Advanced morphologic and spectroscopic characterization of immobilized enzymes and surface analysis

This research is part of the EU-ITN INTERfaces project and it is focused on the development of novel methodologies for the advanced characterization and rational optimization of immobilized enzymes. The approach involved the correlation between the physical and chemical properties of the carriers with the conformational and experimental behavior of the enzyme. All activities were performed at the University of Trieste, EnginZyme (Stockholm) and ELLETRA Sincrotrone (Trieste). The results contributed to the establishment of optimal protocols for immobilized transaminases for industrial use. The first part of the study was dedicated to the characterization of porous glass carriers (EziG) and four immobilized ω-transaminases that were cloned and expressed in the laboratory. FTIR microspectroscopy, confocal microscopy, and μ-ATR FTIR spectroscopy enabled to visualize the enzyme distribution in the carrier. The spectroscopic methods were also used for describing the conformational modifications of the enzymes upon immobilization and after exposure to solvents. Molecular dynamics were carried out to simulate the conformations of the free enzymes in explicit organic solvents (toluene, 2-methyltetrahydrofuran, dibutyl ether, pinacolone). The second part of the project was focused on evaluating the impact of different post-immobilization treatments, which had the aim of achieving optimally hydrated immobilized transaminases for applications in organic solvents. The measurement of water activity was carried out in the reaction systems, allowing for a comparison of enzyme performance across different conditions. The investigation of 70 different enzymatic formulations provided data in accordance with the spectroscopic analyses, indicating a correlation between the performance of the immobilized enzymes and the pre-treatment protocols. In the third part of the thesis, the enzymatic synthesis of bio-based aromatic polyamides was explored. Two different microbial lipases, either native or immobilized, were tested in solvent-free and organic solvent media. The polymerization process was successful in both cases, although leading to low molecular weight oligomers as a consequence of the high viscosity and low solubility of furan derivatives. FTIR, ESI MS, and NMR, confirmed the formation of the polyamides that were used in the investigation of the biodegradability of furan-based polyamides in marine environment.