Identification, characterization and engineering of glutathione-dependent, lignin-degrading enzymes

Lignin is one of the most abundant polymers in nature. Due to this it is a very interesting source for the provision of aromatic platform chemicals and biofuels. Until now, however, lignin is not industrially used since the required lignin depolymerization is very challenging. Beta-etherases and glutathione lyases are enzymes with proven potential in depolymerizing lignin and one possible solution for this problem. However, the number of these enzymes is still rather limited. Using classical public database mining in combination with the peptide pattern recognition algorithm, 96 putatively novel beta-etherases have been identified in this thesis, some even originating from bacteria outside the order Sphingomonadales. Of these, a subset of 13 enzymes was chosen for further biochemical characterization. All tested enzymes are active beta-etherases and some enzymes displayed up to three-fold higher activity compared to the previously known beta-etherases. Furthermore, the sequence diversity within beta-etherase family was increased and detailed information about conserved sequence areas of beta-etherases was generated. In combination with performed structural analyses, this is a great step in understanding the beta-etherase mechanism and to identify amino acids essential for their activity. Moreover, through phylogenetic analyses an interesting clustering of beta-etherases was revealed. The gained phylogenetic information will likely also lead to the identification of new types of lignin-degrading beta-etherases in the future. The second part of this thesis dealt with the optimization of glutathione lyase LigG-TD. First, a high-throughput assay for activity screening of glutathione lyases was developed. This assay is based on the NADPH-dependent, glutathione reductase-catalyzed reduction of oxidized glutathione GSSG, one product of the glutathione lyase reaction. Hence, based on the consumption of NADPH the glutathione lyase activity is indirectly measured. This assay was subsequently applied in a protein engineering campaign to increase the activity of the glutathione lyase LigG-TD. Upon screening six site-saturation mutagenesis libraries, several LigG-TD mutants with improved activity could be identified. Overall, with only one round of protein engineering the activity of LigG-TD was increased by 20%. The developed screening assay can be also applied for the protein engineering of other glutathione lyases in order to increase their activity or stability.