Roles of Esterase and Alcohol Acetyltransferase on Production of Isoamyl Acetate in Hansenula mrakii

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Abstract

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

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