Computational and experimental studies on lignin converting enzymes

As a component of lignocellulose, lignin is one of the most abundant aromatic polymers on Earth. Its utilization is therefore considered a valuable alternative to the synthesis of aromatics from mineral oil. Glutathione-dependent β-etherases can cleave β-O-4 aryl ether bonds, which are the most common type of bond found in lignin. Due to their non-radical mechanism, these enzymes show promise as biocatalysts for the selective depolymerization of lignin polymers and oligomers, and for the production of aromatic platform chemicals from renewable raw materials. The first part of this work focused on investigating the catalytic mechanism of β-etherases and identifying important amino acid residues in the active site. Using computer-aided methods, various substrates were first docked into the active site of LigE and LigF, which then served as starting structures for molecular dynamics simulations. During the simulation, parameters were monitored that enabled a nucleophilic attack of the co-substrate glutathione on the beta-carbon of the substrate. For LigF, it was shown that the nucleophilic attack can take place. In addition, those amino acids that interact with the substrate during the simulation were experimentally subjected to an alanine scan in order to estimate the influence of the individual amino acids. Kinetic studies showed that the amino acids W115 and W148 are essential for catalysis, while S111, which is involved in the protonation of the leaving group, is of great importance but not essential. In addition, the simulation revealed a flexible helix-turn-helix motif, from which two proline residues were also replaced by alanine. The substitution of residue P142 led to significantly increased activity, probably due to the influence of increased movement of the domain. Also, LigE was investigated for its catalytic key residues. The results revealed that the crystal structure of LigE corresponds to a catalytic inactive form of the enzyme due to a truncation of 27 amino acids at the C-terminus. The second part of this thesis focused on heterologous production of unspecific peroxygenases and their potential role in lignin degradation. First, the expression of different potential peroxygenase genes in N. crassa and E. coli was investigated. No UPOs could be produced using the N. crassa production system. The situation was different for E. coli. With the help of co-produced chaperones, the short-type UPO from Botryobasidium botryosum could be produced in E. coli. Subsequent biochemical characterization of BboUPO revealed that this enzyme can catalyze numerous reactions and function over a wide pH range. Furthermore, BboUPO tolerates solvents such as acetonitrile and acetone up to a concentration of 10%. Due to its relatively large active site, BboUPO presumably prefers larger substrates than the typical UPO model substrates Finally, the involvement of UPOs in lignin depolymerization was investigated. The tested UPOs were examined using both lignin model substrates and a lignin oligomer mixture. The results showed that UPOs both demethylate and hydroxylate the model substrates. Lignin oligomers were also converted by UPOs to a certain extent, but this did not correspond to depolymerization. Future studies could combine UPOs with other lignin-degrading enzymes to contribute to the valorization of lignin.