Isoamyl acetate is a major determinant of the quality of Japanese sake. The amount of isoamyl acetate in the cultures of Hansenula mrakii and Saccharomyces cerevisiae Kyokai No. 7, which is industrially used in sake fermentation, and the isoamyl acetate-producing activities of each yeast strain were compared to investigate biochemical properties of the producing system of isoamyl acetate in these yeast strains. S. cerevisiae could not produce, or produced an extremely low level of, isoamyl acetate when the cells were cultured under aerobic conditions, while H. mrakii could produce a large amount of isoamyl acetate cultured at both 15 and 30 °C under aerobic conditions. Intact cells of H. mrakii cultured at 15 and 30 °C could produce isoamyl acetate from isoamyl alcohol and acetic acid. Alcohol acetyltransferase activity of H. mrakii was detected in insoluble fractions, while isoamyl acetate-synthesizing esterase was detected only in soluble fractions of the cell extracts. Isoamyl acetate-hydrolyzing esterase was detected in both soluble and insoluble fractions. Expression patterns of esterase were examined by native PAGE followed by activity staining by using 1-naphthyl acetate and Fast Blue B salt. H. mrakii is likely to have several esterases, and their expressions were varied depending upon the growth phase and temperature. Keywords: Hansenula yeast; isoamyl acetate; esterase; alcohol acetyltransferase

Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) inSaccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.

The IAH1 gene of Saccharomyces cerevisiae encodes an esterase that preferentially acts on isoamyl acetate; however, the enzyme has not yet been completely purified from the yeast S. cerevisiae. We constructed the IAH1 gene expression system in Escherichia coli, and purified the IAH1 gene product (Iah1p). The amount of Iah1p produced by recombinant E. coli was more than 40% of total cellular proteins. The molecular size of Iah1p was 28 kDa by SDS-polyacrylamide gel electrophoresis. Judging from the molecular weight estimation by gel filtration of purified Iah1p, the enzyme was thought to be a homodimer. The Km values for isoamyl acetate and isobutyl acetate were 40.3 mM and 15.3 mM, respectively. The enzyme activity was inhibited by Hg2+, p-chloromercuribenzoate, and diisopropylfluorophosphate.

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