Prospecção de queratinases microbianas: produção e caracterização bioquímica funcional
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2020-05-15
Autores
Duffeck, Carlos Eduardo
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Universidade Estadual Paulista (Unesp)
Resumo
Atualmente, a avicultura é um dos setores de grande impacto na economia brasileira. Nos últimos anos, tem sido observado um aumento na produção de frangos de corte, fazendo com que este segmento da indústria gere toneladas de queratina com o descarte de penas. Isso aponta para a necessidade de degradar este material que emerge como um problema ambiental. Neste cenário, as enzimas queratinolíticas têm despertado interesse biotecnológico devido a peculiar capacidade para a degradação de queratina e a possibilidade de aplicar o hidrolisado protéico para suplementação de ração animal e uso como biofertilizantes. Desta forma, neste trabalho, nós propomos prospectar queratinases pela bactéria Citrobacter diversus e o fungo Coriolopsis byrsina e, em seguida, investigar as características bioquímicas destas enzimas, a fim de propor aplicação na degradação de penas de frango. Em nossos resultados, a bactéria C. diversus foi capaz de degradar quase completamente as penas de frango (0,5%) em meio submerso após 36 h de fermentação. O estudo com o extrato enzimático mostrou máxima atividade caseinolítica a pH 9-10,5 e 50-55 ºC, e queratinolítica a pH 8,5-9,5 e 50 ºC. Em destaque, conforme a estabilidade em incubação por 1 h a 50ºC, foi detectado aproximadamente 50% e 100% da atividade queratinolítica e caseinolítica, respectivamente. Sob estabilidade a pH por 48 h a 4ºC, o extrato enzimático manteve maior atividade na faixa de pH 6-8. A atividade caseinolítica foi inibida por EDTA e PMSF, e atividade queratinolítica foi inibida por EDTA. Para o fungo C. byrsina, nós observamos a secreção de diferentes enzimas proteolíticas, cuja atividade caseinolítica atingiu picos a pH 7,0-9,0 e 60-70 ºC, e a pH 10,5 e 55-60ºC; e máxima atividade queratinolítica a pH 7,0-7,5 e 40-55ºC, e pH 9,0 e 55 ºC. Enzimas com melhor atividade caseinolítica na faixa de pH 8,5 demonstraram maior estabilidade a pH 5-6. Enzimas com melhor atividade caseinolítica a pH 10,5 exibiram melhor estabilidade na faixa de pH 6-10. Para atividade queratinolítca, enzimas com melhor atividade a pH 7,5 demonstraram maior estabilidade quando incubadas a pH 10-11, enquanto que enzimas com máxima atividade a pH 9,0 exibiram estabilidade em ampla faixa de pH (5-11). Atividade queratinolítica manteve cerca de 63% de atividade residual em 1 h a 50 ºC. A atividade caseinolítica a pH 10,5 mantém-se estável até 1 h a 50 ºC, em contraste a atividade a pH 8,5, cuja atividade residual foi 50%. A atividade caseinolítica foi inibida somente por PMSF, enquanto a atividade queratinolítca foi reduzida por PMSF e EDTA. Conforme a atividade e estabilidade em pH alcalino, estas enzimas aparecem como atrativos candidatos para uso em indústria de detergente.
Currently, poultry is one of the sectors of great impact on the Brazilian economy. In recent years, there has been an increase in the production of broilers, causing this segment of the industry to generate tons of keratin with the disposal of feathers. This points to the need to degrade this material, which emerges as an environmental problem. In this scenario, keratinolytic enzymes have aroused biotechnological interest due to the peculiar capacity for the degradation of keratin and the possibility of applying protein hydrolyzate to supplement animal feed and use as biofertilizers. Thus, in this work we propose to prospect keratinases for the bacterium Citrobacter diversus and the fungus Coriolopsis byrsina and, next, to investigate the biochemical characteristics of these enzymes, in order to propose application in the degradation of chicken feathers. In our results, the bacterium C. diversus was able to degrade chicken feathers almost completely (0.5%) in submerged medium after 36 h. The study with the enzymatic extract showed maximum caseinolytic activity at pH 9-10.5 and 50-55 ºC, and keratinolytic activity at pH 8.5-9.5 and 50 ºC. Notably, after enzyme pre-incubation for 1 h at 50 ºC, approximately 50% and 100% of keratinolytic and caseinolytic activity were detected, respectively. Under pH stability for 48 h at 4ºC, the enzyme extract maintained greater activity in the pH 6-8 range. Caseinolytic activity was inhibited by EDTA and PMSF, and keratinolytic activity was inhibited by EDTA. For the fungus C. byrsina, we observed the secretion of different proteolytic enzymes, whose caseinolytic activity reached peaks at pH 7.0-9.0 and 60-70 ºC, and at pH 10.5 and 55-60ºC; and maximum keratinolytic activity at pH 7.0-7.5 and 40-55ºC, and pH 9.0 and 55 ºC. Enzymes with better caseinolytic activity at pH 8.5 demonstrated the highest stability at pH 5-6. Enzymes with better caseinolytic activity at pH 10.5 exhibited the highest stability in the pH range 6-10. For keratinolytic activity, enzymes with better activity at pH 7.5 showed the highest stability when incubated at pH 10-11, while enzymes with maximum activity at pH 9.0 exhibited stability at pH range 5-11. Keratinolytic activity maintained approximately 63% of residual activity after incubation for 1 h at 50 ºC. The caseinolytic activity at pH 10.5 remains stable for 1 h up to 50 ºC, in contrast to the activity at pH 8.5, whose residual activity was 50%. Caseinolytic activity was inhibited only by PMSF, while keratinolytic activity was reduced by PMSF and EDTA. Depending on the activity and stability at alkaline pH, these enzymes appear as attractive candidates for use in the detergent industry.
Currently, poultry is one of the sectors of great impact on the Brazilian economy. In recent years, there has been an increase in the production of broilers, causing this segment of the industry to generate tons of keratin with the disposal of feathers. This points to the need to degrade this material, which emerges as an environmental problem. In this scenario, keratinolytic enzymes have aroused biotechnological interest due to the peculiar capacity for the degradation of keratin and the possibility of applying protein hydrolyzate to supplement animal feed and use as biofertilizers. Thus, in this work we propose to prospect keratinases for the bacterium Citrobacter diversus and the fungus Coriolopsis byrsina and, next, to investigate the biochemical characteristics of these enzymes, in order to propose application in the degradation of chicken feathers. In our results, the bacterium C. diversus was able to degrade chicken feathers almost completely (0.5%) in submerged medium after 36 h. The study with the enzymatic extract showed maximum caseinolytic activity at pH 9-10.5 and 50-55 ºC, and keratinolytic activity at pH 8.5-9.5 and 50 ºC. Notably, after enzyme pre-incubation for 1 h at 50 ºC, approximately 50% and 100% of keratinolytic and caseinolytic activity were detected, respectively. Under pH stability for 48 h at 4ºC, the enzyme extract maintained greater activity in the pH 6-8 range. Caseinolytic activity was inhibited by EDTA and PMSF, and keratinolytic activity was inhibited by EDTA. For the fungus C. byrsina, we observed the secretion of different proteolytic enzymes, whose caseinolytic activity reached peaks at pH 7.0-9.0 and 60-70 ºC, and at pH 10.5 and 55-60ºC; and maximum keratinolytic activity at pH 7.0-7.5 and 40-55ºC, and pH 9.0 and 55 ºC. Enzymes with better caseinolytic activity at pH 8.5 demonstrated the highest stability at pH 5-6. Enzymes with better caseinolytic activity at pH 10.5 exhibited the highest stability in the pH range 6-10. For keratinolytic activity, enzymes with better activity at pH 7.5 showed the highest stability when incubated at pH 10-11, while enzymes with maximum activity at pH 9.0 exhibited stability at pH range 5-11. Keratinolytic activity maintained approximately 63% of residual activity after incubation for 1 h at 50 ºC. The caseinolytic activity at pH 10.5 remains stable for 1 h up to 50 ºC, in contrast to the activity at pH 8.5, whose residual activity was 50%. Caseinolytic activity was inhibited only by PMSF, while keratinolytic activity was reduced by PMSF and EDTA. Depending on the activity and stability at alkaline pH, these enzymes appear as attractive candidates for use in the detergent industry.
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Palavras-chave
Bactéria, Detergente, Enzimas, Fungo, Queratina, Peptidase, Protease, Bacteria, Detergent, Enzymes, Fungus, Keratin, Peptidase, Protease