Enzima bifuncional xilanase-esterase obtida do metagenoma do líquido ruminal
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Data
2020-07-28
Autores
Pavarina, Gabriella Cavazzini
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Universidade Estadual Paulista (Unesp)
Resumo
A biomassa lignocelulósica, importante e estratégica fonte de energia renovável, é complexa e heterogênea. Para que ocorra a hidrólise enzimática desta matéria-prima é necessária a ação sinérgica de enzimas degradadoras de celulose, hemicelulose e lignina. As endo-β-xilanases, enzimas capazes de degradar a cadeia principal das xilanas, hemicelulose mais abundante, apresentam sua eficiência reduzida devido à presença de ramificações em sua estrutura, como por exemplo ligações éster com ácido ferúlico e cumárico, as quais podem ser degradadas por esterases. Uma alternativa para aumentar a eficiência seria a hidrólise simultânea destas ramificações e da cadeia principal, o que é possibilitado pelo uso de enzimas bifuncionais. O objetivo do trabalho foi prospectar e expressar uma xilanase-esterase obtida do metagenoma do líquido ruminal. A sequência da xilanase-esterase foi obtida com base na inferência de função por similaridade no metagenoma de rúmen. Foi realizada a amplificação da sequência do DNA metagenômico por PCR, a inserção em vetor pET28a e transformação em Escherichia coli BL21 (DE3). A purificação da enzima recombinante foi realizada utilizando a cromatografia de afinidade por íons metálicos imobilizados e filtração em gel no sistema AKTA. A atividade de endo-β-xilanase foi verificada por zimograma contendo xylan from beechwood e a de esterase pela hidrólise do substrato de p-nitrofenil-éster. Os resultados demonstram a obtenção de uma enzima bifuncional (xilanase-esterase), ativa para ambos os substratos, resistente ao NaCl e solventes orgânicos (etanol, metanol, propanol e DMSO), com potencial para aplicação na degradação de biomassa lignocelulósica.
Lignocellulosic biomass, an important and strategic source of renewable energy, is complex and heterogeneous. For the enzymatic hydrolysis of this raw material to occur, it needs the synergistic action of cellulose, hemicellulose and lignin degrading enzymes. The endo-β-xylanases, enzymes capable of degrading the main chain of xylans, the most abundant hemicellulose, have their efficiency reduced due to the presence of branches in their structure, such as ester bonds with ferulic and cumaric acid, which can be degraded by esterases. An alternative to increase efficiency would be the simultaneous hydrolysis of these branches and the main chain, which is possible using bifunctional enzymes. The aim of this work was to prospect and express a xylanase-esterase achieved by metagenome from the rumen fluid. The xylanase-esterase sequence was obtained based on the inference of function by similarity in the rumen metagenome. The amplification of the metagenomic DNA sequence by PCR was realized, insertion into vector pET28a and transformation into Escherichia coli BL21 (DE3) were made. The purification of the recombinant enzyme was performed using affinity chromatography for immobilized metal ions and gel filtration in the AKTA system. The endo-β-xylanase activity was verified by zymogram containing xylan from beechwood and esterase activity by the hydrolysis of the substrate of p-nitrophenyl ester. The results demonstrate the obtaining of a bifunctional enzyme (xylanase-esterase), active for both substrates, resistant to NaCl and organic solvents (ethanol, methanol, propanol and DMSO), with potential for application in the degradation of lignocellulosic biomass.
Lignocellulosic biomass, an important and strategic source of renewable energy, is complex and heterogeneous. For the enzymatic hydrolysis of this raw material to occur, it needs the synergistic action of cellulose, hemicellulose and lignin degrading enzymes. The endo-β-xylanases, enzymes capable of degrading the main chain of xylans, the most abundant hemicellulose, have their efficiency reduced due to the presence of branches in their structure, such as ester bonds with ferulic and cumaric acid, which can be degraded by esterases. An alternative to increase efficiency would be the simultaneous hydrolysis of these branches and the main chain, which is possible using bifunctional enzymes. The aim of this work was to prospect and express a xylanase-esterase achieved by metagenome from the rumen fluid. The xylanase-esterase sequence was obtained based on the inference of function by similarity in the rumen metagenome. The amplification of the metagenomic DNA sequence by PCR was realized, insertion into vector pET28a and transformation into Escherichia coli BL21 (DE3) were made. The purification of the recombinant enzyme was performed using affinity chromatography for immobilized metal ions and gel filtration in the AKTA system. The endo-β-xylanase activity was verified by zymogram containing xylan from beechwood and esterase activity by the hydrolysis of the substrate of p-nitrophenyl ester. The results demonstrate the obtaining of a bifunctional enzyme (xylanase-esterase), active for both substrates, resistant to NaCl and organic solvents (ethanol, methanol, propanol and DMSO), with potential for application in the degradation of lignocellulosic biomass.
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Enzimas, Biotecnologia, Genética microbiana, Enzymes, Biotechnology, Microbial genetics