Characterization of ulvan polysaccharide degrading enzymes from Wenyingzhuangia fucanilytica

Abstract: The aim of this thesis was to fully characterize the enzyme WFp-105, including bioinformatic analysis, structural modeling, full activity and stability characterization and product determination. As well as for the enzyme WFp-37; optimize protein production in regard to expression strains and cultivation temperature, analyzing the genomic context as well as performing an initial activity detection. From the genome of Wenyingzhuangia fucanilytica two enzymes were cloned, one glucoside hydrolase termed WFp-105 and one polysaccharide lyase termed WFp-37 attributed to polysaccharide lyase family 37. Both were heterologously produced in Escherichia coli and purified using affinity chromatography. WFp-37 showed activity on raw ulvan in the crude cell lysate. However, it was prone to aggregation as pure enzyme. The glucoside hydrolase attributed to glycoside hydrolase family 105 by gene phylogenetic analysis and structural modeling. Recombinant WFp-105 was purified to high concentrations and remained soluble. Enzyme demonstrated activity on unsaturated oligosaccharides from ulvan in the temperature range 10-40°C and pH 5-7.5. WFp-105 activity optima corresponded to algae containing ulvan growth conditions. Hydrolysis product detection with thin layer chromatography as well as product profiling with high-performance anion exchange chromatography - pulsed amperometric detection confirmed generation of monosaccharides from unsaturated oligosaccharides, in particular xylose, as well as three other products that was not successfully attributed to any mono- or oligosaccharide. Further investigation is needed in order to fully investigate potential of studied enzymes to be implemented for biotechnological applications.