Non-invasive and real-time recording of processes in living cells has been limited to detection of small cellular components such as soluble proteins and metabolites. Here we report a multiphase... Show moreNon-invasive and real-time recording of processes in living cells has been limited to detection of small cellular components such as soluble proteins and metabolites. Here we report a multiphase NMR approach using Magic-Angle Spinning NMR to synchronously follow microbial processes of fermentation, lipid metabolism and structural dynamic changes in live microalgae cells. Chlamydomonas reinhardtii green algae were highly concentrated, introducing dark fermentation and anoxia conditions. Single-pulse NMR experiments were applied to obtain temperature-dependent kinetic profiles of the formed fermentation products. Through dynamics-based spectral editing NMR, simultaneous conversion of galactolipids into TAG and free fatty acids was observed and rapid loss of rigid lipid structures. This suggests that lipolysis under dark and anoxia conditions finally results in the breakdown of cell and organelle membranes, which could be beneficial for recovery of intracellular microbial useful products. Show less
Since the discovery of enzymes as biological catalysts, the mechanism of enzymatic reactions has been a key question to enzymologists. Elucidating the reaction kinetics and the nature of enzymatic... Show moreSince the discovery of enzymes as biological catalysts, the mechanism of enzymatic reactions has been a key question to enzymologists. Elucidating the reaction kinetics and the nature of enzymatic intermediates are necessary to understand such reactions. An important challenge in these studies is the limited lifetime of such intermediates, usually on the time scale of milliseconds to seconds. Therefore, a suited trapping method is required. Rapid freeze-quench (RFQ) is a proven technique to trap the intermediates on the time scale of milliseconds. The RFQ technique has been mostly combined with electron paramagnetic resonance (EPR) spectroscopy to reveal the nature of the paramagnetic intermediates involved in enzymatic reactions. In this thesis, we improve and extend the combination of conventional RFQ technique with EPR spectroscopy up to a microwave frequency of 275 GHz to trap and characterize the intermediates involved in the enzymatic reduction of O2 by small laccase (SLAC). Show less
