Co-purificação e caracterização das fosfatase e fitase alcalinas de Rhizopus microsporus var. microsporus produzidas em fermentação submersa
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Data
2017-08-03
Autores
Ornela, Pedro Henrique de Oliveira [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A investigação biotecnológica, acompanhada da aplicação das enzimas, tem sido realizada em microrganismos para a produção de enzimas para fins industriais. Entre estas enzimas, as fosfatases, responsáveis por hidrolisar ésteres e anidridos de ácido fosfórico, e as fitases microbianas, que catalisam a hidrólise do fitato (mio-inositol hexaquisfosfato) em mio-inositol e fosfato inorgânico, têm sido amplamente utilizadas em diferentes setores como, por exemplo, em experimentos de biologia molecular e na alimentação animal. De acordo com o pH ótimo de reação, as fosfatases são divididas em alcalinas (EC 3.1.3.1) e ácidas (EC 3.1.3.2). As fitases são enzimas que também pertencem à classe das fosfatases, hidrolisando, no entanto, de forma específica, o ácido fítico. Em recentes trabalhos, o fungo filamentoso Rhizopus microsporus var. microsporus apresentou potencialidade na produção de fosfatases e fitases. Diante disto, este estudo visou a produção, a purificação e caracterização da fosfatase e da fitase alcalina produzidas por R. microsporus var. microsporus. No processo de otimização em Fermentação Submersa (FSbm), a maior produção enzimática foi em meio Khanna com 0,4 mM de KH2PO3 e adicionado de 0,5% de farinha de centeio por 76 h, 32ºC, pH 6,3, a 100 rpm. Em colunas cromatográficas, a fosfatase alcalina foi purificada 10 vezes e com recuperação de 13%, e a fitase alcalina foi purificada 86 vezes com recuperação de 167%. A massa molecular nativa da fosfatase e da fitase alcalinas produzidas por R. microsporus var. microsporus foi de 122,7 e 55,5 kDa, respectivamente. A fosfatase alcalina purificada apresentou temperatura ótima de atividade de 45ºC e pH ótimo de 8,5, mantendo sua atividade catalítica acima de 60% nas temperaturas de 40 e 45ºC por até 210 min. Manteve sua atividade acima de 50% em pH alcalinos por até 24 horas. A fitase alcalina purificada apresentou temperatura e pH ótimos de atividade de 65ºC e 9,5, respectivamente. A fitase alcalina manteve sua atividade enzimática acima de 60% nas temperaturas de 65, 70 e 80ºC por 270 min. Após incubação da fitase em valores de pH alcalinos por 3 horas, houve aumento da atividade enzimática em até 50%. A fosfatase teve sua atividade aumentada na presença de 0,4 mM de FeCl3, e melhor hidrolisou o substrato p-nitrofenilfosfato, seguido pelo ATP. O Km para a fosfatase alcalina utilizando o substrato p-nitrofenilfosfato na ausência e na presença de FeCl3 foi 0,449 mM e 0,382 mM, respectivamente. Para a fitase alcalina o Km foi 0,413 mM utilizando fitato de sódio como substrato. Deste modo, a fosfatase e a fitase alcalinas produzidas por R. microsporus var. microsporus foram co-purificadas e bioquimicamente caracterizadas, evidenciando-se importantes características de ambas as enzimas possibilitando, assim, futuras aplicações industriais.
Biotechnological research, accompanied by the application of enzymes, has been carried out in microorganisms for production of enzymes for industrial purposes. Among these enzymes, microbial phosphatases, responsible for hydrolyzing phosphoric acid esters and anhydrides, and phytases, which catalyzes the hydrolysis of phytate (myo-inositol hexaquisphosphate) in myo-inositol and inorganic phosphate, have been widely used in different sectors as, for example, in molecular biology experiments and in animal feed. According to the optimum reaction pH, phosphatases are divided into alkaline (EC 3.1.3.1) and acidic (EC 3.1.3.2). Phytases are enzymes that also belong to the class of phosphatases, however, hydrolyzing phytic acid. In recent works, the filamentous fungus Rhizopus microsporus var. microsporus presented potential for production of phosphatases and phytases. In view of this, this study aimed at the production, purification and characterization of phosphatase and alkaline phytase produced by R. microsporus var. microsporus. In the optimization of Submerged Fermentation (FSbm), the highest enzymatic production was in Khanna medium with 0.4 mM KH2PO3 and added with 0.5% rye flour for 76 h, 32ºC, pH 6.3, at 100 rpm. In chromatographic columns, alkaline phosphatase was purified 10 folds and recovered at 13%, and alkaline phytase was purified 86 folds with recovery of 167%. The native molecular mass of alkaline phosphatase and phytase produced by R. microsporus var. microsporus was 122.7 and 55.5 kDa, respectively. The purified alkaline phosphatase had an optimum temperature of 45ºC and optimum pH of 8.5, maintaining its catalytic activity above 60% at temperatures of 40 and 45ºC for up to 210 minutes. It maintained its activity above 50% in alkaline pH for up to 24 h. Purified alkaline phytase presented optimum temperature and pH of activity of 65°C and 9.5, respectively. Alkaline phytase maintained its enzymatic activity above 60% at temperatures of 65, 70 and 80ºC for 270 min. After phytase incubation in alkaline pH for 3 hours, enzymatic activity increased by up to 50%. Phosphatase had increased activity in presence of 0.4 mM FeCl3, and better hydrolyzed the substrate p-nitrophenylphosphate, followed by ATP. The Km for alkaline phosphatase using the substrate p-nitrophenylphosphate in the absence and presence of FeCl3 was 0.449 mM and 0.382 mM, respectively. For alkaline Phytase, the Km was 0.413 mM using sodium phytate as substrate. Thus, alkaline phosphatase and phytase produced by R. microsporus var. microsporus were co-purified and biochemically characterized, evidencing important characteristics of both enzymes, thereby, possibilities of future industrial applications.
Biotechnological research, accompanied by the application of enzymes, has been carried out in microorganisms for production of enzymes for industrial purposes. Among these enzymes, microbial phosphatases, responsible for hydrolyzing phosphoric acid esters and anhydrides, and phytases, which catalyzes the hydrolysis of phytate (myo-inositol hexaquisphosphate) in myo-inositol and inorganic phosphate, have been widely used in different sectors as, for example, in molecular biology experiments and in animal feed. According to the optimum reaction pH, phosphatases are divided into alkaline (EC 3.1.3.1) and acidic (EC 3.1.3.2). Phytases are enzymes that also belong to the class of phosphatases, however, hydrolyzing phytic acid. In recent works, the filamentous fungus Rhizopus microsporus var. microsporus presented potential for production of phosphatases and phytases. In view of this, this study aimed at the production, purification and characterization of phosphatase and alkaline phytase produced by R. microsporus var. microsporus. In the optimization of Submerged Fermentation (FSbm), the highest enzymatic production was in Khanna medium with 0.4 mM KH2PO3 and added with 0.5% rye flour for 76 h, 32ºC, pH 6.3, at 100 rpm. In chromatographic columns, alkaline phosphatase was purified 10 folds and recovered at 13%, and alkaline phytase was purified 86 folds with recovery of 167%. The native molecular mass of alkaline phosphatase and phytase produced by R. microsporus var. microsporus was 122.7 and 55.5 kDa, respectively. The purified alkaline phosphatase had an optimum temperature of 45ºC and optimum pH of 8.5, maintaining its catalytic activity above 60% at temperatures of 40 and 45ºC for up to 210 minutes. It maintained its activity above 50% in alkaline pH for up to 24 h. Purified alkaline phytase presented optimum temperature and pH of activity of 65°C and 9.5, respectively. Alkaline phytase maintained its enzymatic activity above 60% at temperatures of 65, 70 and 80ºC for 270 min. After phytase incubation in alkaline pH for 3 hours, enzymatic activity increased by up to 50%. Phosphatase had increased activity in presence of 0.4 mM FeCl3, and better hydrolyzed the substrate p-nitrophenylphosphate, followed by ATP. The Km for alkaline phosphatase using the substrate p-nitrophenylphosphate in the absence and presence of FeCl3 was 0.449 mM and 0.382 mM, respectively. For alkaline Phytase, the Km was 0.413 mM using sodium phytate as substrate. Thus, alkaline phosphatase and phytase produced by R. microsporus var. microsporus were co-purified and biochemically characterized, evidencing important characteristics of both enzymes, thereby, possibilities of future industrial applications.
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Palavras-chave
Rhizopus, Fosfatase alcalina, Fitase alcalina, Fermentação submersa, Alkaline phosphatase, Alkaline phytase, Submerged fermentation