6_2019(en)

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

"Biotechnologia Acta" V. 12, No 6, 2019
Р. 65-70, Bibliography 14, English
Universal Decimal Classification: 631.46.631.445.41:631.84
https://doi.org/10.15407/biotech12.06.065

IRECTION AND STRENGTH OF MICROBIOLOGICAL PROCESSES IN LAYERS OF GRAY FOREST SOIL UNDER DIFFERENT REGIMES OF MANAGEMENT

Malynovska I. M.

Institute of Agriculture of the National Academy of Sciences of Ukraine, Kyiv

The aim of the work was to study the direction and intensity of mineralization and immobilization processes in different layers of gray forest soil at fallow fields, extensively and intensively used agrosoils. The research included laboratory analysis, microbiological studies, and statistical processing. For the fallow plots, the layers chosen for study were Hd – turf (0–10 cm), He – humus-eluvial (11–40 cm), Hi – humus-illuvial (41–74 cm), Ih – illuvial-humus (75–115 cm), Ip – transitional from the illuvial layer to bedrock (116–156 cm), Pi – bedrock with occasional insertions of illuvial soil (157?191 cm); for the agrosoils of the stationary experiments: He – humus-eluvial (0-10 and 11–40 cm), Hi – humus-illuvial (41–74 cm), Ih – illuvial-humus (75–115 cm), Ip – transitional from the illuvial layer to bedrock (116–56 cm). We found that humus mineralization differed in some layers of the gray forest soil under these management regimes. At fallows, the intensity of humus mineralization tended to decrease with depth, and it was interrupted in the Ih and Ip layers. In the intensively used agrosoil, humus mineralization was more active in Hi- and Ih- layers. Comparatively, the activity of humus mineralization smoothly decreased in the profile of the extensively used agrosoil from the uppermost layer to the lower by 97.2%. The mineralization coefficient of Nitrogen compounds gradually decreased in the fallow ground and extensively used agrosoil, unlike the intensively managed agrosoil, in which the intensity of mineralization-immobilization of nitrogen compounds increased in the He and Hi layers. It was shown that the fallow ground had the more efficient system to transfer substrates and mineral ions down the profile to the lower layers. The difference in biologic activity between the upper and lower layers was maximum in the fallow ground, intermediate in the intensively used agrosoil and minimum in the extensive agrosoil.

Key words: soil horizons, index of pedotrophy, nitrogen mineralization coefficient, humus mineralization activity, fallow ground, agrosoil

References

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5. Mishustin E. N., Runov E. V. Successes in developing the principles of microbiological diagnosis of soil conditions. Uspekhi sovremennoy biotekhnolohii. Moskva, AN SSSR. 1957, 44, 256–267. (In Russian).

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9. Rusakova, I. V. Biological properties of sodpodzolic sandy loam soil with prolonged use of straw for fertilizer. Pochvovedeniye. 2013, 1493 (12), 1485. (In Russian).

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11. Malinovska I. M., Tkachenko M. A. The number and physical activity of microorganisms of the genetic horizons of the gray fox area for different purpose. Zbirnyk naukovykh prats Instytutu zemlerobstva. 2015, 4, 13–24. (In Ukrainian).

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13. Gorodetska S. P. Diya Dobriv і crops on the balance of humus in the grain-saponny sіvozmіnі. Zbirnyk naukovykh prats Instytutu zemlerobstva, UAAN, Kyiv. 2003, Issue 1–2, 55–64 р. (In Ukrainian).

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

"Biotechnologia Acta" V. 12, No 6, 2019
Р. 56-64 , Bibliography 29, English
Universal Decimal Classification: 577.21+632.78+57.084.1
https://doi.org/10.15407/biotech12.06.056

GENETIC TRANSFORMATION OF PLANTS CONTAINING THE SYNTHETIC cry1Ab GENE ENCODING RESISTANCE TO LEPIDOPTERAN PESTS

A. M. Taranenko¹, I. O. Nitovska¹, L. H. Velykozhon^{1, 2}, P. D. Maystrov¹, M. V. Kuchuk¹, B. V. Morgun^{1, 2}

¹Institute of Cell Biology and Genetic Engineeringof the National Academy of Sciences of Ukraine, Kyiv
²Institute of Plant Physiology and Genetics of thr National Academy of Sciences of Ukraine, Kyiv

The research was aimed to develop genetic constructs for Agrobacterium-mediated plant transformation, containing the synthetic cry1Ab gene, and their testing through the transformation of tobacco, followed by a molecular genetic analysis of the obtained plants to confirm the transformation event. Basic methods of DNA cloning, Agrobacterium-mediated transformation of Nicotiana tabacum L. by leaf disc method, selection of transformants in vitro, analysis of the transgene presence in plant DNA, detection of cry1Ab gene expression by PCR with reverse transcription were used. In the course of the study, the vectors pCB182 and pCB241 that contained the synthetic gene cry1Ab were constructed. Agrobacterium-mediated transformation of tobacco was carried out by created vectors and regenerant plants containing transgenes in their DNA were obtained. Expression of cry1Ab transgene in the obtained transformants of tobacco by the RT-PCR method was confirmed. As a result of the Agrobacterium-mediated transformation of plants with pCB182 and pCB241 vectors containing the synthetic cry1Ab lepidopteran resistance gene it is possible to obtain transgenic plants with expression of the transgene.

Key words: cry1Ab, Nicotiana tabacum L., Agrobacterium-mediated transformation, PCR-analysis, transgenesis.

References

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6. Ye G. Y., Yao H. W., Shu Q. Y., Cheng X., Hu C. High levels of stable resistance in transgenic rice with a cry1Ab gene from Bacillus thuringiensis Berliner to rice leaffolder, Cnaphalocrocis medinalis (Guen?e) under field conditions. Crop. Protect. 2003, 22 (1), 171–178.https://doi.org/10.1016/S0261-2194(02)00142-4

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8. Valldor P., Miethling-Graff R., Martens R., Tebbe C. C. Fate of the insecticidal Cry1Ab protein of GM crops in two agricultural soils as revealed by 14C-tracer studies. Appl. Microbiol. Biotechnol. 2015, 99 (17), 7333–7341. https://doi.org/10.1007/s00253-015-6655-5

9. ISAAA. Global status of commercialized biotech/GM crops in 2017: Biotech crop adoption surges as economic benefits accumulate in 22 years. Ithaca, NY: The International Service for the Acquisition of Agri-biotech Applications. 2018. http://www.isaaa.org/resources/publications/briefs/53/download/isaaa-brief-53-2017.pdf

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18. Morgun B. V., Stepanenko O. V., Stepanenko A. I., Taranenko A. M., Kuchuk M. V. Detection of transgenic herbicide resistant rapeseed plants in Kyiv region. Visnyk ukr. tov. henetykiv i selektsioneriv. 2014, 12 (2), 166–173.

19. Box M. S., Coustham V., Dean C., Mylne J. S. Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis. Plant Meth. 2011, 7 (1), 7. https://doi.org/10.1186/1746-4811-7-7

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25. Wu G., Cui H., Ye G. Inheritance and expression of cry1Ab gene in Bt (Bacillus thuringiensis) transgenic rice. Theoret. Appl. Genet. 2002, 104, 727. https://doi.org/10.1007/s001220100689

26. Zhao Q., Liu M., Tan M., Gao J., Shen Z. Expression of Cry1Ab and Cry2Ab by a Polycistronic Transgene with a Self-Cleavage Peptide in Rice. PLoS ONE. 2014, 9 (10), e110006. https://doi.org/10.1371/journal.pone.0110006

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29. Kohli A., Twyman R. M., Abranches R., Wegel E., Stoger E. Transgene integration, organization and interaction in plants. Plant Mol. Biol. 2003, 52 (2), 247–258. https://doi.org/10.1023/A:1023941407376

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

"Biotechnologia Acta" V. 12, No 6, 2019
Р. 46-55, Bibliography 30, English
Universal Decimal Classification: 579.663
https://doi.org/10.15407/biotech12.06.046

POST-HARVEST TREATMENT OF VEGETABLES WITH EXOMETABOLITES OF Nocardia vaccinii IMV B-7405, Аcinetobacter calcoaceticus IMV В-7241 AND Rhodococcus erythropolis IMV Ас-5017
TO EXTEND THEIR SHELF LIFE

T. P. Pirog, B. S.Geichenko, A. O. Zvarych

National University of Food Technologies, Kyiv, Ukraine

The aim of the work was to study the possibility of Nocardia vaccinii IMV B-7405, Acinetobacter calcoaceticus IMV B-7241 and Rhodococcus erythropolis IMV Ac-5017 supernatants usage with various concentrations of surfactants for post-harvest processing of vegetables.

N. vaccinii IMV B-7405, A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 were grown on used sunflower oil and ethanol. For vegetables treatment, supernatants of the culture fluid with surfactant concentration of 0.01–0.5 g/l were used. The concentration of surfactants was determined by the gravimetric method after extraction with Folch mixture. The total number of heterotrophic bacteria and fungi on the surface of vegetables was determined by the Koch method on meat-peptone agar and wort agar, respectively.

It was shown that treatment of broccoli, Brussels sprouts, sweet pepper and tomatoes with N. vaccinii IMV B-7405, A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 supernatants was accompanied by 6–17 and 8–50 times decrease of bacteria and fungi number on their surface, respectively, compared with that on the surface of vegetables washed with tap water. The possibility of double use of the same supernatant for various batches of vegetables washing was established. Non-treated and water-washed vegetables spoiled faster than those treated with surfactant-containing supernatants did.

N. vaccinii IMV B-7405, R. erythropolis IMV Ac-5017 and A. calcoaceticus IMV B-7241 exometabolites used for treating vegetables to extend their shelf life have the following advantages in comparison with known microbial surfactants: they exhibit high antimicrobial activity when the surfactant concentration was sometimes lower and in the form of supernatant, which lets you exclude the expensive stage of isolation and purification of the target product from the technological process. In addition, surfactant-containing supernatants are highly effective in their repeated use.

Key words: storage of vegetables, microbial spoilage, surfactants.

References

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2. Mnif I., Ghribi D. Glycolipid biosurfactants: main properties and potential applications in agriculture and food industry. J. Sci. Food Agric. 2016, 96 (13), 4310–4320. https://doi.org/10.1002/jsfa.7759

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4. De Graeve M., De Maeseneire S. L., Roelants S. L. K. W., Soetaert W. Starmerella bombicola, an industrially relevant, yet fundamentally underexplored yeast. FEMS Yeast Res. 2018, 18 (7). https://doi.org/10.1093/femsyr/foy072

5. Jimoh A. A., Lin J. Biosurfactant: A new frontier for greener technology and

environmental sustainability. Ecotoxicol. Environ. Saf. 2019, 184, 109607. https/doi.org/10.1016/j.ecoenv.2019.109607.

6. Naughton P. J., Marchant R., Naughton V., Banat I. M. Microbial biosurfactants: current trends and applications in agricultural and biomedical industries. J. Appl. Microbiol. 2019, 127 (1), 12–28. https://doi.org/10.1111/jam.14243

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9. Sharma V., Garg M., Devismita T., Thakur P., Henkel M., Kumar G. Preservation of microbial spoilage of food by biosurfactantbased coating. Asian J. Pharm. Clin. Res. 2018, 11 (2), 98–101. https://doi.org/10.22159/ajpcr.2018.v11s2.28592

10. Romanazzi G., Feliziani E., Ba?os S. B., Sivakumar D. Shelf life extension of fresh fruit and vegetables by chitosan treatment. Crit. Rev. Food Sci. Nutr. 2017, 57 (3), 579–601. https://doi.org/10.1080/10408398.2014.900474

11. Romanazzi G., Feliziani E., Sivakumar D. Chitosan, a biopolymer with thrice action on postharvest decay of fruit and vegetables: eliciting, antimicrobial and film-forming properties. Front. Microbiol. 2018, 9, 2745. https://doi.org/10.3389/fmicb.2018.02745

12. Barbosa A. A. T., Mantovani H. C., Jain S. Bacteriocins from lactic acid bacteria and their potential in the preservation of fruit products. Crit. Rev. Biotechnol. 2017, 37 (7), 852–864. https://doi.org/10.1080/07388551.2016.1262323

13. Sinumvayo J. P., Ishimwe N. Agriculture and food applications of rhamnolipids and its production by Pseudomonas aeruginosa. J. Chem. Eng. Process. Technol. 2015, 6 (2), 223. https://doi.org/10.4172/2157-7048.1000223

14. Pirog T., Beregova K., Geichenko B., Stabnikov V. Application of surface-active substances produced by Nocardia vaccinii ІМV В-7405 for the treatment of vegetables. Ukrainian food journal. 2019, 8 (1), 99–109. https://doi.org/10.24263/2304-974X-2019-8-1-11

15. Pirog T. P., Konon A. D., Sofilkanich A. P., Iutinskaia G. A. Effect of surface-active substances of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017, and Nocardia vaccinii K-8 on phytopathogenic bacteria. Appl. Biochem. Microbiol. 2013, 49 (4), 360–367. https://doi.org/10.1134/S000368381304011X

16. Pirog T. P., Lutsay D. A., Kliuchka L. V., Beregova K. A. Antimicrobial activity of surfactants of microbial origin. Biotechnologia Acta. 2019, 12 (1), 39–57. https://doi.org/10.15407/biotech12.01.039

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18. Pirog T., Sofilkanych A., Konon A., Shevchuk T., Ivanov S. Intensification of surfactants’ synthesis by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV В-7241 and Nocardia vaccinii K-8 on fried oil and glycerol containing medium. Food. Bioprod. Proces. 2013, 91 (2), 149–157. https://doi.org/10.1016/j.fbp.2013.01.001

19. Dilarri G., da Silva V.L., Pecora H. B., Montagnolli R. N., Corso C. R., Biddia E. D. Electrolytic treatment and biosurfactants applied to the conservation of Eugenia unflora fruit. Food Sci. Technol. 2016, 36 (3). https://doi.org/10.1590/1678-457X.00516

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21. Mart?nez-Hern?ndez B. G., Art?s-Hern?ndez F., G?mez P. A., Formica A. C., Arles F. Combination of electrolysed water, UV-C and superatmospheric O2 packaging for improving fresh-cut broccoli quality. Postharv. Biol. Technol. 2013, 76, 125–134. https://doi.org/10.1016/j.ifset.2013.11.004

22. Ben-Fadhel Y., Ziane N., Salmieri S., Lacroix M. Combined post-harvest treatments for improving quality and extending shelf-life of minimally processed broccoli florets (Brassica oleracea var. italica). Food Bioprocess. Technol. 2017, 11 (1), 84–95. https://doi.org/10.1007/s11947-017-1992-2

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ISSN 2410-7751 (Print)

"Biotechnologia Acta" V. 12, No 6, 2019
Р. 35-45, Bibliography 27, English
Universal Decimal Classification: 577.151.6
https://doi.org/10.15407/biotech12.06.035

METALLOPROTEASE FROM THE CULTURAL LIQUID OF Pleurotus osreatus

Sakovich V. V.¹, Stohnii Ye. M^.2, Zhernosekov D. D.¹, Rebriev A. V.², Korolova D. S.², Marunych R. Yu.², Chernyshenko V. O^.2

The aim of this work was to identify and to study physical and chemical properties of the enzyme preparation which was obtained from the cultural liquid of Pleurotus ostreatus.

The protease containing fraction was obtained from the cultural liquid by sodium chloride precipitation followed by dialysis and concentration procedures. Gelatinase and milk-clotting activity were defined by standard methods. The content of the protein component of the fraction was analysed by HPLC, Laemmli electrophoresis and MALDI-TOF analysis. Protease activity was proved by enzyme-electrophoresis. To identify the protease, mass-spectrometry was carried out using the MatrixScience database. To study the specificity of protease action, the series of chromogenic substrates were used: S2238, S236, S2251, S2765, Leu-pNa, Ala-pNa and S2302. The inhibitory analysis was carried out using EDTA, benzamidine, PMSF, PCMB.

The obtained fraction possessed maximal protease activity at 45 °C. Meanwhile maximal milk-clotting activity was observed at 35 °C. The highest milk-clotting activity was shown at pH 5.0 and less than 3.0. The highest protease activity was shown at pH 6.0. Using HPLC method, it was found the main protein component and some minor proteins. According to the electrophoresis results, the main protein component of the fraction had molecular mass 45 kDa. Enzyme electrophoresis demonstrated that protease activity of the fraction was present in the zone corresponding to 45 kDa. When identifying trypsinolysis products, no homology was found with other known proteinases. It was shown that the protease hydrolyzed peptide bonds which were formed by carboxyl group of amino acids with hydrophobic side chains. The enzyme was inhibited by EDTA (ІС50 = 2.5 mМ). The maximal enzyme activity towards gelatin and Leu-pNa was shown in the presence of 5 mM calcium chloride.

The new calcium-dependent metalloprotease with molecular weight 45 kDa was found in the cultural liquid of Pleurotus ostreatus. The enzyme had no homology with other known proteases and hydrolyzes peptide bonds formed by carboxyl groups of amino acids with hydrophobic side chains.

Key words: basidiomycetes, proteolytic enzymes, milk-clotting activity, physical and chemical properties.

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Details: Hits: 123

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

Biotechnologia Acta V. 12, No. 6, 2019
P. 25-34,Bibliography 80, English.
Universal Decimal Classification: 582.26: 574.62 : 574.55
https://doi.org/10.15407/biotech12.06.025

BIOTECHNOLOGICAL PROSPECTS OF MICROALGAE

Kirpenko N., Leontieva T.

Institute of Hydrobiology of the National Academy of Sciences of Ukraine, Kyiv

The current state and perspectives of biotechnological use of microscopic algae were analyzed. The main directions of algobiotechnology, due to the physiological and biochemical features of these organisms, the volume of algae production in the world, the types of microalgae that had already been used or had practical prospects, ways of biomass obtaining and productivity increasing of industrial algae cultivation were given. The state of this problem, expediency of algobiotechnology development and prospects of microalgae cultivation in Ukraine were discussed.

Key words: algobiotechnology, microalgae, industrial cultivation, aquaculture.

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