Construction of energy-independent producing system of sake flavor by Hansenula yeasts

乙酸异戊酯是决定日本清酒质量的最重要因素之一。酿酒酵母(S.cerevisiae)在异戊醇和乙酸乙酯存在下，通过AATFase的作用合成乙酸异戊酯，该酶被认为是结合在细胞膜上的。该酶在高温(30ﾟC)下不稳定，最高活性温度为10ﾟC，在不饱和脂肪酸存在下也不稳定。这些就是为什么银杏清酒是用高度抛光的大米在低温下发酵的一些原因。另一方面，汉塞努拉酵母一直被认为是一种强有力的酯生产菌。通过对几株汉逊酵母对异戊醇、乙醇和乙酸乙酯的抗性筛选，发现两株酵母菌对异戊醇、乙醇和乙酸乙酯的抗性较强，分别为H.mrakii IFO0895和H.onomala IFO014…对这些醇类和酯类的抗性越强，抗性越强。其中，H.mrakii对异戊醇表现出较高的抗性，异戊醇是AATFase合成乙酸异戊酯的前体，也是酯酶逆转反应的前体。此外，mrakii酵母菌培养物中乙酸异戊酯的稳态含量为33.9ppm，而异型H.mrakii菌培养物中乙酸异戊酯的稳态含量为27ppm，因此，作者选择H.mrakii菌株作为研究汉逊酵母的酯酶，并比较了工业上用于清酒发酵的H.mrakii和S.cerevisiae Kyokai No.7培养物中乙酸异戊酯的含量。酿酒酵母在好氧培养条件下不能产生乙酸异戊酯，而米氏酵母在15ﾟC和30ﾟC的好氧培养条件下仍能产生大量的乙酸异戊酯。然后，我们用15ﾟC和30ﾟC培养的mrakii完整细胞测定了异戊醇和冰醋酸或异戊醇和乙酰辅酶A合成乙酸异戊酯的情况。由于酿酒酵母的AATFase被认为是与细胞膜结合的，mrakii的细胞匀浆在100,000xg下离心2小时，以分离可溶组分和不溶组分。Mrakii的AATFase活性仅在不溶组分中检测到，而乙酸异戊酯合成酶仅在可溶组分中检测到。乙酸异戊酯的可溶组分和不溶性组份均有一定的水解性。然后，我们用高浓度的盐和洗涤剂处理不溶部分。在高浓度洗涤剂(2%Triton X-100)的作用下，Hmrakii的AATFase被溶解，因此认为Hmrakii的AATFase与细胞膜紧密结合。经感官检验，发酵产品(清酒)具有果味(醋酸异戊酯，2.11ppm)。酿酒酵母Kyokai 7号产乙酸异戊酯的浓度为2.49ppm。两株酵母菌产生的乙酸异戊酯具有可比性，因此我们认为H.mrakii可能适用于生产清酒香精乙酸异戊酯。较少

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 … More 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