Browsing by Subject "vierasproteiinien tuotto"
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(2018)Glucuronoyl esterase (GE) is an enzyme produced by plant biomass degrading basidiomycete and ascomycete fungi. GEs have been found to hydrolyse a specific ester bond between lignin alcohols and xylan hemicellulose, which is one of the crosslinks in plant biomass. GEs are the only known enzymes capable of hydrolysing both glycosidically bound and free methyl esters of 4-O-methyl-D-glucuronic acid. In biodegradation of plant biomass, fungal GEs are assumed to cleave the bonds between aromatic lignin alcohols and 4-O-methyl-D-glucuronic acid of hemicellulose. Due to these characteristics, GEs are promising candidates for future industrial applications, such as biofuel production. The genome of white-rot bacidiomycete Dichomitus squalens contains two GE encoding genes. The aim of this work was to express one wild-type GE from D. squalens, DsGE1, and its site-directed mutants as recombinant proteins in the yeast Pichia pastoris. The catalytic activity of the wild-type and mutated recombinant DsGE1 enzymes was examined in optimum conditions with synthetic benzyl glucuronate as a substrate. Recombinant GEs were then exposed to varying of temperature and pH values as well as organic solvents. The aim was to assess whether the mutated DsGE1 enzymes retained a higher GE activity than the wild type enzyme in the tested conditions. Hypothesis of this work was that mutated recombinant DsGE1 enzymes catalyse the hydrolysis of a synthetic benzyl glucuronase. The mutated enzymes were expected to have improved thermal resistance in temperatures higher to the optimum when compared to the wild-type GE. In addition, another hypothesis was that the DsGE1 mutants retain their specific activity better than the wild-type enzyme when pH changes from optimum as well as when the enzymes are exposed to organic solvents. The first research hypothesis was confirmed, while the results of the work disproved the following two hypotheses.
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(2020)Usp45 is the major secreted protein in Lactococcus lactis. Protein’s 27-aa signal peptide (SPUsp45) is widely used for increased secretion and improved yields of heterologous proteins. L. lactis, previously used mainly in food production, has gained increasing popularity in gene technology and, thanks to extensive research, became a Gram-positive model organism alongside Bacillus subtilis. Despite the widespread use of its signal peptide, the biological role of Usp45 protein remains largely a mystery. The aim of this study was to test whether decreased translation of the naturally highly secreted Usp45 protein would lead to improved secretion of desired heterologous proteins. The hypothesis was that high levels of secreted Usp45 cause the Sec translocon becoming a bottleneck and by reducing this strain on the secretion route, the capacity to secrete other proteins could increase. Based on literature, usp45 is not part of the L. lactis core genome and was assumed to be non-vital. To study this, the L. lactis strain NZ9000 was transformed with the plasmid pLEB805 which contains a nisin inducible antisense-usp45 gene (ASusp45) resulting in strain LAC455. The effect of antisense-RNA mediated silencing on growth and morphology of the cells was observed as well as the changes in quantity and quality of secreted proteins in ASusp45 induced cells. The secretion of heterologous proteins was tested with bacteriocins leucocin A and C that were introduced to the cells in expression vectors. This study brought new information on the function of the usp45 gene in L. lactis. The results show that the silencing of the usp45 gene leads to retarded growth rate, multifold ingrowth of the cell wall, aggregation of the cells and the leakage of cytoplasmic proteins leading to loss of viability of the cells. These results demonstrate that Usp45 is vital for the structure of the cell wall, cell separation and normal chain formation, and it probably acts as a vital peptidoglycan hydrolase.
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