Caracterização de lacases produzidas por fungos basidiomicetos de origem marinha e terrestre e aplicação biotecnológica na descoloração de corante têxtil
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2019-02-28
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Universidade Estadual Paulista (Unesp)
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As lacases têm sido alvo de investigação devido à capacidade de oxidar compostos aromáticos fenólicos e não fenólicos com a concomitante redução do oxigênio à agua. Embora possam ser sintetizadas por diversos organismos, sua produção se destaca em fungos de podridão branca. As lacases tem sido amplamente aplicada no campo industrial e ambiental. A fim de comparar o potencial catalítico das lacases dos fungos de ambiente marinho e terrestre, no presente estudo as lacases produzidas pelos basidiomicetos de origem marinha (Peniophora sp. CBMAI 1063 e Marasmiellus sp. CBMAI 1062) e terrestre (Peniophora cinerea CCIBt 2541 e Marasmiellus colocasiae CCIBt 3388) foram parcialmente purificadas (ultrafiltração e cromatografia de troca iônica) e caracterizadas bioquimicamente. Para a quantificação das laccases foram utilizados os substratos enzimáticos 2,2’-azino-bis (3- etilbenzotiazolina-6-sulfónico) (ABTS) e siringaldazina (SYG). Os fungos analisados apresentaram produção de lacases no meio utilizado, o qual foi previamente otimizado para a produção de lacases para o Peniophora sp. CBMAI 1063. Nestas condições de cultivo, as enzimas ligninoliticas Manganês Peroxidase (MnP) e Lignina Peroxidase (LiP) não foram produzidas. Com relação ao processo de purificação, a cada passo houve perda no rendimento, em proporções diferentes para o conjunto enzimático de cada fungo, com melhor recuperação para as lacases produzidas pelos fungos do gênero Marasmiellus. O zimograma revelou que Peniophora sp. CBMAI 1063 produz duas isoenzimas de lacases, P. cinerea CCIBt 2541 cinco, Marasmiellus sp. CBMAI 1062 duas e M. colocasiae CCIBt 3388 uma. O conjunto enzimático foi caracterizado de modo aparente. O pH ótimo (4,0 para ABTS e 5,5 para SYG) e temperatura ótima (55 °C) foram iguais para os dois fungos do gênero Peniophora. Para os fungos do gênero Marasmiellus o pH ótimo foi o mesmo (5,0 para ABTS e 6,5 para SYG) porém a temperatura ótima foi diferente (55 °C para Marasmiellus sp. CBMAI 1062 e 50 °C para M. colocasiae CCIBt 3388). As lacases de todas as espécies estudadas não apresentarem atividade na presença de EDTA e L-cisteína, evidenciando a importância do Cu2+ e do aminoácido cisteína no sítio ativo. Diferentes íons metálicos ativam as atividades das lacases. De modo geral, a caracterização do conjunto enzimático do P. cinerea CCIBt 2541 apresentou melhores resultados que o do Peniophora sp. CBMAI 1063. No conjunto enzimatico M. colocasiase CCIBt 3388 íons Mn2+ aumentou a atividade (117%), entretanto Marasmiellus sp. CBMAI 1062 apresentou estabilidade maior frente a todos os íons testados, apresentando aumento da atividade na presença de Al3+ (126%). Quanto à salinidade, o I50 para NaCl foi mais eficiente para as lacases do P. cinerea CCIBt 2541, enquanto que Marasmiellus sp. CBMAI 1062 apresentou maior resistência a 1000 mM de NaCl. De acordo com os valores de KM e Vmax há uma maior afinidade das lacases dos fungos de origem terrestres pelos substratos ABTS e SYG. As lacases parcialmente purificadas apresentaram as seguintes porcentagens de descoloração do corante têxtil marinho reativo (MR) (40 mg/L) após sete dias de incubação: P. cinerea CCIBt 2541 67%, Peniophora sp. CBMAI 1063 61% e M. colocasiae CCIBt 3388 27%. A descoloração do MR pelas lacases dos fungos do gênero Peniophora foi similar à obtida para a lacase industrial de Trametes versicolor (66%). Devido à quantidade insuficiente de lacases do Marasmiellus sp. CBMAI 1062 o teste de descoloração não foi realizado. Testes de toxicidade mostraram que as lacases de P. cinerea CCIBt 2541 deixa praticamente inalterada a toxicidade no tratamento. Entretanto o conjunto de lacases do Peniophora sp. CBMAI 1063 e M. colocasiae CCIBt 3388 apresentam um leve aumento de toxicidade após o período de reação. A lacase pura de T. versicolor não alterou a toxicidade inicial. Os resultados do presente estudo ampliam o conhecimento sobre as lacases produzidas por fungos de origem marinha e abrem novas perspectivas de estudos na área da biotecnologia ambiental.
Laccases have been under investigation due to their capacity to oxidize phenolic and non-phenolic aromatic compounds with concomitant reducing of oxygen to water. Although laccases can be synthetized by several organisms; their production is notable in white rot fungi. Laccases have been wide applied in industrial and environmental fields. Aiming to compare the catalytic potential of laccases in fungi from marine and terrestrial environments, in the present work laccases produced by marine basidiomycetes (Peniophora sp. CBMAI 1063 and Marasmiellus sp. CBMAI 1062) and the terrestrial (Peniophora cinerea CCIBt 2541 and Marasmiellus colocasiae CCIBt 3388) were partially purified (ultrafiltration and ionic chromatography) and biochemically characterized. For the laccases quantification were used 2,2’-azino-bis (3- ethilbenzotiazolina-6-sulphnic) (ABTS) and syringaldazine (SYG) as substrates. All studied fungi showed satisfactory laccases productions in the utilized medium, which was previously optimized to laccases production by Peniophora sp. CBMAI 1063. In these culture conditions ligninolytic enzymes Manganese Peroxidase (MnP) and Lignin Peroxidase (LiP) were not produced. Regarding the purification process, there was yield loss in each step, in different proportions to the enzymatic pool for each fungi, with better yielded the laccases produced by the fungi from Marasmiellus genus. Zymogram revealed that Peniophora sp. CBMAI 1063 has two laccase isoforms, P. cinerea CCIBt 2541 has five, Marasmiellus sp. CBMAI 1062 has two and M. colocasiae CCIBt 3388 has one. The enzymatic pools were apparently characterized. The optimum pH (4.0 to ABTS and 5.5 to SYG) and optimum temperature (55 °C) were the same to both Peniophora fungi. The fungi from the genus Marasmiellus had the same optimum pH (5.0 to ABTS and 6.5 to SYG), however, the optimum temperature was distinct, 55 °C to the Marasmiellus sp. CBMAI 1062 and 50 °C to the M. colocasiae CCIBt 3388). EDTA and L-cysteine completely inhibit all enzymatic pools, showing the importance of Cu2+ and the cysteine aminoacid in the active site of laccases. Different metallic ions acted empowering the laccase activities. The enzymatic pool obtained with the fungus P. cinerea CCIBt 2541 showed better results in comparison with Peniophora sp. CBMAI 1063. In the enzymatic pool of M. colocasiase CCIBt 3388 Mn2+ ions enhanced laccase activity (117%), but the pool of Marasmiellus sp. CBMAI 1062 showed better stability with all tested ions, enhancing its activity in the presence of Zn2+ (102%) and Al3+ (126%). Regarding salinity, the I50 of NaCl was most efficient to the laccases of P. cinerea CCIBt 2541, while laccases from Marasmiellus sp. CBMAI 1062 were more resistant to 1000 mM of NaCl. According to the KM e Vmax values, there is a better affinity with laccases from terrestrial fungi to the substrates ABTS and SYG. The partially purified laccases exhibited the following percentage of the Marine Reactive (MR) (40 mg/L) textile dye decolorization after seven days of incubation: P. cinerea CCIBt 2541 67%, Peniophora sp. CBMAI 1063 61% and M. colocasiae CCIBt 3388 27%. Decolorization of MR by Peniophora fungal laccases was similar to the pure industrial laccase from Trametes versicolor (66%). Due to the little amount of laccases of Marasmiellus sp. CBMAI 1062 discoloration test was not carried out. Toxicity tests revealed that laccases from P. cinerea CCIBt 2541 did not change the dye toxicity during the treatment. However, the pool of Peniophora sp. CBMAI 1063 and M. colocasiae CCIBt 3388 slightly increased the toxicity after the treatment. T. versicolor pure laccase did not change the initial toxicity. Results from the present work increase the knowledge about the laccases produced by fungi from marine origin and open new perspectives of studies in the area of environmental biotechnology.
Laccases have been under investigation due to their capacity to oxidize phenolic and non-phenolic aromatic compounds with concomitant reducing of oxygen to water. Although laccases can be synthetized by several organisms; their production is notable in white rot fungi. Laccases have been wide applied in industrial and environmental fields. Aiming to compare the catalytic potential of laccases in fungi from marine and terrestrial environments, in the present work laccases produced by marine basidiomycetes (Peniophora sp. CBMAI 1063 and Marasmiellus sp. CBMAI 1062) and the terrestrial (Peniophora cinerea CCIBt 2541 and Marasmiellus colocasiae CCIBt 3388) were partially purified (ultrafiltration and ionic chromatography) and biochemically characterized. For the laccases quantification were used 2,2’-azino-bis (3- ethilbenzotiazolina-6-sulphnic) (ABTS) and syringaldazine (SYG) as substrates. All studied fungi showed satisfactory laccases productions in the utilized medium, which was previously optimized to laccases production by Peniophora sp. CBMAI 1063. In these culture conditions ligninolytic enzymes Manganese Peroxidase (MnP) and Lignin Peroxidase (LiP) were not produced. Regarding the purification process, there was yield loss in each step, in different proportions to the enzymatic pool for each fungi, with better yielded the laccases produced by the fungi from Marasmiellus genus. Zymogram revealed that Peniophora sp. CBMAI 1063 has two laccase isoforms, P. cinerea CCIBt 2541 has five, Marasmiellus sp. CBMAI 1062 has two and M. colocasiae CCIBt 3388 has one. The enzymatic pools were apparently characterized. The optimum pH (4.0 to ABTS and 5.5 to SYG) and optimum temperature (55 °C) were the same to both Peniophora fungi. The fungi from the genus Marasmiellus had the same optimum pH (5.0 to ABTS and 6.5 to SYG), however, the optimum temperature was distinct, 55 °C to the Marasmiellus sp. CBMAI 1062 and 50 °C to the M. colocasiae CCIBt 3388). EDTA and L-cysteine completely inhibit all enzymatic pools, showing the importance of Cu2+ and the cysteine aminoacid in the active site of laccases. Different metallic ions acted empowering the laccase activities. The enzymatic pool obtained with the fungus P. cinerea CCIBt 2541 showed better results in comparison with Peniophora sp. CBMAI 1063. In the enzymatic pool of M. colocasiase CCIBt 3388 Mn2+ ions enhanced laccase activity (117%), but the pool of Marasmiellus sp. CBMAI 1062 showed better stability with all tested ions, enhancing its activity in the presence of Zn2+ (102%) and Al3+ (126%). Regarding salinity, the I50 of NaCl was most efficient to the laccases of P. cinerea CCIBt 2541, while laccases from Marasmiellus sp. CBMAI 1062 were more resistant to 1000 mM of NaCl. According to the KM e Vmax values, there is a better affinity with laccases from terrestrial fungi to the substrates ABTS and SYG. The partially purified laccases exhibited the following percentage of the Marine Reactive (MR) (40 mg/L) textile dye decolorization after seven days of incubation: P. cinerea CCIBt 2541 67%, Peniophora sp. CBMAI 1063 61% and M. colocasiae CCIBt 3388 27%. Decolorization of MR by Peniophora fungal laccases was similar to the pure industrial laccase from Trametes versicolor (66%). Due to the little amount of laccases of Marasmiellus sp. CBMAI 1062 discoloration test was not carried out. Toxicity tests revealed that laccases from P. cinerea CCIBt 2541 did not change the dye toxicity during the treatment. However, the pool of Peniophora sp. CBMAI 1063 and M. colocasiae CCIBt 3388 slightly increased the toxicity after the treatment. T. versicolor pure laccase did not change the initial toxicity. Results from the present work increase the knowledge about the laccases produced by fungi from marine origin and open new perspectives of studies in the area of environmental biotechnology.
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Palavras-chave
Lacase, Basidiomicetos, Purificação enzimática, Biotecnologia ambiental, Laccase, Basidiomycetes, Enzymatic purification, Environmental biotechnology