Avaliação da termoestabilidade e aplicação biotecnológica de endo-xilanases GH11 obtidas por otimização in silico
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Data
2017-06-30
Autores
Ramos, Felipe Cardoso [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O processamento da biomassa lignocelulósica para a produção do etanol de segunda geração (E2G) e outras aplicações requer enzimas termoestáveis capazes de operar sob altas temperaturas e longos períodos de incubação. Nesse contexto, reportamos aqui a validação experimental preliminar de uma abordagem in silico para a obtenção enzimas hidrolíticas termoestáveis. Simulações computacionais da endoxilanase GH11 de Bacillus subtilis (XBS) usando a metodologia de otimização de interações eletrostáticas (EIO) indicaram três resíduos (K99, R122 and K154) críticos para o aumento da termoestabilidade através da substituição de cargas. Assim, analisamos os efeitos da mutação K99E em diversas propriedades da XBS selvagem (XBSWT) e também de uma XBS obitida in vitro através de evolução dirigida (XBSM6). Após expressão heteróloga em células de Escherichia coli BL21 e purificação através de cromatografia de afinidade, realizamos a caracterização bioquímica e biofisica das enzimas. Além disso, conduzimos estudos de termotolerância incubando as enzimas em condições industriais (pH 4,8 e 50 °C). Finalmente, realizamos testes de suplementação de um coquetel enzimático comercial esperando obter melhoras no processo de hidrólise da biomassa. Os resultados mostraram que a mutação K99E foi capaz de aumentar a temperatura de desnaturação térmica (Tm) das enzimas em aproximadamente 1 °C e melhorar a termotolerância de XBSWT e XBSM6 sem alterar suas condições ótimas. Somado a isso, a enzima XBSM6+K99E apresentou melhor performance nos ensaios de suplementeção. Estes resultados sugerem que a abordagem de EIO pode ser usada como estratégia inicial para o desenho de novas endo-xilanases GH11 termoestáveis, capazes de suportar as condições operacionais empregadas na industria de E2G.
Processing of lignocellulosic biomasses for second-generation bioethanol (SGB) production and others applications requires thermostable enzymes able of operating under high temperatures and long incubation periods. In this context, we report here the preliminary experimental validation of an in silico approach for engineering hydrolytic enzymes towards thermostability. Previous computational simulations of GH11 endo-xylanase from Bacillus subtilis (XBS) using the electrostatic interaction optimization (EIO) methodology indicated three amino acid residues (K99, R122 and K154) critical for increasing thermostability through charge replacement. Thus, we analyzed the effects of the K99E mutation on several proprietes of two XBS enzymes: the wild-type XBS (XBSWT) and an XBS obitited in vitro by directed evolution (XBSM6). After heterologous expression in Escherichia coli BL21 cells and purification by affinity chromatography, we performed the biochemical and biophysical characterization of enzymes. We also conducted thermotolerance studies, incubating the enzymes under industrial conditions (pH 4.8 and 50 ° C). Finally, we performed supplementation assays of a commercial enzyme cocktail hoping to obtain improvements in the biomass hydrolysis process. The results showed that the K99E mutation was able to increase the enzymes thermal denaturation temperature (Tm ) by approximately 1 °C and improve thermotolerance of XBSWT and XBSM6 without alter its optimum conditions. In addition, the enzyme XBSM6+K99E showed better performance in the cocktail supplementation trials. These results suggest that the EIO approach can be used as initial strategy for the design of new thermostable GH11 endo-xylanases able of overcome the operating conditions employed in the SGB industry.
Processing of lignocellulosic biomasses for second-generation bioethanol (SGB) production and others applications requires thermostable enzymes able of operating under high temperatures and long incubation periods. In this context, we report here the preliminary experimental validation of an in silico approach for engineering hydrolytic enzymes towards thermostability. Previous computational simulations of GH11 endo-xylanase from Bacillus subtilis (XBS) using the electrostatic interaction optimization (EIO) methodology indicated three amino acid residues (K99, R122 and K154) critical for increasing thermostability through charge replacement. Thus, we analyzed the effects of the K99E mutation on several proprietes of two XBS enzymes: the wild-type XBS (XBSWT) and an XBS obitited in vitro by directed evolution (XBSM6). After heterologous expression in Escherichia coli BL21 cells and purification by affinity chromatography, we performed the biochemical and biophysical characterization of enzymes. We also conducted thermotolerance studies, incubating the enzymes under industrial conditions (pH 4.8 and 50 ° C). Finally, we performed supplementation assays of a commercial enzyme cocktail hoping to obtain improvements in the biomass hydrolysis process. The results showed that the K99E mutation was able to increase the enzymes thermal denaturation temperature (Tm ) by approximately 1 °C and improve thermotolerance of XBSWT and XBSM6 without alter its optimum conditions. In addition, the enzyme XBSM6+K99E showed better performance in the cocktail supplementation trials. These results suggest that the EIO approach can be used as initial strategy for the design of new thermostable GH11 endo-xylanases able of overcome the operating conditions employed in the SGB industry.
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Palavras-chave
Endo-xilanases GH11, Termoestabilidade, E2G, Otimização in silico, GH11 endo-xylanases, Thermostability, SGB, In silico optimization