Перегляд за Автор "Zhdanyuk, Valentina"

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Effect of tryptophane on synthesis of certain exometabolites by bacteria of genus Acinetobacter, Nocardia, and Rhodococcus and their properties
(2022) Pirog, Tatiana; Piatetska, Daria; Zhdanyuk, Valentina; Leonova, Natalia; Shevchuk, Tetiana
The efficiency of integrated microbial biotechnologies for obtaining several practically valuable metabolites in one technological process is determined both by the maximum concentration of these substances and their properties. This is especially true for secondary metabolites, the composition and properties of which vary depending on the cultivation conditions of the producer. Aim. To research the eff ect of tryptophan (a precursor of auxin biosynthesis) in the culture media on the synthesis of certain exometabolites by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241, and Nocardia vaccinii IMV B-7405 as well as their properties. Methods. R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-724, and N. vaccinii IMV B-7405 were cultivated in a medium containing refi ned and waste sunflower oil, biodiesel waste, or ethanol as a carbon source. The concentration of tryptophan in the medium was 300 mg/L. Surfactants were extracted from the supernatant of the cultural liquid with a modified Folch mixture. Phytohormones were isolated from the supernatant by sequential extraction with organic solvents after surfactant extraction. Thin-layer chromatography was used for preliminary purification and concentration of phytohormones. Qualitative and quantitative determination of auxins was performed using high-performance liquid chromatography. The antimicrobial activity of surfactants was analysed by the minimum inhibitory concentration. The activity of enzymes of surface-active glycoand aminolipids biosynthesis (phosphoenolpyruvate synthetase, phosphoenolcarboxykinase, and NADP+-dependent glutamate dehydrogenase) was determined spectrophotometrically during the oxidation of NADH or NADP. Results. It was found that the presence of tryptophan in the culture medium of the strains under study did not affect the number of synthesized surfactants, which was 1.80−1.90, 1.55−1.75, and 1.50−1.65 g/L, respectively. At the same time, cultivation of R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-724, and N. vaccinii IMV B-7405 in the media with tryptophan increased the number of phytohormones: it was higher than the amount of phytohormones synthesized during cultivation without a precursor. The introduction of tryptophan into the culture medium of the strains was accompanied by the formation of surfactants. These compounds showed 2−4 times higher antimicrobial activity against the phytopathogenic bacteria (Agrobacterium tumefaciens UCM B-1000, Pseudomonas syringae UCM B-1027T, Xanthomonas vesicatoria UCM B-1106, Pectobacterium carotovorum UCM B-1075T, Clavibacter michiganensis IMV B-102 and Pseudomonas syringae pv. tomato IMV B-9167) than compounds synthesized on a medium without a precursor. The antimicrobial activity of surfactants synthesized by A. calcoaceticus IMV B-7241 in the presence of tryptophan either did not change compared to that for surfactants obtained without tryptophan, or increased slightly. Data on the activity of surfactant biosynthesis enzymes correlated with the indicators of their antimicrobial activity. In the presence of tryptophan in the culture medium of N. vaccinii IMV B-7405 and R. erythropolis IMV Ac-5017, NADP+-dependent glutamate dehydrogenase activity in the cells of these strains (a key enzyme for biosynthesis of aminolipids responsible for antimicrobial activity) increased almost by 1.4 times compared to that on a tryptophan-free medium. Conclusions. As a result of this work, it was found that the presence of tryptophan in the culture medium of researched strains did not affect the number of surfactants. The antimicrobial activity of surfactants against phytopathogenic bacteria either increased or remained unchanged compared to that established for surfactants synthesized without a precursor of auxin biosynthesis. The obtained data testify to the high efficiency of the potential use of surfactants complex preparations and phytohormones in crop production to stimulate the growth of plants and biocontrol of phytopathogenic bacteria.
Induction of auxins synthesis by Rhodococcus erythropolis IMV Ac-5017 with the addition of tryptophan to the cultivation medium
(2020) Pirog, Tatiana; Leonova, Natalia; Piatetska, Daria; Klymenko, Natalia; Zhdanyuk, Valentina; Shevchuk, Tetiana
The ability of surfactant producers to synthesize phytohormones expands the scope of their practical application and provides prospects for the development of microbial preparations with growth-stimulating properties. The possibility to intensify the phytohormone-stimulants synthesis by bacterial strains increases the efficiency of such preparations. Aim. The aim is to research the possibility of extracellular auxin synthesis induction in the presence of tryptophan in the cultivation medium of surfactant producer Rhodococcus erythropolis IMV Ac-5017 and establish the optimal concentration of tryptophan and time of introduction into the medium to ensure maximum synthesis of auxins. Methods. Biochemical, microbiological, biotechnological. Cultivation was performed in the liquid mineral medium using ethanol and waste sunflower oil as substrates. Tryptophan was added to the medium as a 1 % solution in an amount of 200 or 300 mg/l at the beginning of the cultivation process or at the end of the exponential growth phase. Phytohormones were isolated by triple extraction with organic solvents from the culture broth supernatant after surfactant extraction. Preliminary purification and concentration of phytohormones was performed by thin layer chromatography. Qualitative and quantitative determination of auxins was performed using high performance liquid chromatography. Results. It was found that regardless of the concentration and time of tryptophan introduction to the culture medium of R. erythropolis IMV Ac-5017 with both substrates, a significant increase (by two to three orders of magnitude) was observed in the amount of synthesized auxins compared to tryptophan-free medium. The highest concentration of auxins (5552–5634 μg/l) was achieved by adding 300 mg/l of tryptophan into the culture medium of R. erythropolis IMV Ac-5017 with ethanol, while without the precursor their amount was only 143 μg/l. In contrast to the cultivation of the strain on culture medium with ethanol, where the synthesis of auxins did not depend on the time of tryptophan introduction, R. erythropolis IMV Ac-5017 formed the maximum amount of auxins when 300 mg/l tryptophan was added to the culture medium with waste oil at the end of the exponential growth phase (2398 μg/l compared to 9.8 μg/l on the medium without tryptophan). As auxin compounds were identified: indole-3-acetic acid, indole-3-carboxylic acid and indole-3-butyric acid. However, the highest amount of indole-3-acetic acid was synthesized, the precursor of which is tryptophan. The synthesis of this auxin (the most common plant auxin) in the presence of 300 mg/l of tryptophan increased more than 40 times on ethanol medium and more than 700 times on medium with waste oil. Induction of auxin synthesis by strain R. erythropolis IMV Ac-5017 correlated with the activity of tryptophan transaminase: when cultured on ethanol without tryptophan, it was 138 nmol•min-1•mg-1 of protein, while cultured in the presence of precursor it was increased by 5.2 times (up to 714 nmol•min-1•mg-1 of protein). The obtained results suggest that indole-3-acetic acid biosynthesis by the strain IMV Ac-5017 occurs due to the formation of indole-3-pyruvate. Conclusions. Thus, it was established the possibility of increasing by two or three orders the amount of synthesized auxins in the case of low concentrations of tryptophan introducing to the culture medium of R. erythropolis IMV Ac-5017 not only with ethanol but also with industrial waste (waste oil). The obtained results can be considered as promising for use of exometabolites of R. erythropolis IMV Ac-5017 with growth-stimulating properties in crop production.