| Budget Amount *help |
¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Isoamyl acetate is one of the most important factors that determine the quality of Japanese sake. Synthesis of isoamyl acetate by sake yeast (S.cerevisiae) is performed by the action of AATFase in the presence of isoamyl alcohol and acetyl-CoA.The enzyme is thought to be bound to the cytoplasmic membrane. The AATFase is labile at high temperature (30ﾟC) and most active temperature of this enzyme is 10ﾟC.The enzyme is also unstable in the presence of unsaturated fatty acids. These are some of the reasons why the fermentation of ginjo-sake is performed at low temperature using the rice that are highly polished. On he other hand, the Hansenula yeasts have been known as a potent producer of esters. The author found that H.mrakii IFO 0895 could produce isoamyl acetate by the reverse reaction of esterase in addition to the AATFase.
We screened several Hansenula yeasts for resistance aginst isoamyl alcohol, ethyl alcohol and ethyl acetate, and found that H.mrakii IFO 0895 and H.anomala IFO 014
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9 showed higher resistance against these alcohols and esters. Among them, H.mrakii showed much higher resistance against isoamyl alcohol, a precursor for the synthesis of isoamyl acetate by both of AATFase and reverse reaction of esterase. Futhermore, the steady state level of isoamyl acetate in the culture of H.mrakii was 33.9ppm, whereas that of H.anomala was 27ppm ; therefore, the author selected H.mrakii for the study of esterase in Hansenula yeast.
We compared the amount of isoamyl acetate in the culture of H.mrakii and S.cerevisiae Kyokai No.7, which is industrially used in the sake fermentation. S.cerevisiae could not produce isoamyl acetate when the cells were cultured under the aerobic conditions, while H.mrakii could produce large amount of isoamyl acetate even though the yeast was cultured at both 15ﾟC and 30ﾟC under the aerobic conditions. These results suggested that H.mrakii might have some producing system of isoamyl acetate other than AATFase.
Then we measured the synthesis of isoamyl acetate from either isoamyl alcohol and acetic acid, or isoamyl alcohol and acetyl-CoA using the intact cells of H.murakii cultured at 15ﾟC and 30ﾟC.Intact cells preferably used isoamyl alcohol and acetic acid, i.e., by the reverse reaction of esterase, for the synthesis of isoamyl acetate. Cell homogenates of H.mrakii were centrifuged at 100,000xg for 2h to separate soluble fractions and insoluble fractions, since AATFase of S.cerevisiae is believed to be bound to the cell membrane. AATFase activity of H.mrakii was specifically detected in the insoluble fractions, while isoamyl acetate-synthesizing esterase was detected only in the soluble fractions. Isoamyl acetate-hydrolyzing activity was detected in both soluble and insoluble fractions. We then treated the insoluble fractions by high concentrations of salt and detergents. AATFase was solubilized by high concentrations of detergent (2% Triton X-100) ; thus the AATFase of H.mrakii was thought to be tightly bound to the cell membranc.
We tried to use H.mrakii for the fermentation of sake by a small scale brewing. By sensory test, the fermented product (sake) had a fruit-like flavor (isoamyl acetate, 2.11ppm). Concentration of isoamyl acetate produced by S.cerevisiae Kyokai No.7 was 2.49ppm. The values of isoamyl acetate produced by these two yeast strains were comparable ; thus we though that H.mrakii might be applicable for the production of sake flavor, isoamyl acetate. Less
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