Laccases and other ligninolytic enzymes of the basidiomycetes Coprinopsis cinerea and Pleurotus ostreatus

Abstract

Organisms produce ligninolytic enzymes in order to degrade lignocellulosic materials. Mainly, such enzymes are found in the fungal kingdom and are particularly widespread in the phylum Basidiomycota. The ligninolytic system of fungi consists of peroxidases (manganese peroxidases - MnP, lignin peroxidases - LiP and versatile peroxidases - VP) and laccases which for substrate degradation are secreted by the fungus into the environment. Laccases as main representative of the ligninolytic system can be used for delignification and bleaching in the pulp and paper industry, in the production of wood composites and to alter wood properties. As production and biochemical characterisations of laccases are in the focus of this PhD thesis, the question of how to produce large and stable amounts of this enzyme was a driving force for the studies. Laccases and other ligninolytic enzymes can be produced by cultivating adequate fungi either in submerged fermentation (SmF) or solid state fermentation (SSF). A short introduction to both fermentation techniques is presented in the study with regard to enzymes which are relevant for the wood processing industry.As an example for SSF, the production of ligninolytic enzymes of the white-rot fungus Pleurotus ostreatus during cultivation on wheat straw was experimentally studied. In comparison to SSF, SmF is easier to handle, better reproducible and normally used in industrial production of fungal enzymes and other metabolites. Therefore in this thesis, SmF is discussed in more detail. In liquid cultures during SmF, filamentous fungi can grow in free filamentous or in aggregated forms, known as pellets. The fungus used for liquid cultivation in this PhD thesis, the basidiomycete Coprinopsis cinerea, grew in a pellet like form in all tested liquid fermentation systems (shaken flasks, bioreactor). The study contains detailed analyses of the morphology of C. cinerea in SmF, which give an overview on the influence of different cultivation parameters (pH, temperature, fermentation system) on the growth type of C. cinerea. To analyse native production of laccases in C. cinerea, ten different monokaryotic strains of this fungus were tested and the isoenzyme patterns of the highest laccase producers were analysed. Further experiments were conducted for recombinant production of a single laccase isoenzyme in C. cinerea and to improve the recombinant production of laccase by cultivation parameters. Thereby, in addition to the already known C. cinerea laccase Lcc1, three C. cinerea laccases (Lcc5, Lcc6 and Lcc7) could be produced in bigger amounts. The so produced laccases were purified and characterised according to molecular and biochemical aspects. Thus, a broad picture is given in this thesis starting from the cultivation of basidiomycetes in either SmF or SSF, the production of native and recombinant laccases and their characterisation.