Otimização dos parâmetros de produção para atividade e expressão heteróloga de enzimas celulolíticas pelo fungo de ambiente marinho Trichoderma lixii-5A7
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Data
2022-12-16
Autores
Eguiluz, Alexandra Daniela Barrios
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Universidade Estadual Paulista (Unesp)
Resumo
Na área industrial existem diversos mecanismos (físicos, químicos, enzimáticos, fermentativos) para degradar biomassa e, assim, obter uma ampla variedade de bioprodutos como biocombustíveis, biopigmentos, carotenoides, etc. Entre os componentes dos resíduos naturais de biomassa, a celulose é o mais abundante podendo degradar os resíduos até glicose e gerar produtos de interesse. Desta forma, as celulases têm uma ampla utilização na indústria de alimentos, detergentes, têxteis, biocombustíveis, assim como na área farmacêutica. Na atualidade, são utilizadas cepas de alguns fungos para produzir as enzimas celulases em escala industrial, como exemplo de Aspergillus niger e Trichoderma reesei. Estas enzimas, mesmo tendo um substrato em comum, podem variar sua atividade dependendo da temperatura, pH, especificidade e concentração. Portanto, identificar novos microrganismos com elevado potencial para a obtenção de celulases é de grande importância na estratégia de melhorar a economia do processo e a especificidade para a obtenção do bioproduto. Levando em consideração a diversidade de microrganismos e a possibilidade de obter uma nova fonte para produção da enzima, o presente trabalho objetivou avaliar a atividade de enzimas celulolíticas do fungo de ambiente marinho Trichoderma lixii -5A7, caracterizá-las quanto à produção e identificação da sequência codificadora. Para tanto, foi selecionado o meio D que apresentou uma maior produção enzimática na temperatura de 20°C, contendo em sua composição carboximetilcelulose (CMC;20 g/L) como indutor. Na cinética de produção, na temperatura selecionada, obteve-se no 15º dia de produção o maior pico de atividade enzimática específica para β-glicosidase, endoglucanase, FPAse e exoglucanase (respetivamente 71,84; 65,87; 49,12 e 68,23 UI/mg). Posteriormente, para otimizar a produção das enzimas celulolíticas do fungo Trichoderma lixii – 5A7 foi realizado o planejamento experimental fatorial para o melhor meio de cultura utilizando o delineamento do composto central rotacional (DCCR) e coletando amostras nos dias 13; 14; 15 e 16. A maior produção da enzima β-glicosidase foi no ensaio 23 com 85,91 UI/mg de atividade específica e no ensaio 9 obteve-se uma atividade específica de 125,99; 60,94; 83,60 UI/mg para as enzimas endoglucanase, FPAse e exoglucanase respectivamente, sendo os resultados de ambos experimentos com a maior produção no dia 15, os ensaios mencionados foram obtidos da matriz proposta pelo software utilizado para o DCCR. A fermentação em escala de biorreator (3 L) realizada posteriormente demonstrou um pequeno aumento da atividade enzimática apenas para a β-glicosidase no ensaio 23 e no ensaio 9 para a FPAse, enquanto as demais enzimas mostraram uma atividade similar à obtida na escala de bancada, mas, no entanto, o pico de maior produção foi obtido no quarto e quinto dia respectivamente. Para clonagem da sequência codificadora, foi extraído o RNA total do fungo para cada ensaio (9 e 23), o qual foi utilizado para a síntese do cDNA com a finalidade de amplificar fragmentos das enzimas celulolíticas, utilizando a técnica de Reação em Cadeia da Polimerase (PCR). Em seguida, foi realizado sequenciamento do produto gerado, clonagem e expressão em Escherichia coli para então avaliar a produção e atividade celulolítica. Desta forma, no presente trabalho, determinou-se que a produção das enzimas celulolíticas em T.lixii -5A7 pode ser otimizada reduzindo a temperatura e variando as concentrações dos componentes do meio selecionado representando uma melhora na atividade enzimática especifica entre 80 a 126 UI/mg, tornando esse fungo viável para a produção dessas enzimas. Além disso, foi possível determinar pela primeira vez a sequência codificadora da β-glicosidase desse fungo e caracterizá-la funcionalmente em bactéria.
In the industrial area there are several mechanisms (physical, chemical, enzymatic, fermentative) to degrade biomass and thus obtain a wide variety of bioproducts such as biofuels, biopigments, carotenoids, etc. Among the components of natural biomass residues, cellulose is the most abundant and can degrade the residues to glucose and generate products of interest. Thus, cellulases are widely use in the food, detergent, textile, biofuel, and pharmaceutical industries. Nowadays, strains of some fungi are used to produce cellulase enzymes on an industrial scale, such as Aspergillus niger and Trichoderma reesei. These enzymes, even having a substrate in common, can vary their activity depending on temperature, pH, specificity, and concentration. Therefore, identifying new microorganisms with high potential for obtaining cellulases is of great importance in the strategy to improve the economy of the process and the specificity for obtaining the bioproduct. Taking into account the diversity of microorganisms and the possibility of obtaining a new source for enzyme production, the present work aimed to evaluate the activity of cellulolytic enzymes from the marine fungus Trichoderma lixii -5A7, characterize them in terms of production and identification of the coding sequence. For this purpose, the medium D was selected, which showed the highest enzyme production at a temperature of 20°C, containing carboxymethyl cellulose (CMC;20 g/L) in its composition as an inducer. In the production kinetics, at the selected temperature, the 15th day of production was obtained as the highest peak of specific enzymatic activity for β-glucosidase, endoglucanase, FPAse and exoglucanase (71.84; 65.87; 49.12 and 68.23 UI/mg, respectively). Subsequently, to optimize the production of cellulolytic enzymes from the fungus Trichoderma lixii - 5A7, a factorial experimental design was carried out for the best culture medium using the rotational central compound design (DCCR) and collecting samples on days 13; 14; 15 and 16. The highest production of the enzyme β-glycosidase in assay 23 obtained 85.91 IU/mg of specific enzymatic activity and in assay 9 a specific activity of 125.99; 60.94; 83.60 IU/mg was obtained for the enzymes endoglucanase, FPAse and exoglucanase respectively, with the results of both experiments with the highest production on day 15, the mentioned assays were obtained from the matrix proposed by the software used for the DCCR. The fermentation in bioreactor scale (3 L) carried out after, showed a small increase in enzymatic activity only for β-glucosidase in assay 23 and in assay 9 for FPAse, while the other enzymes showed a activity similar to that obtained on the scale bench, but however, the highest production peak was obtained on the fourth and fifth days, respectively. For cloning of the coding sequence, total RNA was extracted from the fungus for each assay (9 and 23), which was used for cDNA synthesis to amplify fragments of the cellulolytic enzymes using the Polymerase Chain Reaction (PCR) technique. Then, sequencing of the generated product, cloning and expression in Escherichia coli were performed to then evaluate the production and cellulolytic activity. Thus, in the present work, it was determined that the production of cellulolytic enzymes in T.lixii -5A7 can be optimized by reducing the temperature and varying the concentrations of the selected medium components, representing an improvement in the specific enzymatic activity between 80 and 126 UI/mg, making this fungus viable for the production of these enzymes. In addition to, it was possible to determine for the first time the sequence encoding the β-glucosidase from this fungus and to characterize it functionally in bacteria.
In the industrial area there are several mechanisms (physical, chemical, enzymatic, fermentative) to degrade biomass and thus obtain a wide variety of bioproducts such as biofuels, biopigments, carotenoids, etc. Among the components of natural biomass residues, cellulose is the most abundant and can degrade the residues to glucose and generate products of interest. Thus, cellulases are widely use in the food, detergent, textile, biofuel, and pharmaceutical industries. Nowadays, strains of some fungi are used to produce cellulase enzymes on an industrial scale, such as Aspergillus niger and Trichoderma reesei. These enzymes, even having a substrate in common, can vary their activity depending on temperature, pH, specificity, and concentration. Therefore, identifying new microorganisms with high potential for obtaining cellulases is of great importance in the strategy to improve the economy of the process and the specificity for obtaining the bioproduct. Taking into account the diversity of microorganisms and the possibility of obtaining a new source for enzyme production, the present work aimed to evaluate the activity of cellulolytic enzymes from the marine fungus Trichoderma lixii -5A7, characterize them in terms of production and identification of the coding sequence. For this purpose, the medium D was selected, which showed the highest enzyme production at a temperature of 20°C, containing carboxymethyl cellulose (CMC;20 g/L) in its composition as an inducer. In the production kinetics, at the selected temperature, the 15th day of production was obtained as the highest peak of specific enzymatic activity for β-glucosidase, endoglucanase, FPAse and exoglucanase (71.84; 65.87; 49.12 and 68.23 UI/mg, respectively). Subsequently, to optimize the production of cellulolytic enzymes from the fungus Trichoderma lixii - 5A7, a factorial experimental design was carried out for the best culture medium using the rotational central compound design (DCCR) and collecting samples on days 13; 14; 15 and 16. The highest production of the enzyme β-glycosidase in assay 23 obtained 85.91 IU/mg of specific enzymatic activity and in assay 9 a specific activity of 125.99; 60.94; 83.60 IU/mg was obtained for the enzymes endoglucanase, FPAse and exoglucanase respectively, with the results of both experiments with the highest production on day 15, the mentioned assays were obtained from the matrix proposed by the software used for the DCCR. The fermentation in bioreactor scale (3 L) carried out after, showed a small increase in enzymatic activity only for β-glucosidase in assay 23 and in assay 9 for FPAse, while the other enzymes showed a activity similar to that obtained on the scale bench, but however, the highest production peak was obtained on the fourth and fifth days, respectively. For cloning of the coding sequence, total RNA was extracted from the fungus for each assay (9 and 23), which was used for cDNA synthesis to amplify fragments of the cellulolytic enzymes using the Polymerase Chain Reaction (PCR) technique. Then, sequencing of the generated product, cloning and expression in Escherichia coli were performed to then evaluate the production and cellulolytic activity. Thus, in the present work, it was determined that the production of cellulolytic enzymes in T.lixii -5A7 can be optimized by reducing the temperature and varying the concentrations of the selected medium components, representing an improvement in the specific enzymatic activity between 80 and 126 UI/mg, making this fungus viable for the production of these enzymes. In addition to, it was possible to determine for the first time the sequence encoding the β-glucosidase from this fungus and to characterize it functionally in bacteria.
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Palavras-chave
atividade enzimática, biorreator, Botrylloides giganteus, celulases, fungos marinhos, Trichoderma lixii, planejamento fatorial