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The enzymatic production of R-phenylacetylcarbinol (R-PAC), with either whole cells or partially purified pyruvate decarboxylase (PDC) as the biocatalyst, requires high PDC activity and an inexpensive source of pyruvate for an economical feasible biotransformation process. Microbial pyruvate produced by a vitamin auxotrophic strain of Candida glabrata was selected as a potential substrate for biotransformation. With an optimal thiamine concentration of 60 ??g/l, a pyruvic acid concentration of 43 g/l and yield of 0.42 g/g glucose consumed were obtained. Using microbially-produced unpurified pyruvate resulted in similar PAC concentrations to those with commercial pure substrate confirming its potential for enzymatic PAC production. To obtain high activity yeast PDC, Candida utilis was cultivated in a controlled bioreactor. Optimal conditions for PDC production were identified as: fermentative cell growth at initial pH at 6.0 followed by pH downshift to 3.0. Average specific PDC carboligase activity of 392 ?? 20 U/g DCW was achieved representing a 2.7-fold increase when compared to a constant pH process. A mechanism was proposed in which the cells adapted to the pH decrease by increasing PDC activity to convert the accumulated internal pyruvic acid via acetaldehyde to ethanol thereby reducing intracellular acidification. The effect of pH shift on specific PDC activity of Saccharomyces cerevisiae achieved a comparable increase of specific PDC carboligase activity to 335 U/g DCW. The effect of pyruvic acid at pH 3.0 on induction of PDC activity was confirmed by cultivation at pH 3 with added pyruvic acid. Using microarray techniques, genome-wide transcriptional analyses of the effect of pH shift on S. cerevisiae revealed a transient increased expression of PDC1 after pH shift, which corresponded to the increase in specific PDC activity (although the latter was sustained for a longer period). The results showed significant gene responses to the pH shift with approximately 39 % of the yeast genome involved. The induced transcriptional responses to the pH shift were distinctive and showed only limited resemblance to gene responses reported for other environmental stress conditions, namely increased temperature, oxidative conditions, reduced pH (succinic acid), alkaline pH and increased osmolarity.

viii, 104 leaves : ill., photos., tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1979

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Enzyme concentrates are available for use in commercial wineries to aid in wine processing, or to enhance wine quality. However, pectolytic enzyme remains the sole product routinely used in most wineries. One disadvantage of some of the products currently available commercially is they contain enzymes sourced from microorganisms not usually associated with grape juice or wine, typically fungi such as Aspergillus species. As a result, enzymes are inefficient catalysts under the harsh oenological conditions. In addition, some products contain secondary, and potentially undesirable, contaminant enzyme activities. Clearly there is the potential to develop enzyme preparations specifically for use in grape juice and wine. A potential source of such enzymes are the lactic acid bacteria (LAB), the organisms more commonly associated with the conduct of the malolactic fermentation (MLF) during vinification. In this study, the production of cell-associated enzymes with potential oenological applications by LAB was investigated. A screening of 50 LAB isolates for the production of lipases, esterases, tannases, and polysaccharide-degrading enzymes revealed wine LAB can produce enzymes of oenological importance. In general, activity towards polysaccharide substrates was more frequent among the lactobacilli and pediococci strains. Lipase activity was observed in three lactobacilli, and all strains were found to have tannase activity. Similarly, all strains displayed some esterase activity, although the activity was markedly stronger among the Oenococcus oeni. On the basis of the initial screen, a more detailed characterisation of the esterase activity of selected LAB isolates was conducted. Esterase activity was examined across a range of pH, temperature, and ethanol concentrations - all important oenological parameters. In addition, substrate specificity was determined using six ester substrates. In general, activity was maximal at pH values close to 6.0, and temperatures close to 40°C, although exceptions were observed with some strains. Increases in ethanol concentration resulted in lower activity for most lactobacilli and pediococci, but stimulated the esterase activity of all O. oeni. Work conducted with dairy LAB isolates has suggested esterases may be capable of both hydrolysing and synthesising esters. In the wine industry, the results of some volatile-profiling studies tend to support this theory, with concentrations of esters being reported to both increase and decrease during MLF. Malolactic fermentation trials were conducted in wine with six strains of O. oeni and GCMS was used to quantify particular esters before and after MLF. Some esters were found to increase in concentration during MLF, while others were found to decrease. These findings suggest LAB esterases are in fact capable of both synthesising and hydrolysing ester substrates in wine. To further dissect the esterase make-up of selected LAB strains, attempts to clone and heterologously express three structural genes for these enzymes were made. Three putative esterase genes were identified in O. oeni and cloned. Sequencing was completed and alignment with published esterase sequences used to reveal theoretical proteins of the O. oeni genes with high homology with those from other organisms. Of note, key features, such as active site motifs, were conserved in each O. oeni sequence. Expression of the recombinant proteins in E. coli resulted in higher esterase activity in one of the clones compared to the host. These results indicate that the open reading frame of one esterase gene in 0. oeni has been identified. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297212 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2007

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Limin Kung, Jr., Dept. of Animal and Food Sciences. Includes bibliographical references.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 157-184). Also available via the Internet.

Thesis (M.Sc.). / Written for the Dept. of Chemistry. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisor: Junghuei Chen, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisors: Robert W. Mason and Robert A. Sikes, Dept. of Biological Sciences. Includes bibliographical references.