Produção de quitinases pelo fungo Aspergillus niveus em fermentação submersa e em estado sólido utilizando resíduos da indústria pesqueira do camarão e análise do potencial antifúngico
Carregando...
Data
2022-03-10
Autores
Ornela, Pedro Henrique de Oliveira [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
As indústrias utilizam enzimas em diversos setores como o farmacêutico e alimentício, as quais em sua maior parte, são produzidas por microrganismos. Dentre essas enzimas, as quitinases, que clivam as ligações β-1,4 entre as unidades de N-acetilglicosaminas da quitina, têm destaque quanto a sua aplicação biotecnológica como, por exemplo, no controle de fungos fitopatógenos e pragas herbívoras e no tratamento de efluentes ricos em quitina. A quitina é o segundo biopolímero mais abundante do planeta, sendo encontrado no exoesqueleto de artrópodes e parede celular de fungos. Resíduos da indústria pesqueira de crustáceos são ricos em quitina e não têm sido aproveitados para outras finalidades, sendo descartado no meio ambiente, poluindo-o. Com isso, o objetivo deste trabalho foi determinar as melhores condições de cultivo para a máxima produção de quitinases por A. niveus em fermentação submersa (FSbm) e em substrato sólido (FSS), utilizando resíduos da indústria pesqueira do camarão, e avaliar o potencial de aplicação da enzima como antifúngico. No processo de otimização da FSbm, a maior produção quitinásica (1,4 U mL-1) foi obtida utilizando cascas de camarão-de-sete-barbas na granulometria entre 1 e 2 mm², a 35,5 ºC, 150 rpm, 110 h e inóculo inicial de 107 esporos mL de meio-1; já para a FSS (5 U g de substrato-1) as melhores condições foram cascas de camarão-de-sete-barbas (entre 2 e 10 mm²), a 31 ºC, por 144 h, inoculado de 106 esporos, umidificado com água de torneira na proporção de 1:2 (m/v). Ambos filtrados brutos apresentaram acima de 80% de atividade quitinásica residual por até 90 dias armazenados em geladeira (4 ºC). As quitinases obtidas da FSbm e FSS foram purificadas 40 e 2 vezes, respectivamente, com massas moleculares de 49,3 e 47,6 kDa, sendo monômeros. A quitinase produzida em FSbm apresentou máxima atividade catalítica no pH 6,0, e a produzida em FSS no pH 5,0. A melhor temperatura para a atuação de ambas as formas enzimáticas foi 65 ºC, mantendo a estabilidade a 30 e 40 ºC por até 6 h. Ambas as formas foram resistentes ao KCl, ao SDS e ao β-mercaptoetanol. Os parâmetros cinéticos foram determinados, sendo o Km e a Vmáx de 2,7 mmol L-1 e 12,6 U mg de prot.-1, respectivamente, para a quitinase da FSbm, e de 6,1 mmol L-1 e 16,0 U mg de prot.-1 para a enzima da FSS, sugerindo que se tratam de diferentes isoformas. O filtrado bruto da FSbm contendo quitinase inibiu o crescimento dos fungos Cladosporium herbarium, Rhizopus microsporus var. microsporus e Fusarium lateritium. O filtrado bruto da FSS inibiu o crescimento do fungo Penicillium purpurogenum em concentrações menores que as observadas para o uso de fluconazol e anfotericina B. Assim, é possível concluir que a utilização das cascas de camarão-de-sete-barbas para produção de quitinases por A. niveus em FSbm e FSS, é viável, obtendo-se uma biomolécula com potencial de aplicação em diferentes setores como o agrícola, farmacêutico e industrial, minimizando também os efeitos poluentes desse resíduo no meio ambiente.
Industries use enzymes in various sectors such as pharmaceuticals and food, which for the most part are produced by microorganisms. Among these enzymes, chitinases, which cleave the β-1,4 bonds between the N-acetylglucosamine units of chitin, are highlighted in terms of their biotechnological application, for example, in the control of phytopathogenic fungi and herbivorous pests, and in the treatment of effluents rich in chitin. Chitin is the second most abundant biopolymer on the planet, found in the exoskeleton of arthropods and the cell wall of fungi. Residues from the crustacean fishing industry are rich in chitin and have not been used for other purposes, being discarded in the environment, polluting it. Thus, the objective of this study was to determine the best cultivation conditions for maximum production of chitinases by A. niveus in submerged fermentation (SbmF) and in solid-state fermentation (SSF), using residues from the shrimp fishing industry, and to evaluate the potential application of the enzyme as an antifungal. In the SbmF optimization process, the highest chitinase production (1.4 U mL-1) was obtained using seven-bearded shrimp shells with granulometry between 1 and 2 mm², at 35.5 ºC, 150 rpm, 110 he initial inoculum of 107 spores ml of medium-1; for the SSF (5 U g of substrate-1) the best conditions were seven-bearded shrimp shells (between 2 and 10 mm²), at 31 ºC, for 144 h, inoculated with 106 spores, humidified with water of faucet in a ratio of 1:2 (m/v). Both crude filtrates showed above 80% residual chitinase activity for up to 90 days stored in a refrigerator (4 ºC). The chitinases obtained from SbmF and SSF were purified 40 and 2 times, respectively, with molecular masses of 49.3 and 47.6 kDa, being monomers. The chitinase produced in SbmF showed maximum catalytic activity at pH 6.0, and that produced in SSF at pH 5.0. The best temperature for the performance of both enzymatic forms was 65 ºC, maintaining stability at 30 and 40 ºC for up to 6 h. Both forms were resistant to KCl, SDS and β-mercaptoethanol. The kinetic parameters were determined, being the Km and Vmax of 2.7 mmol L-1 and 12.6 U mg of prot.-1 for the chitinase of SbmF, and of 6.1 mmol L-1 and 16.0 U mg of prot.-1 for the enzyme of SSF, suggesting that they are different isoforms. The SbmF crude filtrate containing chitinase inhibited the growth of the fungi Cladosporium herbarium, Rhizopus microsporus var. microsporus and Fusarium lateritium. The SSF crude filtrate inhibited the growth of the fungus Penicillium purpurogenum at concentrations lower than those observed for the use of fluconazole and amphotericin B. Thus, it is possible to conclude that the use of seven-bearded shrimp shells for the production of chitinases by A. niveus in SbmF and SSF, is feasible, obtaining a biomolecule with potential application in different sectors such as agriculture, pharmaceutical and industrial, also minimizing the polluting effects of this residue in the environment.
Industries use enzymes in various sectors such as pharmaceuticals and food, which for the most part are produced by microorganisms. Among these enzymes, chitinases, which cleave the β-1,4 bonds between the N-acetylglucosamine units of chitin, are highlighted in terms of their biotechnological application, for example, in the control of phytopathogenic fungi and herbivorous pests, and in the treatment of effluents rich in chitin. Chitin is the second most abundant biopolymer on the planet, found in the exoskeleton of arthropods and the cell wall of fungi. Residues from the crustacean fishing industry are rich in chitin and have not been used for other purposes, being discarded in the environment, polluting it. Thus, the objective of this study was to determine the best cultivation conditions for maximum production of chitinases by A. niveus in submerged fermentation (SbmF) and in solid-state fermentation (SSF), using residues from the shrimp fishing industry, and to evaluate the potential application of the enzyme as an antifungal. In the SbmF optimization process, the highest chitinase production (1.4 U mL-1) was obtained using seven-bearded shrimp shells with granulometry between 1 and 2 mm², at 35.5 ºC, 150 rpm, 110 he initial inoculum of 107 spores ml of medium-1; for the SSF (5 U g of substrate-1) the best conditions were seven-bearded shrimp shells (between 2 and 10 mm²), at 31 ºC, for 144 h, inoculated with 106 spores, humidified with water of faucet in a ratio of 1:2 (m/v). Both crude filtrates showed above 80% residual chitinase activity for up to 90 days stored in a refrigerator (4 ºC). The chitinases obtained from SbmF and SSF were purified 40 and 2 times, respectively, with molecular masses of 49.3 and 47.6 kDa, being monomers. The chitinase produced in SbmF showed maximum catalytic activity at pH 6.0, and that produced in SSF at pH 5.0. The best temperature for the performance of both enzymatic forms was 65 ºC, maintaining stability at 30 and 40 ºC for up to 6 h. Both forms were resistant to KCl, SDS and β-mercaptoethanol. The kinetic parameters were determined, being the Km and Vmax of 2.7 mmol L-1 and 12.6 U mg of prot.-1 for the chitinase of SbmF, and of 6.1 mmol L-1 and 16.0 U mg of prot.-1 for the enzyme of SSF, suggesting that they are different isoforms. The SbmF crude filtrate containing chitinase inhibited the growth of the fungi Cladosporium herbarium, Rhizopus microsporus var. microsporus and Fusarium lateritium. The SSF crude filtrate inhibited the growth of the fungus Penicillium purpurogenum at concentrations lower than those observed for the use of fluconazole and amphotericin B. Thus, it is possible to conclude that the use of seven-bearded shrimp shells for the production of chitinases by A. niveus in SbmF and SSF, is feasible, obtaining a biomolecule with potential application in different sectors such as agriculture, pharmaceutical and industrial, also minimizing the polluting effects of this residue in the environment.
Descrição
Palavras-chave
Antifúngico, Fermentação, Quitinase, Otimização, Casca de camarão, Resíduos industriais, Enzimas de fungos, Quitina