Project Title: Elucidation of the oxidative mechanism of lytic polysaccharide monooxygenases
Supervisor: Prof Morten Sørlie, Deparment of Chemistry, Norwegian University of Life Sciences
Objectives: The main objectives are the elucidation of structural determinants of substrate specificity, oxidative regioselectivity, and industrial performance of lytic polysaccharide monooxygenases (LPMO). The study of LPMOs and the exploitation of these powerful enzymes in industrial applications is only in its infancy. Enzymatic degradation of recalcitrant polysaccharides is thought to occur through the synergistic action of glycoside hydrolases (GHs) that have complementary activities. Endo-acting GHs make random scissions on the polysaccharide chains, whereas exo-acting processive GHs mainly target single reducing and non-reducing chain ends. LPMOs targets crystalline regions using an activated dioxygen to cleave glycosidic bonds, creating new chain ends for exo-acting GHs and by this greatly enhances rate of depolymerization. A key goal is to determine conditions that allows for maximum rate enhancement through a detailed understanding of the LPMO mechanism.
Methodology: The work will involve various molecular biology, biochemical, and physicochemical techniques. Enzymes can be expressed both in bacteria (i.e. Escherichia coli) as well as in fungi (i.e. Pichia pastoris). Enzyme kinetics will be assessed using stopped-flow spectrophotometry and calorimetry. Advanced mass spectrometry and liquid chromatography will be central in the determination of substrate and product profiles, optimal co-substrate delivery, and ideal conditions when interplaying with GHs. Moreover, the use of protein crystallography and spectroscopy, i.e. electron paramagnetic resonance among others, allows for structure – function correlations.
Expected Results: (i) Detailed characterization of selected LPMOs by physicochemical and analytical chemical analyses. (ii) Optimized conditions for LPMO action from laboratory to pilot scale.