Polyketide enzymes and genes in penicillium urticae

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dc.contributor.advisorGaucher, G. Maurice
dc.contributor.authorFedeshko, Ronald W.
dc.date.accessioned2005-08-05T17:00:00Z
dc.date.available2005-08-05T17:00:00Z
dc.date.issued1992
dc.descriptionBibliography: p. 390-401.en
dc.description.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.
dc.format.extentxxxiii, 401 leaves : ill. ; 30 cm.en
dc.identifier.citationFedeshko, R. W. (1992). Polyketide enzymes and genes in penicillium urticae (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/15229en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/15229
dc.identifier.isbn0315752025en
dc.identifier.lccQP 752 P65 F43 1992en
dc.identifier.urihttp://hdl.handle.net/1880/31271
dc.language.isoeng
dc.publisher.institutionUniversity of Calgaryen
dc.publisher.placeCalgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.
dc.subject.lccQP 752 P65 F43 1992en
dc.subject.lcshPenicillium urticae
dc.subject.lcshCell metabolism
dc.subject.lcshEnzymes
dc.subject.lcshPolyketides
dc.titlePolyketide enzymes and genes in penicillium urticae
dc.typedoctoral thesis
thesis.degree.disciplineUniversity Biochemistry Group
thesis.degree.grantorUniversity of Calgary
thesis.degree.nameDoctor of Philosophy (PhD)
ucalgary.item.requestcopytrue
ucalgary.thesis.accessionTheses Collection 58.002:Box 817 520535200
ucalgary.thesis.notesoffsiteen
ucalgary.thesis.uarcreleaseyen
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