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

Abstract

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.