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    Studies on the non-specific esterases of Saccharomyces cerevisiae : a thesis in partial fulfillment [sic.] of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1984) Dick, Bruce
    Twenty wine-making and three laboratory strains of Saccharomyces cerevisiae were examined for non-specific esterases by Polyacrylamide Gel Electrophoresis. All wine-making strains contained the fast alleles of the Est 1 and Est 2 loci, confirming there is a selective advantage for the Est 1f and Est 2f genes in these strains. Only one wine-making strain carried the Est 3 and Est 4 genes, which was a much lower frequency than that published. The three laboratory strains all contained the Est 1f and Est 2s genes. A new non-specific esterase band, labelled Est 5, was identified by using a modified staining technique, which was apparently of low molecular weight as it travelled with the tracking dye front. Fast and slow alleles of Est 1 and Est 2 were determined to be charge allozymes. Est 2 proteins were considered to be polymeric, probably dimeric, and the Est 1 proteins to undergo post-translational modification. Difficulty in resolving the Est 4 band was overcome by adding Triton X-100 to cell suspensions before disruption, indicating this esterase protein may be particulate bound. Molecular weights were determined by Ferguson Plots to be 51,000 ± 10,000 daltons (Est 2), 60.000 ± 12,000 daltons (Est 3), 73,000 ± 15,000 daltons (Est l), and 113,000 ± 23,000 daltons (Est 4). No isolates of S. cerevisiae for comparison of allele frequencies could be made from mature locally-grown grapes, indicating that this species is rare in the New Zealand environment, which is in accordance with published studies. No "inducible" non-specific esterases were found in strains examined at different stages in the life cycle, or by growth in different media. The level of esterase activity in cells increased throughout aerobic growth in liquid media, but was quickly lost during fermentation. Esterase activity during sporulation also decreased. A non-specific esterase mutant was induced by ethyl methane-sulfonate and detected by the hydrolysis of α-naphthyl acetate incorporated into solid medium. This mutant lost expression of both Est If and Est 2s , as did subsequent mutants produced by hybridisation. Segregation of esterase-deficient to esterase-proficient spores after hybridisation, showed that two unlinked loci were involved in esterase suppression, both genes being unlinked to ade 1, Est 1 and mating type locus MAT. It is hypothesised these genes are a suppressor (SUP) and a mutated regulator (Reg Est− ). Gas Liquid Chromatography was used to quantitatively determine volatile ester concentrations produced during fermentation. Selected wine-making strains and diploid strains produced by micromanipulation and having different non-specific esterase compositions were fermented to the limit of their ethanol tolerance in Reisling Sylvaner grape juice and Complete Defined Medium. Ethyl acetate, ethyl propanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, 2-phenethyl acetate, n-hexyl acetate and iso-pentyl acetate were all quantitated. A maximum error of ±30% was determined for differences in ester concentration between two fermentations using the same strain. Correction for differences in fermentation ability by different strains was attempted, and the resulting ester concentrations compared qualitatively. Results indicate that differences in volatile ester concentrations between strains are not due to the esterase composition. The non-specific esterases probably have little if any influence on wine bouquet as the majority of ester production is late in fermentation when esterase activity has ceased.
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    Glycerol production by various strains of Saccharomyces cerevisiae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1987) Munster, Rosalind Evelyn Gordon
    The influence of yeast strain, fermentation procedure and media on cell growth and the production of glycerol and ethanol was studied. Two fermentation procedures were compared (a) fermentation at a constant temperature of 15°C and (b) fermentation at higher temperatures (15–20°C) maintaining a constant rate of sugar utilization. Three wine-making yeasts and three high glycerol producing hybrid yeasts were fermented on two types of grape juice and a synthetic [control] media. The effect of the fermentation procedure on glycerol, ethanol production and cell growth was variable and appeared to depend on the yeast strain. Comparison of the yeast strains showed glycerol production to vary considerably depending on the yeast this effect was also dependent on the media. The yeast strain is important for maximum fermentation efficiency in a specific grape juice. Selective hybridisation of pure culture wine yeasts was employed to develop yeast strains capable of maximum glycerol yield, without jeopardising ethanol production in Muller Thurgau and in Chenin Blanc grape juices. Improved yields were achieved, but those yeasts selected for fermentation in one type of grape juice did not give outstanding yields when fermented in the other type of grape juice. This suggests that for wine-making it is possible to tailor yeasts for fermentation in specific grape juices. The addition of sulphur dioxide [0–300 ppm] and its influence on glycerol and ethanol production was studied using a wine-making yeast and a high glycerol producing hybrid. The effect was strain dependent and as expected, the addition of sulphur dioxide to the wine-making yeast showed enhanced glycerol production and depressed ethanol production. However, the converse was apparent with the high glycerol producing hybrid. The addition of glycerol to the media prior to fermentation at levels of 0 to 20 g/1 was tested in an attempt to simulate the conditions of grapes attacked by the fungus Botrytis cinerea [noble rot]. No inhibition or stimulation of glycerol or ethanol production was apparent by either the wine-making yeast or the high glycerol producing hybrid yeast tested.
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    Fluoride inhibition of wine yeasts : a thesis presented in partial fulfilment of the requirements for the degress of Master of Science in Microbiology at Massey University
    (Massey University, 1997) Clayton, Miranda Gaye
    Stuck or slowed fermentations are costly in time and money to winemakers. There are many variables that can interrupt fermentation. One of the lesser known factors is the effect of fluoride on grape juice fermentations. Winemakers in California have had problems with slow or stuck fermentations with grapes that have been treated with the insecticide Cryolite, which contains fluoride. A selection of 6 yeasts, 3 commercial strains and 3 natural strains, commonly associated with winemaking were used in this study. Preliminary experiments investigated a wide range of fluoride challenge with different pH and cell densities on solid and liquid media. The effectiveness of fluoride was compared between sodium fluoride and Cryolite, as the fluoride source. The effect of fluoride was more potent with sodium fluoride, as the fluoride source. The minimum inhibitory concentration of fluoride for the yeast strains was recorded. The most sensitive commercial yeast was Saccharomyces cerevisiae RS1, the most resistant commercial yeast was Saccharomyces bayanus RS2. The most sensitive yeast overall was Hansenula saturnus AWRI-354. The next stage examined the effect of fluoride on the selected yeast in small scale grape juice fermentations. Within this investigation the effect of different media sources and heat treatments was included. Fluoride concentrations reflected levels of fluoride found in grape musts and wines. During this study we found that the effect of fluoride on yeasts is increased with lower pH and lower cell densities. The effect of fluoride on yeast growth and fermentation was also strain dependent.
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    Investigations on malic acid utilisation in Schizosaccharomyces species : a thesis presented in partial fulfilment of the requirements for the degree in Doctor of Philosophy in Genetics at Massey University
    (Massey University, 1990) Cambourn-Theewis, Wilhelmina Margaretha
    The aims of this investigation were: to determine whether alterations in uptake, or metabolism, of glucose and malate was the cause of malate dependence; to determine the number of genes involved in malate dependence; and to clone the gene(s) involved. A malate dependent mutant, i.e. a mutant that requires both malate and glucose for growth, (mutant 11) of Schizosaccharomyces malidevorans 442 was characterised. Malic enzyme activity was increased almost ten-fold. The Vmax of malate uptake was increased four-fold compared to S. malidevorans 442. Uptake of glucose was significantly lower in mutant 11 than the wild-type. The kinetics of glucose uptake by S. malidevorans 442 and mutant 11 suggested the presence of two glucose transporters, a high affinity and a low affinity transporter. Only the low affinity transport was apparently altered in mutant 11 compared to the wild-type. Genetic analysis indicated that the malate dependent mutation is recessive and is the result of a single mutational event. Crosses involving derivatives of mutant 11 and Schizosaccharomyces pombe strains did not yield the expected segregation of markers. Tetrad analysis showed that the spore viability was very low. It was not possible, therefore, to determine linkage of the malate dependence locus and any other loci. All malate dependent strains were apparently homothallic although linkage between the mating-type locus and malate dependence could not be established. The isolation of similar mutants from homothallic strains of S. pombe, but not from heterothallic strains, provided strong support for the requirement of homothallism for malate dependence. The pulse field gel electophoresis karyotypes of mutant 11 and derivatives of mutant 11 suggested the presence of a large chromosomal rearrangement of chromosome 2 that cosegregated with malate dependence. Malate dependent mutants were not obtained from homothallic Saccharomyces cerevisiae MD26. A malate dependent mutant (WT 6) was isolated from S. pombe WT 4 and found to have characteristics similar but not identical to those of mutant 11. WT 6 demonstrated increased utilisation of malate and decreased utilisation of glucose. Malic enzyme activity was not altered in WT 6 compared to the wild-type. Malate uptake was not affected. The karyotype of WT 6 suggested that a chromosomal rearrangement had occurred, but it is not identical to the rearrangement in mutant 11. The differences in the characteristics of mutant 11 and WT 6 suggested the mutations in these mutants may not be identical. The finding that mutant 11 and WT 6 belong to different complementation groups could explain these differences. Although differences were found in the uptake of malate and glucose, the inability of malate dependent mutants to grow on glucose implicates a defect in glucose metabolism.