Publicação
Glycanzyme: characterisation of novel enzymes for protein glycoengineering
| Resumo: | One of the most common protein post-translational modifications occurring in eukaryotes is glycosylation, which is critical for several biological processes. There are different enzymes involved in the processing of N-glycans in eukaryotes, among them endo-β-N-acetylglucosaminidases (ENGases), whose physiological roles are still poorly understood. ENGases of the glycoside hydrolase (GH) family 85 are a class of enzymes (EC 3.2.1.96) that, in addition to hydrolytic activity against the diacetylchitobiose core of N-glycans, can also display transglycosylation activity. Given their usefulness for the analysis and glycoengineering of glycopeptides and glycoproteins, these enzymes have become a focus of biotechnological interest. Envisioning the identification of novel GH85 ENGases with useful action, this thesis focused on the characterization of two new putative ENGases of this family, one from the filamentous fungus Ashbya gossypii (AgENGase) and another from the yeast Zygosaccharomyces rouxii (ZrENGase). Previous experimental results hinted at the existence of ENGase activity in these organisms and in their genome one homologue gene exists that should code for a putative GH85 ENGase (AFR597W in A. gossypii and ZYRO0B07216g in Z. rouxii). Bioinformatic approaches were initially used to assess the potential activity of these putative ENGases and important residues for the hydrolytic and transglycosylation activity were found to be conserved in AgENGase. Only the putative 3D model of ZrENGase presented the typical (β8/α8)-TIM-barrel structure of ENGases. To characterise these proteins, their coding genes were cloned into an Escherichia coli expression plasmid and their recombinant production was optimized by testing different E. coli expression strains, production media and induction conditions. However, the recombinant proteins produced remained mainly in the insoluble fraction after cell lysis and further improvements in their production and purification will be required to generate enough protein for activity studies and eventual application. Nevertheless, different protocols were already optimized in the scope of this thesis for the future evaluation of the deglycosylation activity of these recombinant ENGases. Considering the bioinformatic analysis and the results from enzymatic assays performed in crude cell extracts of wild A. gossypii, the AgENGase action could be located in the intracellular space. Envisioning the understanding of the physiological role of the putative AgENGase, a gene with no homolog in Saccharomyces cerevisiae and with unknown function, the disruption of the A. gossypii AFR597W was also performed. Thereby, AgENGase was found to be important for sporulation, as the generated afr597w mutants lost the ability to sporulate. Nevertheless, further studies will be needed to fully understand the physiological and enzymatic function of this enzyme. |
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| Autores principais: | Baptista, Marlene Isabel Cerqueira Montenegro |
| Assunto: | Ashbya gossypii endo-β-N-acetylglucosaminidases Recombinante protein production Produção de proteínas recombinantes Engenharia e Tecnologia |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | One of the most common protein post-translational modifications occurring in eukaryotes is glycosylation, which is critical for several biological processes. There are different enzymes involved in the processing of N-glycans in eukaryotes, among them endo-β-N-acetylglucosaminidases (ENGases), whose physiological roles are still poorly understood. ENGases of the glycoside hydrolase (GH) family 85 are a class of enzymes (EC 3.2.1.96) that, in addition to hydrolytic activity against the diacetylchitobiose core of N-glycans, can also display transglycosylation activity. Given their usefulness for the analysis and glycoengineering of glycopeptides and glycoproteins, these enzymes have become a focus of biotechnological interest. Envisioning the identification of novel GH85 ENGases with useful action, this thesis focused on the characterization of two new putative ENGases of this family, one from the filamentous fungus Ashbya gossypii (AgENGase) and another from the yeast Zygosaccharomyces rouxii (ZrENGase). Previous experimental results hinted at the existence of ENGase activity in these organisms and in their genome one homologue gene exists that should code for a putative GH85 ENGase (AFR597W in A. gossypii and ZYRO0B07216g in Z. rouxii). Bioinformatic approaches were initially used to assess the potential activity of these putative ENGases and important residues for the hydrolytic and transglycosylation activity were found to be conserved in AgENGase. Only the putative 3D model of ZrENGase presented the typical (β8/α8)-TIM-barrel structure of ENGases. To characterise these proteins, their coding genes were cloned into an Escherichia coli expression plasmid and their recombinant production was optimized by testing different E. coli expression strains, production media and induction conditions. However, the recombinant proteins produced remained mainly in the insoluble fraction after cell lysis and further improvements in their production and purification will be required to generate enough protein for activity studies and eventual application. Nevertheless, different protocols were already optimized in the scope of this thesis for the future evaluation of the deglycosylation activity of these recombinant ENGases. Considering the bioinformatic analysis and the results from enzymatic assays performed in crude cell extracts of wild A. gossypii, the AgENGase action could be located in the intracellular space. Envisioning the understanding of the physiological role of the putative AgENGase, a gene with no homolog in Saccharomyces cerevisiae and with unknown function, the disruption of the A. gossypii AFR597W was also performed. Thereby, AgENGase was found to be important for sporulation, as the generated afr597w mutants lost the ability to sporulate. Nevertheless, further studies will be needed to fully understand the physiological and enzymatic function of this enzyme. |
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