平成23年度 研究成果

Ethyl carbamate concentrations in oak barrel-aged ume (Prunus mume) liqueurs were measured, and possible explanations for elevated levels were examined. The average concentration was 0.30 mg/L, significantly higher than in ume liqueurs not aged in oak (0.08 mg/L). Oak powder extracts were prepared from both untoasted and toasted oak powder by extraction with aqueous ethanol, and these were used to make ume liqueurs. Relative to a no-oak control, the ethyl carbamate concentrations were 3.8 and 11 times higher in the ume liqueur made with the untoasted and toasted oak powder extracts respectively. Ethyl carbamate was formed when lignin was added to a 40% aqueous ethanol solution that contained potassium cyanide. These observations suggest that lignin or fragments thereof promote the formation of ethyl carbamate.

Modern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p in Saccharomyces cerevisiae sake yeast. The HSE-lacZ activity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. Since HSF1 allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a △ppt1 mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entire PPT1 gene locus. We confirmed that the expression of laboratory yeast-derived functional PPT1 recovered the HSE-mediated stress response of sake yeast. In addition, deletion of PPT1 in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of the PPT1 gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains.

The swelling degree, gelatinization temperature, and protein content of rice flour prepared from the ventral and dorsal parts of rice grains used for sake brewing (larage grains) and staple food (small grains) were examined. The swelling degree of rice flour was higher in the order: ventral part of large grains >> ventral part of small grains > dorsal part of large grains ≒ dorsal part of small grains. The rice flour obtained from the ventral part of large grains also exhibited the highest swelling power, lowest gelatinization temperature, and lowest protein content. The white precipitate remaining after the measurement of swelling power was subjected to iodine and Berlin blue reactions and observed microscopically. From these findings, we speculate that rice flour containing large amounts of protein may be difficult to gelatinize because protein firmly adheres to starch, resulting in reduced water absorption and consequently, reduced gelatinization.

We screened 4908 non-essential gene deletion mutant yeast strains for uranium sensitivity and low accumulation by growth in agar medium containing uranium. All mutant strains grew successfully on agar media containing 0 or 0.5 mM uranium for one week at 30℃. Thirteen strains with single gene deletions showed reduced growth in the agar medium containing 0.5 mM uranium and were identified as uranium-sensitive mutant strains. The phosphate transporter genes of PHO86, PHO84, PHO2, and PHO87 were among the deleted genes in the uranium-sensitive mutant strains, suggesting that genes concerned with phosphate transport contribute to uranium tolerance. Seventeen single-deletion strains showed lower uranium accumulation than the wild-type after exposure to agar medium containing 0.5 mM uranium, and were identified as mutant strains with low uranium accumulation. Among the deleted genes in these strains were cell membrane proteins, phospholipid-binding proteins, and cell wall proteins, suggesting that cell surface proteins contribute to uranium accumulation.

Hygromycin B is an aminoglycoside antibiotic that inhibits protein synthesis in prokaryotes and eukaryotes. Twenty-four hygromycin B-resistants mutants were isolated from sake yeast, and were divided into three different degrees of strength according to hygromycin B resistance. Three of four hygromycin B strongly resistant mutants produced increased amounts of isoamyl acetate in sake brewing test, although isoamyl alcohol levels remained unchanged. Many hygromycin B-resistants mutants showed higher E/A ratios than K-701 in culture with koji extract medium. Strain HMR-18 produced the largest amount of isoamyl acetate, and its alcohol acetyltransferase (AATFase) activity was 1.3-fold that of K-701. DNA microarray analysis showed that many genes overexpressed in HMR-18 were involved in stress responses (heat shock, low pH, and so on) but HMR-18 showed thermo- and acid-sensitivity. It was strongly resistant to hygromycin B and another aminoglycoside antibiotic, G418.

Yeast host-vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host-vector system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host-vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5 times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately 15 times greater than that of the wild type.

Lipomyces starkeyi is an oleaginous yeast, and has been classified in four distinct groups, i.e., sensu stricto and custers α, β, and γ. Recently, L. starkeyi clusters α, β, and γ were recognized independent species, Lipomyces mesembrius, Lipomyces doorenjongii, and Lipomyces kockii, respectively. In this study, we investigated phylogenetic relationships within L. starkeyi, including 18 Japanese wild strains, and its related species, based on internal transcribed spacer sequences and evaluated biochemical characters which reflected the phylogenetic tree. Phylogenetic analysis showed that most of Japanese wild strains formed one clade and this clade is more closely related to L. starkeyi s.s. clade including one Japanese wild strain than other clades. Only three Japanese wild strains were genetically distinct from L. starkeyi. Lipomyces mesembrius and L. doorenjongii shared one clade, while L. kockii was genetically distinct from the other three species. Strains in L. starkeyi s.s. clade converted six sugars, D-glucose, D-xylose, L-arabinose, D-galactose, D-mannose, and D-cellobiose to produce high total lipid yields. The Japanese wild strains in subclades B, C, and D converted D-glucose, D-galactose, and D-mannose to produce high total lipid yields. Lipomyces mesembrius was divided into two subclades. Lipomyces mesembrius CBS 7737 converted D-xylose, L-arabinose, D-galactose, and D-cellobiose, while the other L. mesembrius strains did not. Lipomyces doorenjongii converted all the sugars except d-cellobiose. In comparison to L. starkeyi, L. mesembrius, and L. doorenjongii, L. kockii produced higher total lipid yields from D-glucose, D-galactose, and D-mannose. The type of sugar converted depended on the subclade classification elucidated in this study.

A lipase from Aspergillus niger MTCC 2594 was purified 53.8-fold to homogeneity by hydrophobic interaction chromatography using octyl sepharose and the enzyme showed two protein bands with apparent molecular mass of 35 and 37 kDa respectively. The lipase exhibited maximum activity at pH 7.0 and 37 ℃ and was stable between pH 4.0 and 10.0 and temperatures up to 50 ℃. The values of Km and Vmax were 3.83 mM and 32.21 μmol/min/mg respectively, using olive oil as substrate. Lipase encoding gene, lipA, coded for 297 amino acid residues with conserved pentapeptide sequence, G-H-S-L-G, was cloned and expressed in Pichia pastoris. Although lipA showed high homology with the known Aspergillus lipases, it exhibited differences in putative lid domain. Both native and recombinant lipases have potential for degradation of poly(lactic acid) and poly(ε-caprolactone), and the present study will serve as a baseline of initial studies for its exploitation in polymer degradation.

清酒は食中酒であり、食品との相性は重要な品質特性である。しかし相性には少なからず曖昧さが伴うため、信頼性を高めるには、精度と再現性を有する相性評価法の確立が不可欠である。そこで、食品と清酒との相性評価に影響する要因を検証し、以下の結果を得た。（1）食品と清酒を飲食するインターバルは5秒以内が目安と考えられた。（2）食品と清酒との相性は、清酒を味わってから食品を味わうときの相性感性に比べ、食品を味わってから清酒を味わうときの相性感性に強く影響された。その影響度は約1対3と見積もられた。（3）味覚意識の置き方による相性への影響は軽微であった。これは検証の仕方に問題があると考えられた。（4）食品を味わってから清酒を飲むとき、高い頻度で食品から清酒への味覚リクエストが生じた。味覚リクエストは相性評価に影響すると考えられた。（5）相性に関する先入観は相性の良否に直結する清酒の品質に影響した。（6）検証結果を踏まえ、食品と清酒との相性評価法を設定した。

1. 清酒に含まれる成分と生理的嗜好の関係を絶食条件下のマウスを用いて二瓶選択試験で調べた。
2. 嗜好性の異なる純米酒を用い、嗜好性と各種清酒成分量（グルコース、アミノ酸、有機酸、香気成分）との順位相関解析及びCE-TOFMSによる一斉成分分析によって、嗜好性の上昇及び低下に関与する成分を推定した。
3. 嗜好性を上昇させると推定した成分を純米酒に添加し、マウスによる二瓶選択試験でグルコース、リジン、ヒスチジンが嗜好性を上昇させることを示した。
4. リジン、ヒスチジンを単独で添加しても、嗜好性が変化しない清酒も存在し、単一成分の添加で清酒の嗜好を完全に説明するのは難しいことがわかった。
5. 嗜好性を低下させると推定した成分を純米酒に添加し、マウスによる二瓶選択試験でグルタミン、オクタン酸、ピルビン酸、酢酸イソアミルが嗜好性を低下させることを示した。
6. 高嗜好性純米酒摂取後のマウスは低嗜好性純米酒よりも有意に血糖値が高かった。また、低嗜好性純米酒へのリジン添加が血糖値を上昇させ、高嗜好性と血糖値上昇の関連が示唆された。

A technique to determine the geographic origin of wine was examined using 99 samples from Japan and 4 other countries. The contents of 21 elements (Al, B, Ba, Ca, Cd, Cr, Cs, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Sr, V, and Zn) were determined by inductively coupled plasma optical emission spectrometry. A linear discriminate analysis (LDA) model was created to identify wine from Japan and other countries. With these parameters, 99% correct classification was achieved. We developed an LDA model calibrated with the analytical results of 5 elements (B, Ca, Cr, K, and Mg) chosen by a backward stepwise procedure. This LDA model identified the origin of wines as Japan with 91% certainty. This result suggests that discriminating by inorganic element composition may be useful to identify Japanese wine for import clearance.

酒造用原料米の無機元素(K, P, Mg, S, Si, Fe, Ca, Mn, Na, Co, Ni )の含有量について解析した。Co、Niは玄米でも0.1ppm以下と低レベルであった。五百万石、おくほまれ、兵庫北錦のK、P、Mg含有量は他の品種と比較して高い傾向にあった。山田錦のS含量は今回調べた品種の中では最も低く、同一圃場で栽培された日本晴と比較してもFe、S含量は低かった。 精米による変化について調べたところ、K、P、Ca、Mg、Mn、Siは精米歩合70%まで急激に減少しその後横ばいとなったが、Sの減少は精米歩合30%まで直線的に減少した。山田錦と五百万石について心白粒と無心白粒の無機元素含有量を精米歩合別で比較したところ、顕著な違いはみられなかった。SEM/EDXの観察から、P、K、Mg、Siはアリューロン層や果皮に多く分布することが明らかとなった。

The industrial fungus Aspergillus luchuensis is used to produce a distilled spirit in Okinawa Island, Japan. Recently, the genome sequence of A. luchuensis RIB2604 (Aspergillus awamori NBRC 4314) was revealed and many functional genes are now expected to be analysed. Gene targeting is necessary for analysing the function of a gene; however, gene targeting frequencies in A. luchuensis are very low. To develop a highly efficient gene-targeting system for A. luchuensis, we disrupted A. luchuensis ligD (ALligD) encoding the human DNA ligase IV (ligIV) homologue using an Agrobacterium mediated gene transformation method. Deletion of ALligD dramatically improved homologous recombination efficiency (reached 100%) compared to that in the wild-type strain (0.8%), when 1000-bp homologous flanking regions were used. The ALligD disruptant showed no apparent defect in vegetative growth, and it exhibited increased sensitivity to phleomycin and high methyl methanesulphonate concentrations compared to the wild-type strain. Furthermore, using this ALligD disruptant, we disrupted ALpksP encoding an Aspergillus fumigatus polyketide synthase P (alb1/pksP) orthologue. The ALpksP disruptant displayed a decolourized conidial phenotype. This result indicated that ALpksP is a key factor for conidial black pigmentation in A. luchuensis. Our results indicate that the ALligD mutant is an efficient host for targeted gene disruption in A. luchuensis.

Cellular and subcellular morphology reflects the physiological state of a cell. To determine the physiological nature of sake yeast with superior fermentation properties, we quantitatively analyzed the morphology of sake yeast cells by using the CalMorph system. All the sake strains examined here exhibited common morphological traits that are typically observed in the well-characterized whiskey (whi) mutants that show accelerated G₁/S transition. In agreement with this finding, the sake strain showed less efficient G₀/G₁ arrest and elevated expression of the G₁ cyclin gene CLN3 throughout the fermentation period. Furthermore, deletion of CLN3 remarkably impaired the fermentation rate in both sake and laboratory strains. Disruption of the SWI6 gene, a transcriptional coactivator responsible for Cln3p-mediated G₁/S transition, also resulted in a decreased fermentation rate, whereas whi mutants exhibited significant improvement in the fermentation rate, demonstrating positive roles of Cln3p and its downstream signalling pathway in facilitating ethanol fermentation. The combined results indicate that enhanced induction of CLN3 contributes to the high fermentation rate of sake yeast, which are natural whi mutants.

Takeshi Akao, Isao Yashiro, Akira Hosoyama, Hiroshi Kitagaki, Hiroshi Horikawa, Daisuke Watanabe, Rinji Akada, Yoshinori Ando, Satoshi Harashima, Toyohisa Inoue, Yoshiharu Inoue, Susumu Kajiwara, Katsuhiko Kitamoto, Noriyuki Kitamoto, Osamu Kobayashi, Satoru Kuhara, Takashi Masubuchi, Haruhiko Mizoguchi, Yoshihiro Nakao, Atsumi Nakazato, Masahiro Namise, Takahiro Oba, Tomoo Ogata, Akinori Ohta, Masahide Sato, Seiji Shibasaki, Yoshifumi Takatsume, Shota Tanimoto, Hirokazu Tsuboi, Akira Nishimura, Koji Yoda, Takeaki Ishikawa, Kazuhiro Iwashita, Nobuyuki Fujita, and Hitoshi Shimoi

The term 'sake yeast' is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.

An aspartic protease (Cap1) was purified from basidiomycetous yeast Cryptococcus sp. S-2 (FERM ABP-10961) using HiTrap DEAE FF column and HiTrap Q HP column chromatography with azocasein as a substrate. Cap1 has a molecular mass of 34 kDa on SDS-PAGE. It was stable up to 50°C with maximum activity at 30°C. Maximum proteolytic activity was observed at pH 5.0. Cap1 was stable in the pH range 3.0-7.0. Its enzyme activity was strongly inhibited by pepstatin A, an inhibitor of aspartic proteases, indicating that Cap1 is an aspartic protease. Cap1 hydrolyzed protein substrates, including BSA, hemoglobin, α-casein, β-casein, and κ-casein. It showed activity on synthetic substrates, such as MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH₂ and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH₂. Hydrolysis of the oxidized insulin B chain followed by amino acid sequencing analysis of the cleavage products revealed that 9 of its 30 peptide bonds were hydrolyzed by Cap1. This result was similar to that observed with pig pepsin A and human pepsin A. Cap1 also exhibited milk-clotting activity. We cloned the cDNA of CAP1 gene, which contained a 1254 bp open reading frame encoding a protein of 417 amino acid residues. Homology search in the NCBI database revealed that the amino acid sequence of Cap1 showed less than 39% identity to other known proteins. Therefore, we proposed that Cap1 is a novel aspartic protease.

We examined the sensitivity of the 2-thiobarbituric acid (TBA) reaction for measuring thermal stress in beer, happoshu (a beer-like beverage with <67% malt content), and new genre beverages (beer-like beverages without malt or happoshu diluted with spirit). The thiobarbituric acid index (TBI) analytical procedure has been used to measure thermal stress in malt, wort, and beer. However, the TBI method is not as sensitive for beer. We compared the TBA reaction method used in shochu (a traditional Japanese distilled alcoholic beverage) with the TBI method. We confirmed that the TBA reaction method used for shochu is more suitable for measuring thermal stress in beer and beer-like beverages, whereas the TBI method is more suitable for measuring thermal stress in wort. The difference in the suitability of these methods seemed to be partly owing to the difference in the reaction of TBA with 5-hydroxymethylfurfural and 3-deoxyglucosone, both of which are major intermediates in the Maillard reaction. We concluded that the shochu TBA reaction method could produce a sensitive and practical index of thermal stress inflicted during storage of beer and beer-like beverages.

The amount of protein in food is calculated from total nitrogen. The conventional Kjeldahl method has several disadvantages: the time required for quantitative determination and the use of dangerous reagents such as concentrated sulfuric acid, etc. In contrast, the combustion method is capable of determining total nitrogen in a short time and safely, without the use of dangerous reagents.

This subcommittee was charged with evaluating the combustion method for analysis of total nitrogen in beer, low-malt beer, and third-category beer. The collaborative work was carried out by 11 collaborators. Both repeatability and reproducibility were calculated from the results of the collaborative work.

1. Relative repeatability standard deviation (RSD_r) and repeatability limit (r₉₅) for determination of total nitrogen by the combustion method ranged from 0.8 to 4.1% and from 12.6 to 34.5 mg/L, respectively, and were judged acceptable.
2. Relative reproducibility standard deviation (RSD_R) and reproducibility limit (R₉₅) for determination of total nitrogen by the combustion method ranged from 3.0 to 11.9% and from 36.6 to 115.4 mg/L, respectively, and were judged acceptable.

平成21酒造年度全国新酒鑑評会（第98回鑑評会）は、当該年度生産清酒を全国的に調査研究することにより、製造技術と酒質の現状及び動向を明らかにし、もって清酒の品質及び酒造技術の向上に資するとともに、国民の清酒に対する認識を高めることを目的として、日本酒造組合中央会と共催した。

審査は、酒類総合研究所広島事務所において、平成22年4月21日（水）から23日（金）の3日間に予審を行い、5月11日（火）及び12日（水）に決審を行った。また、5月26日（水）に東広島運動公園体育館で製造技術研究会を、6月16日（水）に東京のサンシャインシティ・ワールドインポートマート展示ホールで公開きき酒会を開催した。

出品の内訳は、第Ⅰ部（山田錦の使用割合が50%以下の部）が145点、第Ⅱ部（山田錦の使用割合が50%超の部）が750点の合計895点であった。審査の結果、優秀と認められた457点を入賞酒とし、さらに、決審において特に優秀と認められた242点に金賞を授与した。また、出品酒を公開する製造技術研究会及び公開きき酒会には、全国から、それぞれ、1,392人及び4,227人が来場した。

出品酒については、酒質の現状及び動向を調査研究するため、調査表の内容を集計するとともに成分分析を行った。

単式蒸留しょうちゅうの品質を全国的に調査研究することにより、製造技術と酒質の現状及び動向を把握するとともに製造業者の参考とするため、第33回本格焼酎鑑評会を日本酒造組合中央会と共催で開催した。出品資格として、単式蒸留しょうちゅう製造免許を有する製造者で、日本酒造組合中央会の組合員に限定した。官能審査は平成22年6月3日（木）及び4日（金）に行い、公開きき酒会は6月25日(金)に当所で開催し、出品関係者の参考に供した。

出品酒の官能審査と成分分析を行ったので、その結果の概要について報告する。

各種の酒母を用いた市販清酒（41点）に含まれるアミンの分析を行った。アグマチンは全ての試料から検出され、平均値は276.6 mg/lであった。フェネチルアミンも全ての試料から検出されたものの、その濃度は最大でも2.31 mg/lであった。ヒスタミン、チラミン、トリプタミン、イソアミルアミン、プトレシン、カダベリン及びスペルミンについては、いずれかの試料において検出されているものの、その濃度は最大でも11.78 mg/lであった。使用した酒母により含有量に有意な差が見られたのは、フェネチルアミン、およびアグマチンで、いずれも山廃酛で含有量が高かった。フェネチルアミンは山廃酛と純米酒、山廃酛と普通酒の含有量に、アグマチンは山廃酛と普通酒の含有量に有意な差が見られた。それ以外のアミンについては、使用した酒母による差は認められなかった。清酒中のアミン含有量は、アグマチンを除けば、ワイン、ビールと同程度またはそれ以下であった。

2,4,6-トリクロロフェノール（TCP）の2,4,6-トリクロロアニソール（TCA、カビ臭の要因）への変換に関与する麴菌のO-メチルトランスフェラーゼ遺伝子を探索するために、我々はAspergillus oryzaeの遺伝子破壊を行い、その変換能が低下した破壊株を選抜した。はじめに、Aspergillus oryzae RIB40ゲノムデータベースよりSAM依存性メチルトランスフェラーゼあるいはO-メチルトランスフェラーゼと推定されている76遺伝子を抽出した。このうち、34遺伝子を個別に破壊した。次に、破壊株を用いてTCPの存在下で製麴を行った。AO080521000231の遺伝子破壊株の麴におけるTCPのTCAへの変換能は非破壊株の麴における変換能に比べて非常に低かったが（12%）、麴はα-アミラーゼ、グルコアミラーゼ、酸性カルボキシペプチダーゼ、酸性プロテアーゼの十分な生産性を保持していた。これらの結果より、AO080521000231がTCPのTCAへの変換に関与する主要なO-メチルトランスフェラーゼをコードしている可能性が示唆された。

Sake yeast strains produce a high concentration of ethanol during sake brewing compared to laboratory yeast strains. As ethanol fermentation by yeast cells continues even after cell growth stops, analysis of the physiological state of the stationary phase cells is very important for understanding the mechanism of producing higher concentrations of ethanol. We compared the physiological characteristics of stationary phase cells of both sake and laboratory yeast strains in an aerobic batch culture and under sake brewing conditions. We unexpectedly found that sake yeast cells in the stationary phase had a lower buoyant density and stress tolerance than did the laboratory yeast cells under both experimental conditions. These results suggest that it is difficult for sake yeast cells to enter a quiescent state after cell growth has stopped, which may be one reason for the higher fermentation rate of sake yeast compared to laboratory yeast strains.

In small-scale sake brewing tests, progression of ethanol fermentation is examined by monitoring carbon dioxide emission using the Fermograph, an automated multi-channel gas monitor instrument. The Fermograph system enables automatic measurements of fermentation profiles with high accuracy, reproducibility, and resolution, and facilitates comprehensive and quantitative sake fermentation kinetic analyses.

Some oligopeptides and amino acids have a strong influence on the sensory qualities of sake, but the formation process of such compounds in sake mash has not been well elucidated. In this study, we investigated the formation process of bitter-tasting peptides derived from rice proteins in sake mash, because knowledge about their formation may contribute to the quality control of sake. We analyzed rice protein hydrolysates in sake mash, as well as in the enzymatic digest of steamed rice grains digested by either sake-koji or by crude enzyme extracted from sake- koji. SDS-PAGE showed that a smaller amount of polypeptides (>M.W. 10,000) accumulated in the supernatant of sake mash than in either enzymatic digest. The concentration of peptides in the supernatant of sake mash increased gradually from the early stages of fermentation. Five bitter-tasting peptides (No. 9, <QLFNPS; No. 13, <QLFNPSTNP; No. 17, <QLFNPSTNPWH; No. 18, <QLFNPSTNPWHSP; No. 20, <QLFGPNVNPWHNP), which were previously found in sake mash, were not found in significant amounts in sake- koji. On the other hand, these peptides accumulated at the early stages of both sake mash fermentation and the enzymatic digests, although the levels in sake mash were higher than those in the digests. The present study demonstrated that the 5 bitter-tasting peptides formed in high concentrations when steamed rice grains were digested under conditions of sake mash fermentation with yeast.

Free medium-chain fatty acids (MCFAs) can negatively influence the fermentation process and taste quality in alcoholic beverages. Ethyl hexanoate is important in providing a fruit-like flavour to drinks, particularly in Japanese sake. In this study, we developed a direct injection method for a gas chromatography-flame ionization detector following the semi-purification of chemical components, such as esters, alcohols and MCFAs in alcoholic beverages. Evaluation of MCFAs by this method gave a limit of detection on the order of sub-ppm and relative standard deviations less than 10% in standard solution. Good repeatability and recovery rates against MCFAs and ethyl hexanoate were also obtained in non-distilled real alcoholic beverages. Because this method enabled us to simultaneously quantify the concentrations of MCFAs and ethyl hexanoate, the proportion of ester against MCFAs was proposed as a quality control index. This method could be suitable for routine analysis in the alcohol beverage industry.

The accumulation and change in the chemical states of Co have been studied to elucidate the role of the yeast Saccharomyces cerevisiae in the migration of radioactive cobalt in the environment. The yeast was grown in a solution containing Co(II) ions, a carbon source, and essential elements (metabolically active condition). For comparison, an adsorption experiment of Co(II) ions on the yeast cells under resting condition without essential elements was performed. Time courses of Co concentration in the solution, in the cells, and chemical states of the accumulated Co were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), particle-induced X-ray emission (PIXE), and X-ray absorption fine structure (XAFS) analyses. The time courses of Co concentration in the solution showed that a higher amount of Co was accumulated by the yeast cells under the metabolically active condition than under the resting one. PIXE analyses showed the concurrent accumulation of Fe with Co accumulation under the metabolically active condition, suggesting the intracellular accumulation of Co. XAFS analyses showed that the k³-weighted extended-XAFS functions and the radial structural function of Co accumulated by the yeast cells under the metabolically active condition are similar to those under the resting condition, indicating that the chemical states of the accumulated Co were nearly the same between both conditions. These results indicate that the yeast performs better retardation of the migration of Co under the metabolically active condition than under the resting one.

To assess the position of Kuro-Koji molds in black Aspergillus, we performed sequence analysis of approximately 2500 nucleotides of partial gene fragments, such as histone 3, on a total of 57 Aspergillus strains, including Aspergillus kawachii NBRC 4308, 12 Kuro-Koji molds isolated from awamori breweries in Japan, Aspergillus niger ATCC 1015, and A. tubingensis ATCC10550. Sequence results showed that all black Aspergillus strains could be classified into 3 types, type N which includes A. niger ATCC 1015, type T which includes A. tubingensis ATCC 10550, and type L which includes A. kawachii NBRC 4308. Phylogenetic analysis showed these three types belong to different clusters. All 12 Kuro-Koji molds isolated from awamori breweries were classified as type L, thus we concluded type L represents the industrial Kuro-Koji molds. We found all type L strains lack the An15g07920 gene which is required for ochratoxin A biosynthesis in black Aspergillus. This sequence is present in the genome of A. niger CBS 513.88 and has homology to the polyketide synthase fragment of A. ochraceus which is involved in ochratoxin A biosynthesis. Based on the industrial importance and the safety of Kuro-Koji molds, we propose to classify the type L strains as Aspergillus luchuensis, as initially reported by Dr. Inui.

Aspergillus oryzae, although closely related to Aspergillus flavus, does not produce aflatoxin (AF). A. oryzae RIB strains can be classified into three groups (group 1-3) based on the structure of the AF biosynthesis gene homolog cluster (AFHC). In group 1 strains, where AFHC is present, the expression level of the aflR gene is extremely low and there is no expression of the other four AF homologue genes (avnA, verB, omtA and vbs). We conducted a detailed structural comparison of AFLR ORF and AFLJ ORF from A. oryzae and A. flavus and identified several amino-acid substitutions. If these substitutions induce inactivation of AFLR and AFLJ, AF biosynthesis of A. oryzae will be doubly inhibited at the transcriptional and translational level. In this study, we transferred aflR and aflJ to A. oryzae RIB67, a group 2 strain where more than half of AFHC is missing. Under control of the pgkA promoter, aflR and aflJ was expressed and avnA, verB, omtA and vbs gene expression were monitored by RT-PCR. We prepared six types of forced-expression vectors, including aflR (from A. oryzae RIB40 or its three mutants) or aflJ (from A. oryzae RIB40 or A. flavus RIB4011). RT-PCR analysis showed that transformants containing aflJ from A. oryzae displayed no expression of AF biosynthetic homologue genes, whereas aflR substitutions had no such effect. These results strongly suggest that the amino-acid substitutions in AFLJ of A. oryzae induce inactivation at the protein level.

To investigate 3'-end-processing signals in Aspergillus oryzae, we created a nucleotide sequence data set of the 3'-untranslated region (3'UTR) plus 100 nucleotides (nt) sequence downstream of the poly(A) site using A. oryzae expressed sequence tags and genomic sequencing data. This data set comprised 1065 sequences derived from 1042 unique genes. The average 3'UTR length in A. oryzae was 241 nt, which is greater than that in yeast but similar to that in plants. The 3'UTR and 100 nt sequence downstream of the poly(A) site is notably U-rich, while the region located 15-30 nt upstream of the poly(A) site is markedly A-rich. The most frequently found hexanucleotide in this A-rich region is AAUGAA, although this sequence accounts for only 6% of all transcripts. These data suggested that A. oryzae has no highly conserved sequence element equivalent to AAUAAA, a mammalian polyadenylation signal. We identified that putative 3'-end-processing signals in A. oryzae, while less well conserved than those in mammals, comprised four sequence elements: the furthest upstream U-rich element, A-rich sequence, cleavage site, and downstream U-rich element flanking the cleavage site. Although these putative 3'-end-processing signals are similar to those in yeast and plants, some notable differences exist between them.

Poly(lactic acid) (PLA) depolymerases are categorized into protease-type and lipase-type. Protease-types can hydrolyze poly(L-lactic acid) (PLLA) but not poly(D-lactic acid) (PDLA). Lipase-types, including cutinase-like enzyme (CLE) from Cryptococcus sp. strain S-2 preferentially hydrolyze PDLA. Both enzymes degraded not only PLA emulsion but also PLA film, in which amorphous region is preferentially attacked, but crystalline region can be also attacked. Stereocomplex PLA (sc-PLA) formed by 50:50 blending of PLLA and PDLA included no homo crystals, but a tiny homo crystallization peak appeared and crystallinity increased by 5% when attacked by CLE, although no significant change of molecular weight and crystalline size was found. Enantioselective degradation must occur in amorphous region of PLLA/PDLA film and preferentially hydrolyzed PDLA, resulting in a slightly excess amount of PLLA remained, which must be crystallized.

Microtubules are central to eukaryotic cell morphogenesis. Microtubule plus-end tracking proteins (CTIPs) transport polarity factors to the cell cortex, thereby playing a key role in both microtubule dynamics and cell polarity. However, the signaling pathway linking CTIPs to cell polarity control remains elusive. Here we show that the fission yeast checkpoint kinase Cds1 (Chk2 homologue) delays the transition of growth polarity from monopolar to bipolar (termed NETO; new-end take-off). The CTIPs CLIP170 homologue Tip1 and kinesin Tea2 are responsible for this delay, which is accompanied by a reduction in microtubule dynamics at the cell tip. Remarkably, microtubule stabilization occurs asymmetrically, prominently at the non-growing cell end, which induces abnormal accumulation of the polarity factor Tea1. Importantly, NETO delay requires activation of calcineurin, which is carried out by Cds1, resulting in Tip1 dephosphorylation. Thus, our study establishes a critical link between calcineurin and checkpoint-dependent cell morphogenesis.

A novel aminopeptidase, Aminopeptidase T (APase T), was purified from porcine skeletal muscle following successive column chromatography: twice on DEAE-cellulose, hydroxyapatite, and Sephacryl S-200 HR using Leu-β-naphthylamide (LeuNap) as a substrate. The molecular mass of the enzyme was 69 kDa on SDS-PAGE. The optimum pH towards LeuNap of the enzyme was about 7. The enzyme activity was strongly inhibited by bestatin and was negatively affected by ethylenediaminetetraacetic acid (EDTA). Chlorine-activated APase T liberated Leu, Ala, Met, Pro, and Arg from Nap derivatives. The APase T gene consisted of an ORF of 1,836 bp encoding a protein of 611 amino acid residues. The APase T was highly homologous to bovine, human, and mouse Leukotriene A(4) hydrolase (LTA(4)H), a bifunctional enzyme which exhibits APase and epoxide hydrolase activity.

Hansenula fabianii J640 highly expresses an extracellular glucoamylase (GA). Here, we purified the GA and showed that it has pH and temperature optima of 5.0 and 50 ℃, is stable at temperatures up to 50 ℃, is inhibited by Ag²⁺, Hg²⁺ and Cu²⁺. The gene was found in an expression library with anti-GA antibodies. A cDNA was found to encode 491 amino acids, including a putative signal peptide of 21 amino acids. Because of the gene's high expression, we used its promoter and terminator regions to improve a previously developed H. fabianii J640 expression system.

We previously developed a host vector system for the wastewater treatment yeast Hansenula fabianii J640. The promoter and terminator regions of the gene encoding glucoamylase from H. fabianii J640 were used for a new expression vector, pHFGE-1. The performance of pHFGE-1 was compared with that of the widely used pG-1 transformant vector. H. fabianii J640 (HF-TAMY) cells were transformed with pHFGE-1, and Saccharomyces cerevisiae YPH-499 (SC-TAMY) cells were transformed with pG-1, both of which carried the Taka-amylase. Expression of Taka-amylase by HF-TAMY showed higher than that by SC-TAMY. By using this new system, we bred the new wastewater treatment yeast that shows α-amylase activity. This yeast appears to grow well under experimental wastewater conditions, and is effective in treating model wastewater containing soluble and insoluble starch.