М. B.

National University of Food Technologies, Kyiv, Ukraine

The aim of the work was to establish the cultivation conditions of Acinetobacter sp. IMV B-7005 for providing the maximum indicators of the exopolysaccharide ethapolan synthesis on the mixture of acetate and sunflower oil, as well as to study the possibility of replacing the refined oil in the mixture with acetate on a waste one.

The optimal molar ratio of concentrations for refined sunflower oil and acetate in the mixture was calculated theoretically according to Babel’s concept of “auxiliary substrate”. The EPS concentration was determined gravimetrically after precipitation with isopropanol. The EPS-synthesizing ability was calculated as the ratio of the EPS concentration to the concentration of biomass and expressed in g EPS/g biomass.

Based on theoretical calculations of energy requirements for EPS synthesis and biomass of Acinetobacter sp. B-7005 on energy-deficient substrate (acetate) it was found that molar ratio for the concentrations of sodium acetate and oil in the mixture, at which the maximum EPS synthesis was achieved, should be 1:0.13. It was experimentally confirmed that at this ratio of monosubstrate concentrations and the use of the inoculum grown on refined oil, the synthesis rates of ethapolan were higher than at the other ratios of acetate and oil concentrations in the mixture. However, the assimilation of sodium acetate through the symport with proton led to an increase in pH of the culture liquid to 9.0–9.3, which is not optimal for EPS synthesis. Decrease of the medium alkaline component and fractional introduction of substrates enabled not only to stabilize pH at the level of 7.8–7.9, but to increase the amount of synthesized ethapolan to 16–17 g/l, which was achieved regardless of the type of used oil (refined or mixed waste) in the mixture with acetate.

Key words: Acinetobacter sp. IMV В-7005, mixed substrates, waste oil, fractional substrate introduction.

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6. Hussain A., Zia K. M., Tabasum S., Noreen A., Ali M., Iqbal R., Zuber M. Blends and composites of exopolysaccharides; properties and applications: A review. Int. J. Biol. Macromol. 2017, 94 (Pt. A), 10?27. https://doi.org/10.1016/j.ijbiomac.2016.09.104

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ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)

Biotechnologia Acta V. 13, No 4, 2020
Р. 60-70 , Bibliography 25, English
Universal Decimal Classification: 541.49:546.791.6 +546.73
https://doi.org/10.15407/biotech13.04.060

BIODEGRADATION AND ANTIMICROBIAL ACTIVITY OF GUANIDINE-CONTAINING POLYETHYLENE OXIDE HYDROGEL

G. O. Iutynska ¹, M.Ya. Vortman², D. R. Abdulina ¹, Zh. P. Kopteva¹, A. Ye. Kopteva ¹, A. V. Rudenko³, V. V. Tretyak ³, V. N. Lemeshko ², V. V. Shevchenk o ².

¹Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine,
²Institute of Macromolecular Chemistry of the National Academy of Sciences of Ukraine,
³Institute of Urology, of the National Academy of Medical Sciences of Ukraine

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.

Key words: Guanidine-containing polyethylene oxide hydrogel, antibacterial properties, gram-positive and gram-negative bacteria, catalase and lipase activities, biodegradation, IR spectra.

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ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)

Biotechnologia Acta V. 13, No 4, 2020
Р. 49-59 , Bibliography 31, English
Universal Decimal Classification: 579.695
https://doi.org/10.15407/biotech13.04.049

BIOREMOVAL OF TOXIC CHROMIUM(VI) VIA DARK HYDROGEN FERMENTATION OF MULTICOMPONENT ORGANIC WASTE

V .M. Hovorukha, O. A. Havryliuk, G. V. Gladka, O. B. Tashyrev

Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, Kyiv,

Thermodynamic calculations allow determining optimal metabolic pathways for microbial extraction of toxic soluble hexavalent chromium compounds from contaminated sewage.

The purpose was to predict theoretically and confirm experimentally the possibility of hazardous Cr(VI) removal by hydrogen producing microbiome with simultaneous destruction of multicomponent organic waste and hydrogen synthesis.

The gas composition was determined by the standard gas chromatography method. The redox potential (Eh) and рН of the medium were measured potentiometrically. The Cr(VI) concentration was measured by a photocolorimetric method.

The multicomponent organic waste was effectively destroyed by hydrogen producing microbiome at the absence of chromium. The hydrogen fermentation cycle was not significantly inhibited by addition of Cr(VI). After complete microbial reduction of soluble CrO₄^2– to insoluble Cr(OH)₃·nH2O↓ the metabolic parameters returned to initial values.

The optimal pathway of microbial detoxification of toxic Cr(VI) compounds was thermodynamically predicted and experimentally confirmed. The high efficiency of Cr(VI) removal by strict anaerobic hydrogen producing microbiome via dark hydrogen fermentation of multicomponent organic waste was demonstrated. The obtained results can be used for development of novel environmental biotechnology of chromium-containing sewage purification and simultaneous destruction of environmentally hazardous organic waste as well as obtaining of eco-friendly energy carrier biohydrogen.

Key words: thermodynamic prediction, environmental biotechnologies, hydrogen fermentation, biohydrogen synthesis, toxic chromium(VI) compounds, microbial reduction of chromate, multicomponent organic waste destruction.

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ISSN 2410-776X (Online)

Biotechnologia Acta V. 13, No 4, 2020
Р. 39-48, Bibliography 33, English
Universal Decimal Classification: 571.27: 581.6
https://doi.org/10.15407/biotech13.04.039

FATTY ACID COMPOSITION OF PURSLANE SEED WATER EXTRACT AND ITS EFFECT ON METABOLIC PROFILE OF MURINE PERITONEAL MACROPHAGES

M. Gahramanova ^{1, 2}, A. Ostapchuk ³, O. Molozhava ², V. Svyatetska ², M. Rudyk ², Y. Hurmach ⁴, R. Dovhyi², L. Skivka ²

¹Nargiz Medical Center, Baku, Azerbaijan
² Educational and Scientific Center "Institute of Biology and Medicine", Taras Shevchenko National University of Kyiv, Ukraine
³ Mechnikov Odesa National University, Ukraine
⁴ Bogomolets National Medical University, Kyiv, Ukraine

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.

Key words: purslane seed water extract, peritoneal macrophages, reactive oxygen species, phagocytosis, arginine metabolism.

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Details: Hits: 120

ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)

Biotechnologia Acta V. 13, No 4, 2020
Р. 26-38, Bibliography 26, English
Universal Decimal Classification: 577.151.6:577.161.11
https://doi.org/10.15407/biotech13.04.026

NON-COENZYME PROPERTIES OF THIAMINE: EVALUATION
OF BINDING AFFINITY TO MALATE DEHYDROGENASE ISOFORMS

O. Mezhenska ¹, A. Rebriev ¹, O. Kobzar ², N. Zlatoust ¹, A. Vovk ², Yu. Parkhomenko ¹

¹ Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv
² Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine, Kyiv

The aim of this work was evaluation of binding affinity of thiamine to malate dehydrogenase isoforms. The methods of affinity chromatography, SDS PAGE electrophoresis, and MALDI-TOF mass spectroscopy, as well as molecular modeling in silico were used in the study. Affinity sorbent (T-AS) contained C2-conjugated thiamine fragment as an anchor which was bound to activated Sepharose 4B via a spacer composed of N-4-azobenzoylcaproic acid hydrazide. A commercial preparation of MDH from the porcine heart was chosen for the experimental study. Analysis of the protein content in fractions performed by the Bradford method showed that three separate protein peaks with malate dehydrogenase activity were obtained after elution with a thiamine solution. The results of one-dimensional electrophoresis of the initial MDH preparation and pooled fraction of proteins which were eluted from the affinity sorbent with a thiamine solution demonstrated that almost all protein fractions detected in the commercial MDH preparation were also present in eluates obtained by T-AS affinity chromatography. Four isoforms of MDH, including cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2) and its isoform, and malate dehydrogenase 1B (MDH1B) were specifically bound to the affinity sorbent. According to the molecular docking results, the most preferred for both monomeric and homodimeric MDH1 and MDH2 could be ligand position at the NAD (NADH) binding site. Additional binding site could be located between two subunits of the homodimeric form of enzyme. Our results confirm the previously obtained data and expand an idea of ability of MDH isoforms to interact with the thiamine molecule in vivo. These data can also be useful for identification of thiamine binding protein (ThBP) which was previously isolated from rat brain, taking into account the possible partial homology of this protein with proteins that show MDH activity.

Key words: thiamine, affinity chromatography, malate dehydrogenase, protein affinity for thiamine, molecular docking, thiamine binding protein.

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