Molecular biology of lignin-degrading enzymes from Phlebia radiata: Dissertation

The ligninolytic enzyme system of the white-rot fungus Phlebia radiata was studied in this thesis by methods of molecular genetics. Chromosomal and cDNA gene libraries were constructed from the fungus and the genomic and cDNA clones encoding one of the lignin peroxidases and the laccase of Phlebia radiata were isolated. The lignin peroxidase gene of Phlebia radiata is 60 % homologous to corresponding genes isolated from Phanerochaete chrysosporium, a related fungus. Putative active site regions of the lignin peroxidase were revealed by sequence comparison to yeast cytochrome c peroxidase. By hydrophobic cluster analysis (HCA) it was shown that the ligninolytic peroxidases of white rot fungi most probably have tertiary structures similar to the yeast cytochrome c peroxidase structure that has been solved by X-ray crystallography. The Phlebia radiata laccase gene shows a homology of 63 % to the laccase gene of Coriolus hirsutus and 30 % to the zucchini ascorbate oxidase that has a known three-dimensional structure. Thus the Phlebia laccase protein most probably folds into a three times repeated ß-barrel as does the ascorbate oxidase. Hypotheses concerning the copper binding residues and a region potentially carrying the cofactor pyrroloquinoline quinone (PQQ) in the active site of the laccase have been made on the basis of its homology to other oxidases. The lignin peroxidase and laccase genes isolated from Phlebia radiata were expressed in the softrot fungus Trichoderma reesei under the major cellulase gene (cbh1) promoter. No lignin peroxidase protein production from Trichoderma could be detected even though the respective mRNA was clearly observed in the transformant strains. The Phlebia radiata laccase was produced by Trichoderma reesei with peak concentrations of 20 mg/l from laboratory fermentations. Expression constructions prepared with the laccase cDNA or chromosomal gene yielded equal levels of expression. The laccase produced by Trichoderma was purified and shown to have equal molecular weight and specific activity to the laccase produced by Phlebia radiata. The ability of the heterologous laccase to remove soluble aromatic compounds from waste lignin derived from the pulping process was demonstrated.