Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Mannoproteins released during the fermentation of Saccharomyces cerevisiae in a chemically defined synthetic grape juice medium were isolated by ethanol precipitation. The proteins from the medium were fractionated by ion exchange chromatography. Fractions containing mannoproteins were identified by their UV absorption spectra and by the presence of polymeric mannose. A mannoprotein designated HPF2 was purified from one of the fractions by gel permeation chromatography, and had a high capacity to reduce heat-induced protein haze formation in white wine. After electrophoretic separation and transfer to nitrocellulose, HPF2 stained positively with an antibody (anti-HPFl) which had been raised against a previously isolated mannoprotein with known haze protective activity designated HPFL. Analysis of the carbohydrate portion of HPF2 using methylation analysis confirmed the presence of (1 -> 2), (1 -> 3) and (1 -> 2,1 -> 6) mannosyl residues and showed that it contained N-linked and possibly O-linked carbohydrate. Digestion of the mannoprotein with PNGase F resulted in a reduction in molecular weight, as measured by SDS-PAOE and confirmed the presence of N-linked carbohydrate. N-linked deglycosylation decreased the ability of HPF2 to protect wine from heatinduced protein haze. Protein sequence analysis of the peptides derived from the HPF2 mannoprotein obtained via digestion with endoproteinase Lys C led to the identification of the HPF2 structural gene in Saccharomyces cerevisiae followed by searching the Yeast Proteome Database for related sequences. Analysis of the deduced amino acid sequence of HPF2 from its structural gene indicated that HPF2 possessed a secretion signal at the NH₂-terminus, a putative OPI anchor site at the COOH-terminus and also contained serine and threonine rich regions at both the NH₂-terminus and COOHterminus. These regions may contain O-linked carbohydrate. There were also 15 potential glycosylation sites, five of which were classified from the peptide mapping data as likely glycosylation sites. These characteristics, combined with the results from the carbohydrate analysis, indicate that HPF2 was a mannoprotein derived from the yeast cell walls. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=678380 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Plant Science, 2000
| Identifer | oai:union.ndltd.org:ADTP/280407 |
| Date | January 2000 |
| Creators | Stockdale, Vanessa Jane |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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