Estudo de hemicelulases através de RT-qPCR e expressão heteróloga
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Data
2021-06-07
Autores
Leonel, Tatiane Fernanda [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A produção de energia e de químicos renováveis a partir de resíduos agroindústrias de origens lignocelulósicas se tornou uma alternativa viável para minimizar os impactos causados pela utilização de combustíveis de origem fóssil. Porém, tal processo envolve altos custos, tendo em vista a recalcitrância da parede celular vegetal, o que dificulta a liberação de açúcares fermentáveis, fazendo-se necessário o uso de etapas de pré-tratamento e hidrólise frequentemente conduzidas em condições de concentração de reagentes químicos, temperatura e pressão elevadas, o que resultam na produção de metabólitos secundários e tóxicos indesejáveis. Assim, diversos estudos estão sendo realizados a fim de otimizar o processo de sacarificação da biomassa lignocelulósica, proporcionando um produto final menos oneroso e menos tóxico. Algumas enzimas de origens microbiológicas já estão sendo estudadas para serem utilizadas nessa etapa, sejam estas de função celulolítica, hemicelulolítica, lignocelulolítica ou mesmo auxiliar, porém a falta de eficiência, aliada ao alto custo de produção tem demandado a busca por novas moléculas para desconstrução da parede celular em formulações de coquetéis enzimáticos mais efetivos. Diante disto, o presente estudo teve por objetivo estudar duas hemicelulases da família GH2 de β-manosidases, obtidas por prospecção in silico e anotadas à partir do genoma da bactéria Chitinophaga sp. CB10 que foi isolada de um consórcio microbiano enriquecido a partir de uma pilha de bagaço de cana-de-açúcar. CB10 demonstrou ser uma fonte rica em carboidrases, com 350 domínios CAZyme previstos, o que pode explicar sua capacidade de se desenvolver a partir de carboidratos de diferentes complexidades estruturais: glicose, carboximetilcelulose, galactomanana, goma Aloe vera e bagaço de cana-de-açúcar. O bagaço da cana-de-açúcar é uma fonte rica em polímeros complexos, de forma que a diversidade de metabólitos liberados pela hidrólise enzimática destes polímeros tem um papel importante para a química verde, incluindo a hidrólise por vias enzimáticas minoritárias, como é o caso da degradação de conjugados de manana. Nesse sentido, CB10 demonstrou níveis de aumento consideráveis de expressão gênica para mananases na presença de bagaço de cana-de-açúcar como única fonte de carbono, quando comparado aos níveis de expressão em glicose (controle). Estes aumentos apresentaram respectivos níveis de 4,8x para o gene CB10-00347, localizado dentro de um agrupamento gênico identificado como “Polysaccharide Utilization Loci” (PUL), e 5,6x de aumento para o gene CB10-153.446, não associado a nenhum cluster enzimático. Essas enzimas compartilharam menos do que 45% de identidade com enzimas caracterizadas do gênero Chitinophaga pertencente ao filo Bacteroidetes. O grau de novidade - conforme demonstrado pela baixa identidade com enzimas caracterizadas anteriormente; a notável capacidade de crescer em diferentes substratos; a confirmação de atividade de mananase, evidenciada pela liberação de oligossacarídeos residuais no cultivo com galactomanano (HPLC-RID, 12,3 mMol); associados à capacidade de expressar mananases em uma baixa concentração de condições indutoras (bagaço-de-cana contém cerca de 0,2% de manose) indicam o alto potencial para a aplicação de CB10 como fonte de enzimas na produção de oligossacarídeos conjugados de manose a partir de biomassa. Além destes resultados, grandes esforços foram empreendidos com o intuito de compreender melhor a atividade de uma das enzimas preditas para β-manosidase (ORF CB10_153.446) utilizando ensaios de expressão heteróloga de proteína recombinante. Entretanto, apesar de diversas tentativas de otimização, não houve êxito na estratégia de expressão proteica em E. coli Bl21 DE3, sendo necessários novos estudos para usufruir do potencial biotecnológico demostrado pelas demais técnicas utilizadas, visando aplicação biotecnológica em biorrefinarias de precursores pré-bióticos e outros oligossacarídeos funcionais focados no indústrias alimentícias e farmacêuticas
The production of renewable energy and chemicals from agribusiness residues of lignocellulosic origins has become a viable alternative to minimize the impacts caused by the use of fossil fuels. However, this process involves high costs, in view of the recalcitrance of the plant cell wall, which hinders the release of fermentable sugars, requiring the use of pre-treatment and hydrolysis steps often carried out under conditions of concentration of chemical reagents’, elevated temperature and pressure, which result in the production of undesirable secondary and toxic metabolites. Thus, several studies are being carried out in order to optimize the process of saccharification of lignocellulosic biomass, providing a less costly and less toxic final product. Some enzymes of microbiological origins are already being studied to be used at this stage, whether they have cellulolytic, hemicellulolytic, lignocellulolytic or even auxiliary functions, but the lack of efficiency, combined with the high cost of production has demanded the search for new molecules to deconstruct the cell wall in more effective enzyme cocktail formulations. Therefore, this study aimed to study two hemicellulases of the GH2 family of β-mannosidases, obtained by in silico prospection and annotated from the genome of the bacterium Chitinophaga sp. CB10 which was isolated from an enriched microbial consortium from a pile of sugarcane bagasse. CB10 proved to be a rich source of carbohydrates, with 350 predicted CAZyme domains, which may explain its ability to develop from carbohydrates of different structural complexities: glucose, carboxymethylcellulose, corn starch, galactomannan, Aloe vera gum and sugarcane bagasse -of sugar. Sugarcane bagasse is a rich source of complex polymers, so the diversity of metabolites released by the enzymatic hydrolysis of these polymers plays an important role in green chemistry, including hydrolysis by minor enzymatic pathways, as is the case of the degradation of mannan conjugates. In this sense, CB10 showed considerable levels of increased gene expression for mannanases in the presence of sugarcane bagasse as the only source of carbon, when compared to expression levels in glucose (control). These increases showed respective levels of 4.8x for the CB10-00347 gene, located within a gene cluster identified as "Polysaccharide Utilization Loci" (PUL), and 5.6x increase for the CB10-153.446 gene, not associated with any enzyme cluster. These enzymes shared less than 45% identity with characterized enzymes from the genus Chitinophaga belonging to the phylum Bacteroidetes. The degree of novelty - as demonstrated by the low identity with previously characterized enzymes; the remarkable ability to grow on different substrates; confirmation of mannanase activity, evidenced by the release of residual oligosaccharides in culture with galactomannan (HPLC-RID, 12.3 mMol); associated with the ability to express mannanases in a low concentration of inducing conditions (cane bagasse contains about 0.2% of mannose) indicate the high potential for the application of CB10 as an enzyme source in the production of oligosaccharides conjugated from mannose to from biomass. In addition to these results, great efforts were undertaken in order to better understand the activity of one of the predicted enzymes for β-mannosidase (ORF CB10_153.446) using heterologous recombinant protein expression assays. However, despite several optimization attempts, the protein expression strategy in E. coli Bl21 DE3 was not successful, and further studies are needed to take advantage of the biotechnological potential demonstrated by the other techniques used, aiming at biotechnological application in biorefineries of prebiotic and xii precursors other functional oligosaccharides focused on the food and pharmaceutical industries.
The production of renewable energy and chemicals from agribusiness residues of lignocellulosic origins has become a viable alternative to minimize the impacts caused by the use of fossil fuels. However, this process involves high costs, in view of the recalcitrance of the plant cell wall, which hinders the release of fermentable sugars, requiring the use of pre-treatment and hydrolysis steps often carried out under conditions of concentration of chemical reagents’, elevated temperature and pressure, which result in the production of undesirable secondary and toxic metabolites. Thus, several studies are being carried out in order to optimize the process of saccharification of lignocellulosic biomass, providing a less costly and less toxic final product. Some enzymes of microbiological origins are already being studied to be used at this stage, whether they have cellulolytic, hemicellulolytic, lignocellulolytic or even auxiliary functions, but the lack of efficiency, combined with the high cost of production has demanded the search for new molecules to deconstruct the cell wall in more effective enzyme cocktail formulations. Therefore, this study aimed to study two hemicellulases of the GH2 family of β-mannosidases, obtained by in silico prospection and annotated from the genome of the bacterium Chitinophaga sp. CB10 which was isolated from an enriched microbial consortium from a pile of sugarcane bagasse. CB10 proved to be a rich source of carbohydrates, with 350 predicted CAZyme domains, which may explain its ability to develop from carbohydrates of different structural complexities: glucose, carboxymethylcellulose, corn starch, galactomannan, Aloe vera gum and sugarcane bagasse -of sugar. Sugarcane bagasse is a rich source of complex polymers, so the diversity of metabolites released by the enzymatic hydrolysis of these polymers plays an important role in green chemistry, including hydrolysis by minor enzymatic pathways, as is the case of the degradation of mannan conjugates. In this sense, CB10 showed considerable levels of increased gene expression for mannanases in the presence of sugarcane bagasse as the only source of carbon, when compared to expression levels in glucose (control). These increases showed respective levels of 4.8x for the CB10-00347 gene, located within a gene cluster identified as "Polysaccharide Utilization Loci" (PUL), and 5.6x increase for the CB10-153.446 gene, not associated with any enzyme cluster. These enzymes shared less than 45% identity with characterized enzymes from the genus Chitinophaga belonging to the phylum Bacteroidetes. The degree of novelty - as demonstrated by the low identity with previously characterized enzymes; the remarkable ability to grow on different substrates; confirmation of mannanase activity, evidenced by the release of residual oligosaccharides in culture with galactomannan (HPLC-RID, 12.3 mMol); associated with the ability to express mannanases in a low concentration of inducing conditions (cane bagasse contains about 0.2% of mannose) indicate the high potential for the application of CB10 as an enzyme source in the production of oligosaccharides conjugated from mannose to from biomass. In addition to these results, great efforts were undertaken in order to better understand the activity of one of the predicted enzymes for β-mannosidase (ORF CB10_153.446) using heterologous recombinant protein expression assays. However, despite several optimization attempts, the protein expression strategy in E. coli Bl21 DE3 was not successful, and further studies are needed to take advantage of the biotechnological potential demonstrated by the other techniques used, aiming at biotechnological application in biorefineries of prebiotic and xii precursors other functional oligosaccharides focused on the food and pharmaceutical industries.
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Palavras-chave
RT-qPCR, Bagaço, Cana de açúcar