Phenoloxidases are absolutely essential for lignin degradation by white-rot fungi. The known phenoloxidases functioning in the process are lignin peroxidase (LiP), manganese peroxidase (MnP), or laccase. Phanerochaete chrysosporium, the model organism for studies of the enzyme mechanisms involved in lignin degradation, produces multiple forms of both LiP and MnP, but laccase is not used as part of this ligninolytic system. It is now becoming evident that the majority of white-rot fungi operate with a different set of phenoloxidases, i.e., laccase in combination with either LiP or MnP or even all three together. To investigate the role of laccase for lignin degradation Pycnoporus cinnabarinus was identified as an excellent organism for further study because it produced only one isoelectric form of laccase. To investigate the importance of laccase, the classical approaches of creating and identifying mutants, i.e., laccase-less (lac-) and peroxidase-less (per-), as well as pleiotropic (i.e., lac-per-), mutants will be used. Substantial efforts will also be expended for the physico-chemical and spectroscopic characterization of the laccase. Materials and procedures earlier developed in this laboratory for cloning of plant laccase genes, will be used to clone the genomic DNA sequence encoding the P. cinnabarinus laccase. This research will provide information on the role of fungal laccases in lignin degradation to be compared with that of the well studied P. chrysosporium system. %%% The oil crisis during the 1970's turned interest towards the utilization of renewable resources, and towards lignocellulosics, in particular. Lignocellulosic materials are a source of fiber, energy and liquid fuels. However, the technologies presently used to take advantage of this raw material are mostly energy consuming and not always environmentally friendly. Since one of nature's most important biological reactions is the degradation of wood and other lignocellulosic material, biotechnical methods for utilization of lignocellulosic materials from agriculture and forestry are both possible and important to develop. Efficient ways to degrade lignin are particularly important in this context. White-rot fungi are the only microorganisms which can degrade lignin to any extent. To efficiently use these organisms for a better utilization of lignocellulosic materials requires an incisive knowledge of the enzyme mechanisms they utilize in the lignin degradation process. The approach we will take in our proposed research will particularly provide information on the role of laccase, i.e., whether or not this is one of the necessary phenoloxidases for lignin degradation.