Publicação
Protein engineering of xynalase XYN10C from Paenibacillus barcinonensis
| Resumo: | Paenibacillus barcinonensis Xyn10C is a modular xylanase composed of an N-terminal tandem CBM22 module, a catalytic domain belonging to GH10 and a C-terminal tandem CBM9 module. The enzyme was previously cloned and a preliminary characterization was performed. In our work several truncated enzymes, containing different combinations of the modules of the xylanase have been constructed, purified and characterized. The activity assays here performed show that the complete enzyme (wild type Xyn10C) and a truncated variant containing only the GH10 catalytic domain are highly active on arabinoxylans, commonly found in cereal hemicelluloses, while show lower activity on glucuronoxylans, typical of hardwood species. Additionally, the diminished activity of the isolated GH10 domain on some arabinoxylans indicate that the CBM22 and CBM9 ancillary tandem domains play a role for the degradation these substrates, probably facilitating hydrolysis to the less accessible insoluble fraction of arabixoxylans. This is strongly supported by the fact that P. barcinonensis was isolated from a rice field. The microcrystalline binding capacity of Xyn10C was also investigated. The results here obtained indicate that the enzyme has binding capacity to insoluble cellulose and that this capacity is mediated through CBM9b, the external domain of the CBM9 tandem, which has been already reported for other enzymes with similar architecture. Together these results point Xyn10C as a potential candidate for application in agro-industrial processes that involve the degradation of straw and cereal biomass, a frequent by product of agriculture, such as bioethanol production, providing at the same time more insights on the biology of modular carbohydratases and on their contribution to xylan degradation in natural habitats. |
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| Autores principais: | Campos, André Nunes, 1989- |
| Assunto: | Biotecnologia Enzimas Xilanos Paenibacillus barcinonensis Teses de mestrado - 2011 |
| Ano: | 2011 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Paenibacillus barcinonensis Xyn10C is a modular xylanase composed of an N-terminal tandem CBM22 module, a catalytic domain belonging to GH10 and a C-terminal tandem CBM9 module. The enzyme was previously cloned and a preliminary characterization was performed. In our work several truncated enzymes, containing different combinations of the modules of the xylanase have been constructed, purified and characterized. The activity assays here performed show that the complete enzyme (wild type Xyn10C) and a truncated variant containing only the GH10 catalytic domain are highly active on arabinoxylans, commonly found in cereal hemicelluloses, while show lower activity on glucuronoxylans, typical of hardwood species. Additionally, the diminished activity of the isolated GH10 domain on some arabinoxylans indicate that the CBM22 and CBM9 ancillary tandem domains play a role for the degradation these substrates, probably facilitating hydrolysis to the less accessible insoluble fraction of arabixoxylans. This is strongly supported by the fact that P. barcinonensis was isolated from a rice field. The microcrystalline binding capacity of Xyn10C was also investigated. The results here obtained indicate that the enzyme has binding capacity to insoluble cellulose and that this capacity is mediated through CBM9b, the external domain of the CBM9 tandem, which has been already reported for other enzymes with similar architecture. Together these results point Xyn10C as a potential candidate for application in agro-industrial processes that involve the degradation of straw and cereal biomass, a frequent by product of agriculture, such as bioethanol production, providing at the same time more insights on the biology of modular carbohydratases and on their contribution to xylan degradation in natural habitats. |
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