Avaliação de parâmetros cinéticos, termodinâmicos e caracterização estrutural in silico de uma endoxilanase heteróloga do fungo termofílico Myceliophthora heterothallica F.2.1.4 expressa em Pichia pastoris.
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Data
2022-06-03
Autores
Zanoni, Jéssica de Araújo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Este trabalho realizou estudos sobre propriedades bioquímicas, estruturais e aplicação de uma endoxilanase pertencente à família GH 11 do fungo termofílico Myceliophthora heterothallica F.2.1.4. Foram avaliadas características estruturais através de análises de bioinformática, identificando uma estrutura composta por 65% de folhas beta, apenas 7% de uma alfa hélice e os outros 27% da estrutura total correspondem a enovelamento aleatório (random coil). Os resíduos catalíticos da enzima foram identificados como glutamatos nas posições 89 e 181. A produção da enzima foi feita por expressão heteróloga em Pichia pastoris linhagem X33 (Komagataella phaffi) durante 144 horas a 28 ºC induzida por metanol. A presença da glicosilação na região N-terminal inibiu a afinidade por íons metálicos imobilizadas da cauda de histidina, por isso o protocolo alternativo de purificação foi estabelecido onde obtivemos um rendimento final de 6,0% da purificação. A enzima purificada foi analisada quanto à influência do glicerol sobre parâmetros termodinâmicos e cinéticos, onde o glicerol amentou significativamente Vmax não apresentando variações significativas sobre Km, diminuiu a energia de ativação da reação e da desnaturação térmica, aumentou os valores de meia vida, entropia e entalpia, porém não promoveu alterações nos valores de energia livre de Gibbs da desnaturação térmica. Simulações de dinâmica molecular evidenciaram que a presença do glicerol promoveu menores valores de RMSD e Rg. Os parâmetros cinéticos também foram avaliados na presença de compostos fenólicos, onde apenas os valores de Vmax foram aumentados na presença de ácido siríngico, ácido ferúlico e ácido vanílico. Análises de docking molecular não identificaram sítios de ligação na superfície da estrutura enzimática. Os efeitos observados podem ser atribuídos à adsorção dos fenólicos sobre as fibras de xilana, favorecida pelo pH do meio reacional. A aplicação de 10 U.g-1 da enzima na maceração enzimática para extração do mosto de uva, proporcionou aumento de 8% no rendimento após 2 horas em 55ºC, diminuição da acidez total e aumento nos valores de pH, sólidos solúveis, açúcares redutores e conteúdo de polifenólicos totais. A qualificação dos produtos de hidrólise da endoxilanase foi feita sobre os substratos xilana beechwood e hemicelulose extraída do bagaço de cana-de-açúcar. Em 24 horas de hidrólise foi possível identificar X1, X2 e X5 como produtos principais para a xilana beechwood, a presença de X5 foi relacionada ao mecanismo de transglicosilação descrito na literatura. No mesmo tempo para a hemicelulose extraída do bagaço de cana-de-açúcar foram identificados X1, X2 e X4. A diferença entre os produtos encontrados deve-se à constituição estrutural dos substratos avaliados.
This work carried out studies on the biochemical and structural properties and application of an endoxylanase belonging to the GH11 family of the thermophilic fungus Myceliophthora heterothallica F.2.1.4. Structural characteristics were evaluated through bioinformatics analyses, identifying a structure composed by 65% of beta sheets, only 7% of an alpha helix and the other 27% of the total structure correspond to random coiling. The enzyme catalytic residues were identified as glutamates at positions 89 and 181. Enzyme production followed by heterologous expression in Pichia pastoris lineage X33 (Komagataella phaffi) for 144 hours at 28°C induced by methanol. The presence of glycosylation in the N-terminal region inhibited the affinity for immobilized metal ions from the histidine tail, so the alternative purification protocol was established where we obtained a final yield of 6.0 %. The purified enzyme was analyzed for the influence of glycerol on thermodynamic and kinetic parameters, where glycerol significantly increased Vmax without showing significant variations over Km, decreased the activation energy of the reaction and thermal denaturation, increased the half-life values, entropy and enthalpy, but did not promote changes in the values of Gibbs free energy of thermal denaturation. Molecular dynamic simulations showed smaller RMSD and Rg on the presence of glycerol. The kinetic parameters were also evaluated in the presence of phenolic compounds, where only the Vmax values were increased in the presence of syringic acid, ferulic acid and vanillic acid. Molecular docking analyzes did not identify binding sites on the surface of the enzymatic structure. The observed effects can be attributed to the adsorption of phenolics on the xylan fibers, favored by the pH of the reaction medium. The application of 10 U.g-1 of the enzyme in the enzymatic maceration to extract the grape must, provided an 8% increase in yield after 2 hours at 55ºC, a decrease in total acidity and an increase in pH values, soluble solids, reducing sugars and of total polyphenols. The qualification of endoxylanase hydrolysis products was performed on beechwood xylan substrates and hemicellulose extracted from sugarcane bagasse. In 24 hours it was possible to identify X1, X2 and X5 as main products for beechwood xylan, the presence of X5 was related to the transglycosylation mechanism described in the literature. At the same time, for hemicellulose extracted from sugarcane bagasse, X1, X2 and X4 were identified. The difference between the products found is due to the structural constitution of the evaluated substrates.
This work carried out studies on the biochemical and structural properties and application of an endoxylanase belonging to the GH11 family of the thermophilic fungus Myceliophthora heterothallica F.2.1.4. Structural characteristics were evaluated through bioinformatics analyses, identifying a structure composed by 65% of beta sheets, only 7% of an alpha helix and the other 27% of the total structure correspond to random coiling. The enzyme catalytic residues were identified as glutamates at positions 89 and 181. Enzyme production followed by heterologous expression in Pichia pastoris lineage X33 (Komagataella phaffi) for 144 hours at 28°C induced by methanol. The presence of glycosylation in the N-terminal region inhibited the affinity for immobilized metal ions from the histidine tail, so the alternative purification protocol was established where we obtained a final yield of 6.0 %. The purified enzyme was analyzed for the influence of glycerol on thermodynamic and kinetic parameters, where glycerol significantly increased Vmax without showing significant variations over Km, decreased the activation energy of the reaction and thermal denaturation, increased the half-life values, entropy and enthalpy, but did not promote changes in the values of Gibbs free energy of thermal denaturation. Molecular dynamic simulations showed smaller RMSD and Rg on the presence of glycerol. The kinetic parameters were also evaluated in the presence of phenolic compounds, where only the Vmax values were increased in the presence of syringic acid, ferulic acid and vanillic acid. Molecular docking analyzes did not identify binding sites on the surface of the enzymatic structure. The observed effects can be attributed to the adsorption of phenolics on the xylan fibers, favored by the pH of the reaction medium. The application of 10 U.g-1 of the enzyme in the enzymatic maceration to extract the grape must, provided an 8% increase in yield after 2 hours at 55ºC, a decrease in total acidity and an increase in pH values, soluble solids, reducing sugars and of total polyphenols. The qualification of endoxylanase hydrolysis products was performed on beechwood xylan substrates and hemicellulose extracted from sugarcane bagasse. In 24 hours it was possible to identify X1, X2 and X5 as main products for beechwood xylan, the presence of X5 was related to the transglycosylation mechanism described in the literature. At the same time, for hemicellulose extracted from sugarcane bagasse, X1, X2 and X4 were identified. The difference between the products found is due to the structural constitution of the evaluated substrates.
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Palavras-chave
Endoxilanase., Cinética enzimática, Termodinâmica, Maceração enzimática, Xilooligossacarídeos, Endoxylanase, Enzyme kinetics, Thermodynamics, Enzymatic maceration, Xylooligosaccharides