Expressão, purificação e caracterização de lipases extracelulares
Data
2023-07-18
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Universidade Estadual Paulista (Unesp)
Resumo
Lipases verdadeiras são caracterizadas pela capacidade de catalisar a liberação de ácidos graxos livres de triacilgliceróis de cadeia longa na interface óleo-água. As lipases bacterianas são as que apresentam maior versatilidade, são mais estáveis e reativas em meio orgânico. Quanto às suas aplicações, destacam-se o uso na produção de biodiesel e na biorremediação de águas residuais. É estimada a existência de 5000 enzimas lipolíticas bacterianas, entretanto, apenas 10% dessas foram estudadas experimentalmente, por isso a importância de buscar por lipases com novas especificidades. Diante disso, o principal objetivo deste trabalho foi identificar e caracterizar novas lipases verdadeiras. Para tanto, 10 isolados bacterianos foram avaliados quanto a atividade lipolítica e, em seguida, quanto a atividade lipolítica no sobrenadante. Aqueles com resultado positivo, tiveram suas lipases parcialmente purificadas por precipitação com sulfato de amônio e posteriormente foi realizado um zimograma para confirmar atividade lipolítica das enzimas. Os microrganismos, por sua vez, tiveram o DNA extraído para o sequenciamento do gene 16S rRNA, seguido de análises filogenéticas. Uma das lipases obtidas foi submetida a um ensaio de cromatografia gasosa para observação do padrão de degradação dos ácidos graxos presentes no azeite de oliva, e a mesma foi caracterizada bioquimicamente. Quatro dos microrganismos avaliados apresentaram halo de degradação em meio de cultura LB suplementado com azeite de oliva e três deles apresentaram evidência de produção de enzimas extracelulares, através da detecção de proteína por eletroforese SDS-Page no sobrenadante filtrado de cultura liquida induzida com azeite. Após a purificação, as lipases obtidas tiveram a atividade lipolítica comprovada através do zimograma. Os microrganismos foram classificados como pertencentes ao gênero Burkholderia. O isolado denominado D15, escolhido para a continuação dos estudos por apresentar o maior halo lipolítico, produz uma lipase com atividade específica de 0.297 U/mg para o substrato C14 em condições iniciais de análise (pH 8, 30 °C), esta enzima também apresentou uma degradação expressiva dos ácidos graxos presentes no azeite, seu pH e temperatura ótimos foram 4,5 e entre 40 e 60 ºC (substrato C12), respectivamente. As características apresentadas pela lipase produzida pelo isolado D15 são muito interessantes do ponto de vista biotecnológico, com potencial de aplicação na indústria alimentícia, por exemplo, devido a preferência por pH ácido e estabilidade em uma ampla faixa de temperatura; e biorremediação, já que apresentou alta capacidade de degradação de ácidos graxos de cadeia longa. Além disso, outras duas lipases bacterianas extracelulares foram obtidas, podendo serem exploradas em estudos posteriores.
True lipases are characterized by their ability to catalyze the release of long-chain free fatty acids from triglycerides at the oil-water interface. Bacterial lipases are the most versatile, stable, and reactive in organic media. Their applications include the production of biodiesel and the bioremediation of wastewater. It is estimated that there are 5,000 bacterial lipolytic enzymes, but only 10% of these have been experimentally studied, highlighting the importance of seeking lipases with new specificities. Therefore, the main objective of this work was to identify new true lipases. To this end, 10 bacterial isolates were evaluated for lipolytic activity and then for lipolytic activity in the supernatant. Those with positive results had their lipases purified by ammonium sulfate precipitation, followed by zymogram analysis to confirm the lipolytic activity of the purified enzymes. The microorganisms, in turn, had their DNA extracted for 16S rRNA gene sequencing, followed by phylogenetic analysis. One of the obtained lipases was subjected to gas chromatography to observe the degradation pattern of fatty acids present in olive oil, and it was biochemically characterized. Four of the evaluated microorganisms showed degradation halo in LB culture medium supplemented with olive oil, and three of them showed evidence of extracellular enzyme production through protein detection by SDS-Page electrophoresis in the filtered supernatant of oil-induced liquid culture. After purification, the obtained lipases had their lipolytic activity confirmed by zymogram analysis. The microorganisms were classified as belonging to the Burkholderia genus. The isolate named D15, chosen for further studies due to its largest lipolytic halo, produces a lipase with a specific activity of 0.297 U/mg for the C14 substrate under initial analysis conditions (pH 8, 30 °C). This enzyme also showed significant degradation of fatty acids present in olive oil, with optimal pH and temperature of 4.5 and between 40 and 60 °C (substrate C12), respectively. The characteristics exhibited by the lipase produced by the D15 isolate are highly interesting from a biotechnological standpoint, with potential applications in the food industry, for example, due to its preference for acidic pH and stability over a wide temperature range, and in bioremediation, as it demonstrated a high capacity for degrading long-chain fatty acids. Furthermore, two other extracellular bacterial lipases were obtained, which could be explored in further studies.
True lipases are characterized by their ability to catalyze the release of long-chain free fatty acids from triglycerides at the oil-water interface. Bacterial lipases are the most versatile, stable, and reactive in organic media. Their applications include the production of biodiesel and the bioremediation of wastewater. It is estimated that there are 5,000 bacterial lipolytic enzymes, but only 10% of these have been experimentally studied, highlighting the importance of seeking lipases with new specificities. Therefore, the main objective of this work was to identify new true lipases. To this end, 10 bacterial isolates were evaluated for lipolytic activity and then for lipolytic activity in the supernatant. Those with positive results had their lipases purified by ammonium sulfate precipitation, followed by zymogram analysis to confirm the lipolytic activity of the purified enzymes. The microorganisms, in turn, had their DNA extracted for 16S rRNA gene sequencing, followed by phylogenetic analysis. One of the obtained lipases was subjected to gas chromatography to observe the degradation pattern of fatty acids present in olive oil, and it was biochemically characterized. Four of the evaluated microorganisms showed degradation halo in LB culture medium supplemented with olive oil, and three of them showed evidence of extracellular enzyme production through protein detection by SDS-Page electrophoresis in the filtered supernatant of oil-induced liquid culture. After purification, the obtained lipases had their lipolytic activity confirmed by zymogram analysis. The microorganisms were classified as belonging to the Burkholderia genus. The isolate named D15, chosen for further studies due to its largest lipolytic halo, produces a lipase with a specific activity of 0.297 U/mg for the C14 substrate under initial analysis conditions (pH 8, 30 °C). This enzyme also showed significant degradation of fatty acids present in olive oil, with optimal pH and temperature of 4.5 and between 40 and 60 °C (substrate C12), respectively. The characteristics exhibited by the lipase produced by the D15 isolate are highly interesting from a biotechnological standpoint, with potential applications in the food industry, for example, due to its preference for acidic pH and stability over a wide temperature range, and in bioremediation, as it demonstrated a high capacity for degrading long-chain fatty acids. Furthermore, two other extracellular bacterial lipases were obtained, which could be explored in further studies.
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Bioquímica, Análise enzimática, Lipase