Investigação sobre associações entre fonte de nitrogênio e produção de compostos bioativos por Streptomyces clavuligerus em cultivos submersos
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Data
2017-02-17
Autores
Souza, Milena Alves de [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Streptomyces clavuligerus produz vários compostos bioativos importantes, como os beta-lactâmicos ácido clavulânico (AC), um inibidor de beta-lactamases utilizado em combinação com antibióticos beta-lactâmicos, e mais vinte clavamas (várias com atividade antifúngica), o antibiótico cefamicina (CefC), utilizado comercialmente para a produção de antibióticos semi-sintéticos, além de bioativos não beta-lactâmicos como tunicamicina e holomicina. Estudos têm mostrado que tanto a natureza como a concentração de fontes de nitrogênio influenciam a produção de bioativos em S. clavuligerus, pois muitos precursores destes biocompostos derivam de intermediários nitrogenados do metabolismo primário. Um aminoácido importante para o cultivo de S. clavuligerus é a lisina, que atua no metabolismo primário via cadaverina aminotransferase, e no metabolismo secundário via 1-piperideina-6-carboxilato, para a produção do raro aminoácido ácido alfa-aminoadípico, um precursor de CefC e de todos os demais antibióticos beta-lactâmicos. Outra fonte de nitrogênio de destaque é o glutamato de sódio, que apresenta efeitos positivos na produção de AC por fornecer átomos de carbono para a molécula deste biocomposto. Estudos têm relatado a produção de bioativos utilizando lisina ou glutamato de sódio nos meios de cultivo, mas não há relatos na literatura sobre a influência conjunta destes aminoácidos. Este trabalho trata da investigação de relações entre, a utilização conjunta de lisina e glutamato de sódio e a produção dos compostos beta-lactâmicos AC e CefC por S. clavurigerus em cultivos submersos empregando-se meios quimicamente definidos. Inicialmente foram realizadas fermentações em batelada em mesa incubadora rotativa (28°C, 260 rpm e um pH inicial de 6,5). O conjunto de resultados em frascos agitados utilizando-se uma ampla faixa de combinações de concentração dos aminoácidos mostraram que não é possível otimizar concomitantemente a produção de AC e CefC. Porém, a combinação de 43,7 mmol/L de lisina e 18 mmol/L de glutamato resultou em produções finais relevantes dos bioativos, 146,6 mg/L de AC e 140,51 mg/L de CefC. Nesta condição, foram obtidos biomassa de 8,44 g/L, pH final igual a 6,8 e concentração de amônio de 487 mg/L. Esta combinação foi, então, utilizada para validação em biorreator de bancada, com controles de pH em 6,5, 1 vvm de vazão de ar, (fluxometro de massa) e de oxigênio dissolvido em 50% da saturação por meio de variação automática da agitação. Nestas condições obtiveram-se concentrações finais de 248,28 mg/L de AC, 125,54 mg/L de CefC, com biomassa máxima e concentração final de amônio de 4,96 g/L e 457 mg/L, respectivamente. Desta forma, as produções especificas dos bioativos ao final do processo em biorreator foram 200% maior de AC e 40% de CefC, quando comparadas com os resultados obtidos em frascos agitados. Foi observado também que a molécula de AC é muito mais instável a pHs alcalinos e a maiores concentrações de amônio do que a molécula de CefC. O aumento do pH mostrou-se diretamente associado ao consumo de glutamato enquanto o acúmulo de amônio manteve uma relação direta com o consumo das fontes de nitrogênio.
Streptomyces clavuligerus produces several important bioactive compounds, such as beta-lactams, clavulanic acid (AC), a beta-lactamase inibitor which is used in combination with beta-lactam antibiotics, and twenty more clavams with antifungal activity, the antibiotic cephamycin (CefC), of great commercial interest for the production of semisynthetic antibiotic, and the non-lactamic bioactives tunicamycin and holomicyn. Studies have shown that both the nature and the concentration of nitrogen sources influence the production of bioactives metabolites in S. clavuligerus, because many precursors of these biocompounds derive from nitrogenous intermediates of the primary metabolism. Lysine is an important amino acid in S. clavuligerus, acting on the primary metabolism via cadaverine aminotransferase, and on the secondary metabolismo via 1-piperidine -6- carboxylate, for the production of the rare amino acid alpha-aminoadipic acid, a precursor of CefC as also of all other beta-lactams antibiotics. Another important source of nitrogen is sodium glutamate, which in S. clavuligerus culture media has positive effects on the production of AC. Studies have reported the production of bioactive compounds employing lysine or sodium glutamate in culture media, but there aren’t reports in the literature on the joint infuence of these amino acids. In this sense, this project proposes to investigate relations between combinations of lysine and sodium glutamate and and the production of beta-lactam compounds AC and CefC by S. clavuligerus in submerged cultures employinh chemically defined media. Initially, batch fermentations were carried out in a rotating incubator at 28°C and 260 rpm, in order to select the best combination of lysine and glutamate concentrations to obtain higher final bioactives compounds yields. This combination was then validated in bioreactor. The set of results in shaken flasks in a wide range of amino acid concentration combinations showed that isin’t possible to concomitantly optimize the production of AC and CefC. However the combination of 47,3 mmol/L lysine and 18 mmol/L sodium glutamate resulted in relevant final productions of the biocompounds, 146, 6 mg/L of AC and 141, 51 mg/L of CefC. In this condition, biomass of 8,44 g/L, final pH of 6,8 and ammonium concentration of 487 mg/L were obtained. This combination of lysine and sodium glutamate was then reproduced in benchtop bioreactor, with pH controlled at 6,5. The final concentration of 248,28 mg/L of AC and 125,54 mg/L of CefC with maximum biomass and final concentration of ca. 5,0 g/L and 457 mg/L, respectively, were obtained. That is, the specific productions of the bioactives at the end of the process in biorreactor were 200% higher for AC and 40% for CefC, when compared with the results obtained in shaken flasks. It has also been observed that the AC melecule is much more unstable at alcaline pH values and at higher ammonium concentrations than the CefC molecule.The increase in pH was directly associated with the consumption of glutamate, while the accumulation of ammonium has a straight relation relation with the consumption of nitrogen sources.
Streptomyces clavuligerus produces several important bioactive compounds, such as beta-lactams, clavulanic acid (AC), a beta-lactamase inibitor which is used in combination with beta-lactam antibiotics, and twenty more clavams with antifungal activity, the antibiotic cephamycin (CefC), of great commercial interest for the production of semisynthetic antibiotic, and the non-lactamic bioactives tunicamycin and holomicyn. Studies have shown that both the nature and the concentration of nitrogen sources influence the production of bioactives metabolites in S. clavuligerus, because many precursors of these biocompounds derive from nitrogenous intermediates of the primary metabolism. Lysine is an important amino acid in S. clavuligerus, acting on the primary metabolism via cadaverine aminotransferase, and on the secondary metabolismo via 1-piperidine -6- carboxylate, for the production of the rare amino acid alpha-aminoadipic acid, a precursor of CefC as also of all other beta-lactams antibiotics. Another important source of nitrogen is sodium glutamate, which in S. clavuligerus culture media has positive effects on the production of AC. Studies have reported the production of bioactive compounds employing lysine or sodium glutamate in culture media, but there aren’t reports in the literature on the joint infuence of these amino acids. In this sense, this project proposes to investigate relations between combinations of lysine and sodium glutamate and and the production of beta-lactam compounds AC and CefC by S. clavuligerus in submerged cultures employinh chemically defined media. Initially, batch fermentations were carried out in a rotating incubator at 28°C and 260 rpm, in order to select the best combination of lysine and glutamate concentrations to obtain higher final bioactives compounds yields. This combination was then validated in bioreactor. The set of results in shaken flasks in a wide range of amino acid concentration combinations showed that isin’t possible to concomitantly optimize the production of AC and CefC. However the combination of 47,3 mmol/L lysine and 18 mmol/L sodium glutamate resulted in relevant final productions of the biocompounds, 146, 6 mg/L of AC and 141, 51 mg/L of CefC. In this condition, biomass of 8,44 g/L, final pH of 6,8 and ammonium concentration of 487 mg/L were obtained. This combination of lysine and sodium glutamate was then reproduced in benchtop bioreactor, with pH controlled at 6,5. The final concentration of 248,28 mg/L of AC and 125,54 mg/L of CefC with maximum biomass and final concentration of ca. 5,0 g/L and 457 mg/L, respectively, were obtained. That is, the specific productions of the bioactives at the end of the process in biorreactor were 200% higher for AC and 40% for CefC, when compared with the results obtained in shaken flasks. It has also been observed that the AC melecule is much more unstable at alcaline pH values and at higher ammonium concentrations than the CefC molecule.The increase in pH was directly associated with the consumption of glutamate, while the accumulation of ammonium has a straight relation relation with the consumption of nitrogen sources.
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Palavras-chave
Streptomyces clavuligerus, Lisina, Glutamato de sódio, Ácido clavulânico, Cefamicina C, Lysine, Sodium glutamate, Acid clavulanic, Cephamycin C