Purification and characterization of an aromatic L-amino acid decarboxylase from Penicillium raistrickii isolate H10BA2, the transformation of this fungus, and investigation of taxa-4(5),11(12)-diene synthase

An aromatic L-amino acid decarboxylase from Penicillium raistrickii isolate H10BA2, isolated from Taxus brevifolia, was purified and characterized. This is the first time this enzyme class has been described in a fungus. The enzyme was purified to homogeneity by chromatography on phenyl-sepharose and carboxymethyl-sepharose media. Purity was demonstrated by the presence of one protein band in a silver stained SDS-PAGE gel. Optimum pH for catalysis by this enzyme is pH 5.0 to 5.6. The enzyme has a broad substrate specificity, utilizing tryptophan (Km = 49 ;A transformation protocol was developed for P. raistrickii isolate H10BA2. This fungus was transformed with the plasmid pAN71, which contains a selectable hygromycin B resistance gene. Putative transformants were isolated on hygromycin B containing media. Conidia, from colonies of putative transformants grown on nonselective media, also gave rise to colonies on selective media. This indicates that stable transformants of P. raistrickii isolate H10BA2 can be developed with the transformation protocol as described. These transformation experiments were performed to test the possibility of future determination of aromatic amino acid decarboxylase function in the fungus.;Attempts to identify taxa-4(5), 11(12)-diene synthase from P. raistrickii isolate H10BA2 with techniques used for identification this enzyme from Taxus brevifolia are described.