The lignin-degrading white rot fungus Phanerochaete chrysosporium secretes a family of lignin-degrading enzymes which can be divided into two classes according to their catalytic activities. The lignin peroxidases catalyze the oxidation of a large number of non-phenolic aromatic substrates. The Mn peroxidases catalyze the oxidation of Mn (II) to Mn (III). Mn (III), in turn, can oxidize a large number of phenolic substrates. The high redox potential of the lignin and Mn peroxidases confers them with the ability to oxidize recalcitrant organopollutants. the first objective of this study is to characterize how the proteins control heme reactivity in these two peroxidases. We will use kinetic techniques in addition to using modern methods of recombinant DNA technology. We have the isolated cDNA for both of these enzymes and will use the insect tissue culture expression system to synthesize recombinant protein. These studies will not only allow us to understand the basis of catalysis but also permit attempt to alter substrate specificity. We will use molecular modeling of these enzymes to predict which residues will alter specificity. The second objective of this proposal is to develop strains of Phanerochaete chrysosporium which overproduce the lignin and Mn peroxidases. The selection procedure is based on the use of lignin peroxidase-dependent release of amino acids from an adduct of the amino acid and a lignin model compound. The release of the free amino acid yields the positive selection. Lignin peroxidase, manganese peroxidase and glyoxal oxidase activities for such a strain under nonlimiting conditions were from 4- to 10-fold higher than those of the wild type under nitrogen-limiting conditions. We propose to use the same selection procedure except we will use the cDNAs for overexpression. The cDNAs encoding the lignin and Mn peroxidase will be placed behind a promoter library and transformed into P. chrysosporium. Transformant which overproduce the lignin peroxidases will be isolated by the positive selection procedure.
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