Our long term objective of this proposal is the utilization of ligninases in the detoxification of environmental pollutants and biotreatment of hazardous waste. Success in this objective will have unmeasurable effects upon the health of the human race. The major obstacle to the assessment studies of the potential use of ligninases for these purposes is the limited availability of these enzymes. Thus our specific aims are to (1) determine, identify, clone and sequence most if not all the ligninase genes in Phanerochaete chrysosporium, (2) determine the effect of glycosylation inhibitors on ligninase activity, (3) overproduce the cloned ligninase gene products in several eucaryotic expression systems. cDNA library will be constructed in phage vector gt10 with mRNAs isolated from P. chrysosporium grown in liquid culture under conditions of nitrogen limitation. Positive plaques are identified with either polyclonal antibody against ligninase isozyme H8 or by differential hybridization with specific synthetic oligonucleotide probes. The ligninase gene clones will then be sequenced by the M13 dideoxy nucleotide chain termination method and their sequences compared. Each of the cloned ligninase will be excised and then subcloned into the multiple cloning sites of several eukaryotic expression vectors and baculovirus. Seven different expression strategies will be applied to overproduce these cloned ligninases in yeast, mammalian cell and insect cell cultures in the presence and absence of glycosylation inhibitors (e.g. 2-deoxy-D- glucose and tunicamycin). The overexpressed ligninases will be concentrated and then purified by FPLC and immunoaffinity columns. Purified ligninases will be characterized biologically and biochemically and then compare with the extracellular ligninases isolated from the culture fluid. The ability of these ligninases singly or in combination as pulping agents and as biodegradative agents of environmental pollutants such as pentachlorophenol, PCBs, DDt, etc. will then be assessed.
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