ARYL-ALCOHOL DEHYDROGENASE FROM THE WHITE-ROT FUNGUS PHANEROCHAETE-CHRYSOSPORIUM - GENE CLONING, SEQUENCE-ANALYSIS, EXPRESSION, AND PURIFICATION OF THE RECOMBINANT ENZYME

A cDNA clone encoding a ligninolytic aryl-alcohol dehydrogenase (AAD; EC 1.1.1.91) from the white-rot basidiomycete fungus Phanerochaete chr ysosporium was isolated and characterized. The nucleotide sequence obt ained reveals an open reading frame encoding a protein of 385 amino ac ids. Substantial homology (49.3% identity and 67.3% similarity, respec tively) was observed between AAD and an open reading frame sequence pr esent on chromosome III of Saccharomyces cerevisiae. A Southern blot a nalysis showed the presence of multiple AAD gene-related sequences in P. chrysosporium and in other white-rot fungi including Bjerkandera ad usta and Fomes lignosus. Northern blot analyses are in line with the v iew that the levels and appearance of AAD mRNA correlate with the leve l and appearance of AAD activity and that, under conditions of nitroge n limitation, the AAD mRNA levels are higher than in carbon limited cu ltures. This is consistent with the regulation of the enzyme by carbon or nitrogen Limitation being at the level of transcription. Moreover, the appearance of AAD-specific transcripts correlates with the appear ance of lignin peroxidase-specific transcripts in the same cultures. T his co-appearance is in line with the proposed synergistic interaction of the two enzymes in lignin biodegradation, which suggests a similar regulation. The AAD encoding cDNA was expressed in Escherichia coli t o yield high levels of active enzyme, and the recombinant enzyme was p urifed by using metal chelate affinity chromatography.