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

乙酸异戊酯是决定日本清酒品质的重要因素之一。清酒酵母（S.cerevisiae）在异戊醇和乙酰辅酶A存在下，通过AATF酶的作用合成乙酸异戊酯。AATF酶在高温（30 ℃）下不稳定，该酶的最大活性温度为10 ℃。该酶在不饱和脂肪酸存在下也不稳定。这就是为什么使用高度抛光的大米在低温下进行银酒发酵的原因。另一方面，已知汉逊酵母是一种有效的酯生产者。通过对几株汉逊酵母的抗异戊醇、乙醇和乙酸乙酯的筛选，发现姆氏汉逊酵母IFO 0895和异常汉逊酵母IFO 014对异戊醇、乙醇和乙酸乙酯的抗性最强 ...更多信息 9对这些醇和酯表现出较高的抗性。其中，麦氏血吸虫对异戊醇表现出更高的抗性，异戊醇是通过AATF酶和酯酶逆反应合成乙酸异戊酯的前体。因此，作者选择了H.mrakii作为汉逊酵母酯酶的研究对象，比较了H. mrakii和工业酿酒酵母Kyokai No.7培养物中乙酸异戊酯的含量，结果表明，H. mrakii培养物中乙酸异戊酯的稳定含量为33.9ppm，而H.anomala为27 ppm。酿酒酵母在好氧条件下不能产生乙酸异戊酯，而姆氏哈氏酵母在15 ℃和30 ℃条件下均能产生大量乙酸异戊酯。这些结果表明，在15 ℃和30 ℃培养的完整细胞中，H.mrakii可能具有AATFase以外的乙酸异戊酯合成系统。我们分别测定了异戊醇和乙酸、异戊醇和乙酰辅酶A合成乙酸异戊酯的能力。利用酯酶的逆反应合成乙酸异戊酯。将姆氏血吸虫的细胞匀浆在100，000 xg下离心2小时以分离可溶性级分和不溶性级分，因为据信酿酒酵母的AATF酶与细胞膜结合。在不溶性组分中特异性地检测到Mrakii的AATF酶活性，而合成乙酸异戊酯的酯酶仅在可溶性组分中检测到。乙酸异戊酯水解活性检测在可溶性和不溶性馏分。然后，我们用高浓度的盐和洗涤剂处理不溶性部分。用高浓度的去污剂（2%TritonX-100）溶解AATFase，认为Mrakii的AATFase与细胞膜结合紧密。经感官评定，发酵产物（清酒）具有果香味（乙酸异戊酯，2.11ppm）。由酿酒酵母Kyokai No.7产生的乙酸异戊酯的浓度为2.49ppm。这两种酵母菌产生的乙酸异戊酯的值相当，因此我们认为H.mrakii可能适用于生产清酒风味剂乙酸异戊酯。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