Polyketide enzymes and genes in penicillium urticae
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AbstractPenicillium urticae is one of the best characterized organisms that produces a secondary metabolite (patulin). All of the steps of the pathway have been elucidated and a number of the enzymes have been characterized. Because these enzymes are notoriously unstable, their purification is often difficult. Nevertheless, success has been achieved for the seventh and eighth enzymes of the pathway, isoepoxydon dehydrogenase and neopatulin synthase. Success with isoepoxydon dehydrogenase depended on optimizing the harvest time for 8 L fermenter cultures and then rupturing the cells in a carefully formulated cell breakage buffer which maximized dehydrogenase activity and longevity. A seven step purification (salt fractionation, Blue-A Sepharose, hydrophobic interaction, two anion exchange, and two size-exclusion chromatography steps) resulted in an~ 120 fold purification and 15% yield of the dehydrogenase. This preparation was greater than 99% pure, and the enzyme was characterized as an a2 dimer (29 kDa /monomer). Partial amino terminal sequence analysis of the dehydrogenase yielded 45 residues and this provided the basis for the construction of oligonucleotide probes. Success with neopatulin synthase was made easier due to it's excellent stability, as compared to other enzymes of the pathway. The harvest time was optimized for 8 L fermentor cultures and the cells ruptured in a carefully formulated breakage buffer. A five step purification (Millipore minitan concentration, isoelectric precipitation, three anion exchange steps) resulted in an ~2700 fold purification with a 12% yield. This enzyme was further characterized as a glycoprotein with a deglycosylated subunit molecular weight of ~20 kDa. Amino terminal sequence analysis of one glycoform yielded 29 residues.
Bibliography: p. 390-401.
CitationFedeshko, R. W. (1992). Polyketide enzymes and genes in penicillium urticae (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/15229
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