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Browsing by Author "Tuomiranta, Petra"

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  • Tuomiranta, Petra (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.