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Research project (§ 26 & § 27)
Duration : 2018-12-01 - 2022-11-30

Wood cellulose is a future super material for replacement many fossil-based products. Modification of the wood-pulp is needed for the preparation of value added products. Enzymes are specific, renewable and biodegradable tools for modification of the pulps in mild reaction conditions. Recently discovered novel types lytic polysaccharide monooxygenases, are enzymes that oxidize cellulose in the crystalline parts, thus representing a novel type of enzyme activity with capability to modify the most recalcitrant celluloses. This project will explore the potential of LPMOs in oxidative modification of cellulosic fibres. The consortium brings together top-class expertise in enzymatic modification of pulp and fibre applications, LPMO enzymology and cellulose analytics
Research project (§ 26 & § 27)
Duration : 2018-12-05 - 2021-12-04

Despite the significant advantages that combining ion mobility mass spectrometry with all-ions QTOFMS, the highly complex nature of samples faced in metabolomics studies still poses great challenges for routine use of such workflows in metabolomics. One approach to address this limitation is the use of drift-time dependent quadrupole transmission profiles to facilitate a “bandpass” selection of precursor ions to be fragmented in the collision cell. In such a workflow, the transmission and/or collision energy applied in the high energy frame can be further directed by the drift separation (i.e. the quadrupole transmission is programmed to suit the drift times of relevant metabolites). This technology has enormous potential for metabolomics, but has not been tested or assessed with relevant compounds or real samples. In this project, we propose to investigate the use of continuous wide-band quadrupole isolation in combination with ion mobility separation to establish optimized acquisition settings and comprehensive datamining workflows with an outlook toward critical applications covering both identification and relative quantification in biological and environmental samples.
Research project (§ 26 & § 27)
Duration : 2018-01-01 - 2020-12-31

Chlorite dismutases (Clds) are able to efficiently decompose chlorite (ClO2-) into harmless chloride (Cl-) and dioxygen (O2) with chlorite being the sole source of dioxygen. Thereby, a covalent oxygen-oxygen bond is formed, a unique biochemical reaction that in addition is only catalyzed by the water-splitting manganese complex of photosystem II of oxygenic phototrophic organisms. The mechanism of cleavage of chlorite is still under heavy debate. Computational studies suggest homolytic cleavage, thereby producing chlorine monoxide (ClO●) and Compound II [Por…Fe(IV)=O] followed by a rebound step and release of chloride and dioxygen, whereas biochemical studies on pentameric (clade 1) Clds suggest heterolytic cleavage of chlorite thereby forming Compound I [Por+●…Fe(IV)=O] and hypochlorite (HOCl/-OCl). However, there is no experimental proof for the generation of Compound I nor is it known why these oxidoreductases have their pH optimum around pH 5 and are inactivated at alkaline pH. In order to elucidate structure-function relationships in Cld and understand the molecular basis of chlorite degradation, we have selected the dimeric Cld from Cyanothece sp. PCC7425 (CCld) as model enzyme for several reasons. CCld can easily be produced in E. coli, allows the generation of crystals of optimum size at all relevant pH values to be probed by both X-ray and neutron crystallography and for the first time showed distinct spectral features of typical heme b Compound II, which was formed immediately after mixing the ferric enzyme with chlorite. Moreover, CCld exhibits further enzymatic features that contradict the proposed heterolytic but favour the homolytic cleavage mechanism of chlorite. Thus it is the aim of this project to fully clarify the molecular mechanism of chlorite cleavage and O2 formation by using a broad set of biochemical and biophysical methods: (i) the recombinant production and purification of the wild-type protein and selected single mutants; (ii) characterization of these iron-proteins by various spectroscopic (resonance Raman, UV-vis) and electrochemical techniques, (iii) analysis of all individual reaction steps of enzyme cycle by multi-mixing stopped-flow spectroscopy and freeze-quench electron paramagnetic resonance (EPR) spectroscopy; and (iv) elucidation of X-ray and neutron crystal structures in the pH range 5-10. The work will be performed in close cooperation with internationally well-known scientists from Austria (X-ray crystallography: Kristina Djinovic-Carugo), Italy (RR spectroscopy: Giulietta Smulevich), Belgium (EPR spectroscopy: Sabine Van Doorslaer) and USA (neutron crystallography: Leighton Coates). Understanding structure-function relationships of Cld will provide the basis for its application in chemical engineering and bioremediation.

Supervised Theses and Dissertations