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Документ 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, TetianaThe 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.Документ Synthesis of gibberellins by surfactant producers Nocardia vaccinii IMV В-7405, Acinetobacter calcoaceticus IMV В-7241 and Rhodococcus erythropolis IMV Ас-5017(2020) Leonova, Natalia; Pirog, Tatiana; Piatetska, Daria; Shevchuk, Tetiana; Kharkhota, Maxym; Iutynska, GalynaIn this study, we established that the producers of surfactants Nocardia vaccinii IMV В-7405, Acinetobacter calcoaceticus IMV B-7241 and Rhodococcus erythropolis IMV Аc-5017 were able to synthesize phytohormones of gibberellic nature during cultivation on the traditional substrates (ethanol, hexadecane) and on the industrial waste sunflower oil and biodiesel production waste). The results of specific biotesting showed that the treatment of cucumber seedlings with phytohormonal extracts of all studied strains at 1:500 and 1:600 dilutions stimulated the extension of the cucumber hypocotyls as compared to the water control in values close to the treatment with gibberellic acid. The results of high-performance liquid chromatography showed N. vaccinii IMV B-7405, A. calcoaceticus IMV B-7241 and R. erythropolis IMV Аc-5017 produce highly active forms of gibberellins GA3 and GA4. The level of its synthesis was nearly the same (6.0-10.0 μg∙L-1) under cultivation of strains on every substrate. The exception was strain N. vaccinii IMV В-7405 which synthesized almost 47.0 μg∙L-1 GA3 and GA4 while is growing on the waste oil from meat frying. The obtained results are the groundwork for the development of an economically profitable technology for the recycling of toxic wastes using N. vaccinii IMV B-7405, A.calcoaceticus IMV B-7241 and R. erythropolis IMV Аc-5017. Such technology will allow us to develop complex microbial preparations with various biological properties in a single process. У цьому дослідженні встановлено, що продуценти ПАР Nocardia vaccinii ІМВ В-7405, Acinetobacter calcoaceticus ІМВ В-7241 та Rhodococcus erythropolis ІМВ Ас-5017 здатні синтезувати фітогормони гіберелової природи при культивуванні на традиційних субстратах (етанол, гексадекан) а також на промислових відходах соняшникової олії та відходах виробництва біодизеля). Результати специфічного біотестування показали, що обробка проростків огірка фітогормональними екстрактами всіх досліджуваних штамів у розведенні 1:500 та 1:600 стимулювала подовження гіпокотилів огірка порівняно з контролем у воді в значеннях, близьких до обробки гібереліновою кислотою. . Результати високоефективної рідинної хроматографії показали, що N. vaccinii IMV B-7405, A. calcoaceticus IMV B-7241 і R. erythropolis IMV Аc-5017 продукують високоактивні форми гіберелінів GA3 і GA4. Рівень його синтезу був приблизно однаковим (6,0-10,0 мкг∙л-1) за культивування штамів на кожному субстраті. Виняток становив штам N. vaccinii ІМВ В-7405, який синтезував майже 47,0 мкг∙л-1 ГА3 та ГА4 під час вирощування на відпрацьованому маслі від смаження м’яса. Отримані результати є основою для розробки економічно вигідної технології переробки токсичних відходів з використанням N. vaccinii ІМВ В-7405, A.calcoaceticus ІМВ В-7241 та R. erythropolis ІМВ Ас-5017. Така технологія дозволить розробляти комплексні мікробні препарати з різними біологічними властивостями в одному процесі.