平成16年度 研究成果

T.Koseki, K.Takahashi, S.Fushinobu, H.Iefuji, K.Iwano, K.Hashizume, and H.Matsuzawa

We cloned the feruloyl esterase A gene from Aspergillus awamori (AwfaeA) and engineered it to study substrate specificity and pH dependence of catalysis. Based on the crystal structures of two type-A feruloyl esterases (FAE-III and AnFAEA) from Aspergillus niger, residues located in the flap region of AwFAEA (Asp71, Thr72, Asp77, and Tyr80) were replaced with corresponding amino acid residues (Ile, Arg, Asn, and Phe), respectively, found in the lid of lipases from Rhizomucor miehei(RmLIP) and Humicola lanuginose(HlLIP). Furthermore, Asp77 of AwFAEA, which is conserved in Aspergillus FAEs and lipases, was replaced with a hydrophobic residue (Ile). Kinetic analysis of the mutant enzymes showed that the higher catalytic efficiency of the D77I and Y80F mutants toward α-naphthylbutyrate (C4) and α-naphthylcaprylate (C8), respectively, was due to a lower Km value. The higher catalytic efficiency of D77N toward C4 substrate was due to combination of decreased Km and considerably increased kcat. The D71I and Y80F mutants showed some activity toward long-acyl chain esters. On the other hand, the D77I mutant had no detectable activity toward phenolic acid methyl esters and feruloylated arabinoxylan. Moreover, the pH optima of the D77I, D77N, and Y80F mutants increased from 5.0 to 7.0-8.0, 7.0, and 6.0, respectively.

奥田将生、櫻尾尚平、沼田美子代、小関卓也、畑冨士夫、蓮尾徹夫、庄嶋修、荒巻功、橋爪克己

収穫後15℃以下で5～7年貯蔵された16種類の米試料の酒造適性を2003年に収穫された日本晴と 比較して検討した。長期貯蔵米のいくつかは、テストミル及び60kgスケールの精米機の試験で高い損失（無効精米） と高い砕粒率を示した。この精米試験における特性は試料間で大きく異なっていた。長期貯蔵米の吸水は新米と比較してやや低くかったが、溶解性は新米とほぼ同じであった。長期貯蔵米はカリウムとヘキサナールを新米と比較して高濃度に含んでいたが、長期貯蔵米を蒸して放冷した後にはヘキサナールは検出されなかった。長期貯蔵米のもろみはわずかに速い発酵速度を示し、その酒化率は新米と比較してわずかに高かった。長期貯蔵米の製成酒は、苦味、雑味が指摘されるものもあったが、官能評価の総合得点は新米の酒と有意な差は見られなかった。小仕込酒のジメチルスルフィドのレベルは全て1ppb未満で、酒における推定閾値（10ppb）よりはるかに低かった。

O.Yamada, K.Sakamoto, M.Tominaga, T.Nakayama, T.Koseki, A.Fujita and O.Akita

We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the α-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.

K.Matsumura, H.Hisada, H.Obata, Y.Hata, A.Kawato, Y.Abe and O.Akita

We cloned a novel gene (aoxA) encoding amine oxidase (AOX) from Aspergillus oryzae. One cDNA clone showing extreme homology to the AOX-encoding genes was found in an expressed sequence tag (EST) library of A. oryzae. Molecular analysis revealed that the aoxA carried four exons interrupted by three introns and had an open reading frame encoding 672 amino acid residues. The deduced amino acid sequence showed about 83.5% identity to the Aspergillus niger AO-I. The strictly conserved residues for co-factor and copper binding in copper/quinine-containing AOXs were also preserved at Tyr 405, His 456, His 458 and His 617 in the cDNA sequence. When the aoxA was overexpressed in the homologous hyperexpression system of A. oryzae, AOX activity in the transformant was enhanced 75-fold. An apparent molecular weight of 159,000 by gel filtration and a subunit molecular weight of 75,000 by SDS-PAGE of the purified enzyme were estimated, suggesting that the enzyme molecule is a homo-dimer similar to other copper/quinine-containing AOXs. The A. oryzae AOXA preferentially oxidized aliphatic monoamines of C2-C6 rather than aromatic amines or diamines. From these results, the aoxA gene product obtained by homologous hyperexpression system of A. oryzae is undoubtedly a functional AOX.

S.Ando, Y.Nishiguchi, K.Hayasaka, Y.Yoshihara, J.Takahashi, H.Iefuji

The in vitro degradability of yeast and the effect of yeast on the in vitro degradability of forage may differ in terms of the specific yeast strains or their incubation conditions. Thus in experiment I, two strains of sake yeast (strainK7 and strainK9) and one strain of bakers' yeast (KY5649) were incubated in an aerobic condition. In experiment 2, aerobically or anaerobically incubated K7 was used for investigating the in vitro degradability of yeast, the effect of yeast on the in vitro degradability of forage, and the degradability of yeast by pepsin and pronase treatment. The in vitro degradability of bakers' yeast was significantly (p<0.05) higher than those of sake yeasts. The in vitro degradability of anaerobically incubated yeast was significantly (p<0.01) higher than that of aerobically incubated yeast. The degradability of bakers' yeast by pepsin treatment was significantly (p<0.01) higher than that of the sake yeasts. The degradability of bakers' yeast by pronase treatment was slightly higher than that of the two sake yeasts, while the degradability of anaerobically incubated yeast by both enzymes, respectively, was significantly (p<0.01) higher than that of aerobically incubated yeast. The degradability of forages was increased significantly (p<0.05) by the addition of yeasts. The degradability of roughage by sake yeast tended to be higher than that by the bakers' yeast. The degradability of roughage was significantly (p<0.05) higher by anaerobically incubated yeast than by aerobically incubated yeast. Given the above results, it seems that in vitro degradability of yeast and the magnitude of the increment of roughage degradation differ among the yeast strains and their incubation conditions.

Y.Kadota, S.Hirose, H.Iefuji, T.Matsushita, Y.Matsumoto, R.Ueoka

Development of a new processing method for the effective use of rice-Shochu distillation remnants is an important subject for the reduction of environmental pollution. In this study, inhibitory effects on the growth of human stomach tumor cells along with the induction of apoptosis were obtained for powder treated with ethanol from freeze-dried supernatant of rice-Shochu distillation remnants in vitro. Furthermore, the powder obtained from rice-Shochu distillation remnants showed no toxicity to normal rats in vivo experiments. These results suggest that rice-Shochu distillation remnants might be effectively used not only as medicine but also as food supplements for health.

野村佳司、水野昭博、木曽邦明

これまでの研究で、高温貯蔵清酒とTBAが反応し、マロンアルデヒドがTBA反応するとできる赤色色素が生成されること、及び清酒のTBA反応は酸化した脂質が原因ではなく、3-デオキシグルコソンが部分的に関与していることを報告した。本報では、ピルビン酸や他のカルボニル成分の影響のない清酒中の3-DG濃度の測定法を開発した。市販酒の3-DG濃度は官能審査による劣化の程度と相関があることを確認した。市販酒のTBA反応値、3-DG濃度及び着色度の多変量解析と清酒の日光照射実験の結果、3-DG濃度のみならず、メラノイジンもTBA反応に影響していることが示唆された。

M.Kaya, K.Matsumura, K.Higashida, Y.Hata, A.Kawato, Y.Abe, O.Akita, N.Takaya and H.Shoun

We cloned and characterized the gene and cDNA of Aspergillus oryzae cytochrome P450nor (Anor). The Anor gene (nicA; CYP55A5) has a different gene structure from other P450nor genes in that it has an extra intron. There were not only two kinds of mRNA but also two sets of TATA-box and CCAAT-box, and it appears that this gene has two expression patterns, like CYP55A1 of Fusarium oxysporum. A reporter analysis using the uidA gene indicated that gene expression of CYP55A5 was induced under anaerobic conditions, like CYP55A1. When the CYP55A5 gene was overexpressed in A. oryzae, a large amount of active Anor were accumulated as intracellular protein. Anor employed both NADH and NADPH as electron donors for reducing nitric oxide to nitrous oxide. Anor measured the amount of NO generated from 3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine (NOC5) with a spectrophotometer. The sensitivity was 10 nmol/ml.

宇都宮仁、磯谷敦子、岩田博

清酒の香味特性の参照標準とする物質及びその添加量を定めるため、エステル（酢酸エチル、酢酸イソアミル、カプロン酸エチル）、アルコール（イソアミルアルコール、フェネチルアルコール）及び有機酸（酢酸、酪酸、イソ吉草酸、カプロン酸、カプリル酸）について検知閾値、認知閾値及び90%認知閾値を求めた。前報で報告したものも含め清酒へ添加した20種類の匂い物質の閾値を、ビールに添加した匂い物質の閾値と比較すると酢酸イソアミル、フェネチルアルコール、イソバレルアルデヒド、DMS、TCA、酢酸、カプロン酸及びカプリル酸では低かったが、他10成分（ソトロン、フルフラールを除く）は大きく異なるものではなかった。

宇都宮仁、磯谷敦子、岩田博

消費者に有用な情報を提供することを目的に、専門家パネルの官能評価結果と分析値を基に重回帰を行い、さらにこの回帰式を単純化して甘辛区分判別式を定義した。
AV = G - A
AV：新甘辛度 、G：グルコース (g/dl) 、A：酸度 (ml)
（AV：0.2以下を辛口、0.3から1.0をやや辛口、1.1から1.8をやや甘口、1.9以上を甘口）

清酒の飲用経験が豊富なパネル及び飲用経験が少ないパネルにより市販清酒の官能評価を行い、甘辛表示と甘辛評価が異ならないことを確認した。

後藤（山本）奈美、万光華、沼田美子代、荒巻功、橋爪克己

Simple Sequence Repeat (SSR) 解析は高感度なDNA多型解析であり、RAPD解析よりも再現性が高いことが知られている。SSR解析はブドウの品種同定に適した方法と考えられるが、SSR断片長は研究グループによって若干異なる場合があり、これは断片の検出方法の違いによるものと推定される。そこで、不明品種のSSRデータと他のグループから報告されている品種のSSRデータの相関係数を求め、SSRデータが同じ傾向を示す品種を選択した上で、共通に分析されている品種のデータの差異を補正し、SSRデータが一致するかどうかを検討した。この方法で、Pinot blancとして植栽されているがPinot noirと異なるSSRデータを示す2サンプル（PB1、PB2）の品種同定を試み、PB1はSauvignon blancとSSRデータが一致すること、及びPB2はMelonと大部分のSSRデータが一致し、Melonに近い品種と推定されることが示された。このように、対照となる品種が供試できない場合でも、既報のSSRデータとの比較で、不明品種の同定、又は類似する品種の推定が可能であった。

A.Miyanaga, T.Koseki, H.Matsuzawa, T.Wakagi, H.Shoun and S.Fushinobu

As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-L-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecule is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cystein residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.

K.Tamura, Y.Gu, Q.Wang, T.Yamada, K.Ito and H.Shimoi

DNA microarray and Northern blot analysis revealed that a sake yeast strain Kyokai no. 7 (K7) showed higher expression of genes encoding proteins involved in ergosterol biosynthesis than a laboratory yeast strain X2180. We hypothesized that these differences in expression levels were caused by a defect of a transcriptional factor Hap1, because strain X2180 contained a Ty1 insertion mutation in the HAP1 gene. To confirm this, we constructed a strain X2180 derivative (strain HX) that contained the wild-type HAP1 genes originating from strain K7. The expression levels of ergosterol-related genes and cellular ergosterol content in strain HX were higher than those in strain X2180 and were almost comparable to those in strain K7. These results suggest that the differences in the expression levels of ergosterol-related genes and ergosterol content between strains K7 and X2180 were largely caused by the hap1 mutation in strain X2180. Involvement of the mutated Hap1 in the differential gene expression between strain K7 and strain X2180 was further confirmed by a lacZ reporter assay of HMG1, one of the Hap1-regulated genes. We also revealed that the HMG1 promoter region between -500 and -376 was important in the transcriptional activation by Hap1.

H.Kitagaki, K.Ito and H.Shimoi

Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a △dcw1 △dfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3△dfg5). When DC61 cells were incubated at 37℃, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37℃.At 37℃, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37℃, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37℃, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (△dcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

水野昭博、篠田典子、野村佳司

我々は、発酵度を制御する方法として固定化α-グルコシダーゼの利用を研究した。固定化α-グルコシダーゼは、70%エタノールによって反応器中で10分間殺菌され、94%の活性が残存した。殺菌後、発酵中の麦汁が発酵の異なる段階でポンプによって反応器を通して循環された。固定化α-グルコシダーゼを含む反応器の使用は、α-グルコシダーゼの反応をさせる発酵の段階に依存して、20%Platoの麦汁の真性発酵度を増加又は減少させた。発酵の開始から48時間麦汁を反応器を通して循環した場合には、α-グルコシダーゼの糖転移反応によるイソマルトース、パノース及びイソマルトトリオースの非発酵性糖の生成が、ビールの発酵度を対照と比較して11%減少させる結果となった。反応器を7日目以後に使用した場合には、α-グルコシダーゼの加水分解反応による多糖類からのグルコースの生成が、ビールの発酵度を10%増加させる結果となった。いずれの場合も、酢酸の生成は減少した。固定化α-グルコシダーゼを使用する方法は、ビールの発酵度を自由に制御し得る手段として有効であるばかりではなく、高濃度醸造における発酵度の低下と酢酸生成の増加の両問題を克服し得ることが判明した。

水野昭博、篠田典子、野村佳司

高濃度醸造は、製造場の効率増加のために広く行われるようになった。しかしながら、高濃度の糖分とエタノール分による酵母への高いストレスのために発酵中に酵母活性を良好に保つことは容易ではない。それ故、発酵度が不十分になる傾向があり、製品ビールは高濃度の酢酸とエステルを含む。

我々は、高濃度醸造における幾つかの課題を克服できる新しい発酵方法を報告した。この方法においては、発酵の最初に添加したα-グルコシダーゼによる糖転移反応と加水分解反応による麦汁の糖組成の変換が、酵母によるエタノール生成と並行して進行する。α-グルコシダーゼの使用によって、我々は上面発酵ビール酵母を用いた高濃度醸造を15℃の発酵温度で成功した。しかし、下面発酵ビール酵母が世界中の商業醸造において、一般的に使用されている。そこで、この報文では、下面発酵ビール酵母を用いたその方法の結果を報告する。我々は、その方法が低温において下面発酵ビール酵母を用いた高濃度麦汁の発酵に問題なく適用できることを確認した。α-グルコシダーゼを20%Platoの麦汁へ800mg/の濃度で添加した場合、下面発酵ビール酵母W34/70を用いて10℃で問題なく発酵できた。その真性発酵度は77%と対照より16%高く、酢酸濃度は200mg/lと対照より85mg/l低かった。α-グルコシダーゼの添加は、デキストリンを含有する麦汁の発酵度の向上にも同様に有効であった。6.2%のデキストリンを含有する20%Platoの麦汁へα-グルコシダーゼを400mg/lの濃度で添加した場合、上面発酵ビール酵母NCYC1245を用いた発酵の最後にはデキストリン含量は3.7%まで減少し、その真性発酵度は75%と対照より19%高かった。α-グルコシダーゼの使用は、糖化力の低い麦芽から製造される発酵性糖含量の低い麦汁から良質のビールを醸造する場合に有効であることが示された。発酵における酵素の使用は、熟成期間の短縮のためのアセト乳酸脱炭酸酵素やドライビール醸造のためのグルコアミラーゼの使用という既存の方法以外にも多くの新しい可能性を広げる。

Y.Manabe, M.Shobayashi, T.Kurosu, S.Sakata, T.Fushiki, H.Iefuji

The process of sake making generates a great deal of secondary product called sake lees. Part of the sake lees is disposed of as waste, so finding ways to recycle it are a matter of concern. Because sake lees contains many nutritious substances derived from both rice and sake yeast, we studied the physiological effects of these materials on rats. Sprague-Dawley rats were fed one of three experimental diets for two weeks. Rats fed diets containing either 20% sake lees or 2% sake yeast (Kyokai 9 yeast) both had higher spontaneous locomotive activity than rats fed a control casein diet. Body weight and food consumption were not different among the three groups. These results suggest that sake lees and sake yeast have a reinforcing effect on spontaneous locomotive activity.

宇都宮仁、磯谷敦子、岩田博

清酒の香味特性の参照標準とする物質及びその添加量を定めるため、アセトアルデヒド、イソバレルアルデヒド、ベンズアルデヒド、ジアセチル、フルフラール、ソトロン、エチルメルカプタン、ジメチルスルフィド、ジメチルトリスルフィド、及び2, 4, 6-トリクロロアニソールについて検知閾値、認知閾値及び90%認知閾値を求めた。イソバレルアルデヒド、ソトロンの検知閾値は、既報に比べ10倍以上低かった。

K.Matsumura, H.Obata, Y.Hata, A.Kawato, Y.Abe and O.Akita

We cloned and characterized a novel gene (abfA) encoding α-L-arabinofuranosidase (α-L-AFase) from Aspergillus oryzae. One clone homologous to the α-L-AFase gene of Thermotoga maritima was found in an expressed sequence tag (EST) library of A. oryzae and a corresponding gene was isolated. Molecular analysis showed that the abfA gene carried six exons interrupted by five introns and had an open reading frame encoding 481 amino acid residues. The amino acid sequence similarity at active sites to the α-L-AFases from other organisms indicated that the α-L-AFase encoded by abfA was classified as a family 51 glycoside hydrolase. When the abfA was overexpressed in the homologous hyperexpression system of A. oryzae, a large amount of α-L-AFase was produced as intracellular protein. The apparent molecular mass of the purified enzyme was estimated to be 228,000 by gel filtration and that of its subunit as 55,000 by SDS-PAGE, suggesting that the enzyme is a tetramer. The enzyme hydrolyzed p-nitrophenyl-α-L-arabinofuranoside but not other p-nitrophenyl glycosides. These results demonstrated that the abfA gene encodes a functional α-L-AFase.

小関卓也、奥田将生、米原由希、八田一隆、岩田博、荒巻功、橋爪克己

原料米の酒造適性に及ぼすイネ登熟期の高温の影響を調べた。登熟期に高温で栽培された山田錦及び日本晴は20分、120分及び蒸米吸水率の上昇がみられたが、消化性(Brix糖度)は低下した。一方、粗蛋白質及びカリウム含量は登熟期の気温とともに日照によっても影響されることが示唆された。また、ラピッドビスコアナライザー(RVA)による粘度特性、示差走査熱量計(DSC)による糊化特性を調べた。RVAパラメーターの糊化開始温度、最高粘度及びブレークダウンとDSCパラメーターの糊化開始温度、ピーク温度及び糊化熱は高温区で高くなり、米澱粉の物理化学的特性は気象条件によって影響を受けることが確認された。これらの結果より気象条件は原料米の酒造適性と相関していることが示唆された。

橋爪克己、大浦宏夫、大沼奈緒子、荒巻功

西洋ナシ、ラ・フランスの芳香に着目したワイン醸造法及び製成されたワインの揮発成分について検討を行った。ワインのGC-臭いかぎ法による分析、及び生果実とワインの香気成分の比較分析により、酢酸n-ブチル及び酢酸n-ヘキシルをラ・フランス果実を想起させる特徴的な香りに寄与している主要な成分として検出した。西洋ナシの醸し発酵は、果汁発酵と比較して、酢酸n-ブチル及び酢酸n-ヘキシルを多く含む、特徴香の強いワインを醸出した。果肉から醸し発酵により得られたワインは、これらエステルのレベルが最も高く試験醸造したワイン中では最も高い評価を得た。果皮と果肉から得られたワインは最も高いフェノールレベルを示し最も褐色がかった色を呈し、また官能評価の成績は低かった。一方、果芯と果肉から得られたワインは最も鮮やかな赤色を呈した。

野村佳司、水野昭博、木曽邦明

前報では、清酒とTBAとの反応で、マロンアルデヒドとTBAとが反応して生成される赤色色素が生成されると報告した。しかし、清酒のTBA反応性物質はまだ究明されていない。一般に過酸化脂質がTBA反応の原因となり、赤色色素が生成されると言われている。我々は清酒中の高級脂肪酸の濃度を分析し、TBA反応は過酸化脂質が原因ではないと判断した。そこで、清酒中に存在する多くのカルボニル化合物のTBA反応性を調べ、それらの中で、3DGのみが反応液の530nmの波長を吸光する着色を示した。そして、3DGとTBAがマロンアルデヒドのTBA反応物である赤色色素と同一の物質を生成することが判明した。

岩田博、磯谷敦子、宇都宮仁、西尾尚道

口噛み酒製造工程へのα-グルコシダーゼの働きを検討するため、生米と炊飯米で口噛み試験を行い、糖組成、α-グルコシダーゼ活性、pH及び有機酸組成の各変化を調べた。

糖組成について、24時間後で、生米はグルコースが5%、マルトースが0%であったのに対し、炊飯米ではマルトースが5%、グルコースが0.8%で両者は全く異なっていた。この理由は、糖組成とα-グルコシダーゼ活性の測定結果から、唾液アミラーゼにより生成されたマルトースが、生米のα-グルコシダーゼによりグルコースに分解されたためと推定された。また、有機酸組成の検討から、微生物が生成した乳酸などによるpH低下と酵素抽出促進効果がおこり、α-グルコシダーゼが有効に機能する環境条件下で口噛み試験の反応が起こっていることが分かった。

以上から、口噛み酒は生米のα-グルコシダーゼを巧みに利用した酒造りであると推定された。

H.Maeda, M.Sano, Y.Maruyama, T.Tanno, T.Akao, Y.Totsuka, M.Endo, R.Sakurada, Y.Yamagata, M.Machida, O.Akita, F.Hasegawa, K.Abe, K.Gomi, T.Nakajima, Y.Iguchi

Aspergillus oryzae is a fungus used extensively in the fermentation industry. We constructed cDNA microarrays comprising 2,070 highly expressed cDNAs selected from the ～6,000 non-redundant expressed sequence tags (ESTs) in the A. oryzae EST database (http://www.aist.go.jp/RIODB/ffdb/index.html). Using the cDNA microarrays, we analyzed the gene expression profiles of A. oryzae cells grown under the glucose-rich (AC) and glucose-depleted (AN) liquid culture conditions used during the construction of the EST database. The sets of genes identified by the cDNA microarray as highly expressed under each culture condition agreed well with the highly redundant ESTs obtained under the same conditions. In particular, transcription levels of most catabolic genes of the glycolytic pathway (EMP) and tricarboxylic acid (TCA) cycle were higher under AC than AN conditions, suggesting that A. oryzae uses both EMP and TCA for glucose metabolism under AC conditions. We further studied the expression of genes encoding hydrolytic enzymes and enzymes involved in energy catabolism by using three industrial solid-phase biomass media, including wheat-bran. The wheat-bran culture gave the richest gene expression profile of hydrolytic enzymes and the lowest expression levels of catabolic genes (EMP,TCA) among the three media tested. The low expression levels of catabolic genes in the wheat-bran culture may release catabolite epression, consequently leading to the rich expression profiles of the hydrolytic enzymes.

Y.Manabe, H.Utsunomiya, K.Gotoh, T.Kurosu, T.Fushiki

Selection of foods by animals largely depends on their physiological condition. In this paper, we reported how physiological changes in rats after ingestion of sake (Japanese rice wine) affected their preference for different kinds of sake. Rats could discriminate among various kinds of sakes in a two-bottle choice test, even after adjustment of the glucose concentration or alcohol concentration of the sake, suggesting that the choice of a sake by animals was based on more than one ingredient. To identify effect of a rat's physiological condition on the selection of sake, we monitored the levels of blood glucose, nonesterified fatty acids (NEFA), and ketone bodies in serum in mice after forced intragastric ingestion of sake that had previously been offered to the rats in a two-bottle choice test. Blood glucose levels in mice were not different between the rats fed the palatable and unpalatable sakes, and the NEFA level and ketone level were high in rats fed the unpalatable sake. To further clarify the relationship between physiological condition and preference for sake, rats were offered eight kinds of Junmai-syu, which is made with only rice and no sub-ingredient. Rats could still discriminate among Junmai-syu. Furthermore, with two exceptions, the preference for a sake was significantly correlated with the ketone level. The order in the ratio of blood insulin level to blood glucagon level, which is an indicator of metabolism, was correlated with the order in preference to sake. These results suggest that some physiological factors besides oral stimulation also are important factors in the selection of a sake.

野村佳司、内藤貴文、小野晃、三上重明、高橋利郎、木曽邦明

近年、清酒はしばしば店頭で蛍光灯に照らされた冷蔵ショーケースに入れて販売される。しかし、この場合、清酒はよく着色していることがある。これは蛍光灯の光線の影響である様だ。我々は清酒の着色に対する蛍光灯の影響を調べた。清酒の着色を6種類の蛍光灯で調べた結果、三波長域発光型が最も清酒を着色させ、次に昼白色が清酒を着色させ、美術博物館用が最も清酒を着色させない。我々は4種類のカラーフィルムの清酒の着色に与える影響を調べた。レッドフィルムが着色防止に最も効果があり、ブルーフィルムが最も効果がなかった。多変量解析の結果、450nm以下の光が清酒の着色に影響するということがわかった。

S.Kobayashi, N.Goto-Yamamoto, H.Hirochika

The transcript of a grape myb-related gene, VvmybA1, was detected in mature berry skins of red-skinned bud sports but not in those of their white-skinned progenitors. The white-skinned progenitors have only one gene (VvmybA1a) for VvmybA1, while the red-skinned sports contain two kinds of the gene (VvmybA1a and VvmybA1b). In VvmybA1a, a retrotransposon, Gret1, was inserted into a 5'-flanking region near the coding sequences of the gene, whereas Gret1 was missing in VvmybA1b, leaving behind its solo LTR. These results show that the bud mutation from white to red in berry skins of the grapes was caused by the deletion of Gret1.

S.T.Jeong, N.Goto-Yamamoto, S.Kobayashi, M.Esaka

Anthocyanins are important grape components for red wine making. Here we examined the effects of plant hormones, abscisic acid (ABA) and naphthaleneacetic acid (NAA), and shading treatments on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins. The accumulation of anthocyanins was enhanced by ABA treatment and suppressed by NAA and shading. ABA enhanced the mRNA accumulation of VvmybA1, a putative regulatory gene of anthocyanin biosynthesis of grape, and all the tested enzyme genes of the anthocyanin biosynthetic pathway, while NAA and shading suppressed and retarded them. The change of the mRNA copy number of VvmybA1 coincided with that of all the tested anthocyanin biosynthetic enzyme genes and the accumulation of anthocyanins. We also showed that there were significant differences in the mRNA copy number among three Chss, between two Chis, and two F3hs during the coloration of grape berry skins.

A.Miyanaga, T.Koseki, H.Matsuzawa, T.Wakagi, H.Shouna and S.Fushinobua

α-L-Arabinofuranosidase (EC 3.2.1.55) is one of the hemicellulases that cleave the gylcosidic bonds between L-arabinofuranoside side chains and various oligosaccharides. In this study, the first crystallization and preliminary X-ray analysis ofα-L-arabinofuranosidase B from Aspergillus kawachii IFO4308 (AkAbfB), a family 54 glycoside hydrolase, is described. Recombinant AkAbfB was expressed in Escherichia coli and Pichia pastoris. The native crystals of recombinant AkAbfB produced by P.pastoris belong to the orthorhombic space group P212121 (unit-cell parameters a=39.5, b=98.2, c=144.0Å) and diffracted X-rays to a resolution of 1.82Å.

T.Itani, M.Tamaki, Y.Hayata, T.Fushimi and K.Hashizume

Aromatic strength of aromatic rice varies with the genetic and environmental conditions. We determined the concentration of 2-acetyl-1-pyrroline (2AP), a key compound of the aroma of aromatic rice, in 62 samples of rice grains (brown rice) from 'Hieri' produced by 17-24 farmers in 3 years in the Kubokawa area of Kochi Prefecture, Japan. Many of them showed similar values and the standard deviations were 27-31%. However, a few samples showed extremely high (200%) or low (60%) 2AP concentrations compared to the individual year averages (100%). The influence of harvest time and temperature during ripening on the 2AP concentration in the brown rice was also examined using two cultivars. During grain development in an early-heading cultivar 'Miyakaori', the 2AP concentration in the brown rice reached a peak at 4 or 5 weeks after heading (WAH) and then decreased rapidly to 20% of the maximum at 7 or 8 WAH. In a late-heading cultivar 'Hieri', the 2AP concentration peaked at 4 WAH then gradually decreased to 40% of the maximum at 8 WAH. The 2AP concentration was higher in brown rice ripened at a low temperature (day:25℃/night:20℃) than that ripened at a high temperature (day:35℃/night:30℃) in both a short-grain cultivar 'Hieri' and a long-grain cultivar 'Sari Queen'.

荒巻功、吉井美華、岩瀬新吾、江辺正英、菅野喜浩、菊永雪絵、奥田将生、小関卓也、橋爪克己

酒造好適米9品種を含む14品種の70%精白米胚乳中のアミロペクチンの構造をイソアミラーゼ加水分解の後にHPAEC-PADにより分析した。酒造好適米のイソアミラーゼ加水分解物について、コシヒカリのそれと比較したところ、重合度7-9付近の短鎖が多く、逆に、山田錦、八反錦1号は比較的長鎖が少なく、また、五百万石、おくほまれ、八反錦1号は中鎖が少なかった。全試料について分析した結果、短鎖（重合度：6-9）は吸水又は消化性との間に正の相関を示し、長鎖（重合度：35-41）は消化性と負の相関を示した。また、長鎖（重合度：43-51）とDSCパラメーター（糊化ピーク温度、糊化終了温度、及び糊化転移熱量）の間には正の相関が存在し、一方に、短鎖（重合度：7-11）とこれらのDSCパラメーターの間には負の相関が存在した。RVAパラメーターは、アミロペクチン側鎖のいくつかのものと比較的高い相関関係を示したが、しかしながら、それらは消化性やDSCパラメーターとの間にみられたような高いものではなかった。

磯谷敦子、宇都宮仁、岩田博

清酒の熟成香に対するソトロンおよびフルフラールのの寄与について検討した。これらの化合物はGC-Olfactometryにより熟成酒から強く検出された。また、これらの化合物の定量を溶媒抽出およびGC-MS分析により行った。ソトロンについては、内部標準物質として3-オクタノールの代わりに¹³C標識したソトロンを用いることで、変動係数が10%以下に減少した。熟成酒中のソトロンの濃度は認知閾値を超えていたが、フルフラールは超えていなかった。33年貯蔵酒におけるソトロンのオダーユニットは45であった。したがって清酒の熟成香にはソトロンが大きく寄与していると考えられた。また、ソトロンの前駆物質と考えられているα-ケト酪酸およびアセトアルデヒドとソトロンとの間には相関がみられたが、他の生成経路の関与の可能性も示唆された。

岩田 博、磯谷敦子、宇都宮仁、西尾尚道

米に含まれるα-グルコシダーゼの清酒醸造の浸漬・蒸きょう工程に及ぼす影響を検討した。浸漬中に生成されるグルコース量は、浸漬時間と浸漬温度の増大により増加した。α-グルコシダーゼの特異的阻害剤であるミグリトールは、浸漬中でのグルコース生成を強く阻害した。α-グルコシダーゼ活性は、浸漬0時を除く、浸漬中に生成されるグルコース量と正の相関があった。消化性試験でミグリトールを加えた場合、蒸米吸水率とBrix値は対照より低かった。これらの結果から、米α-グルコシダーゼは、浸漬・蒸きょう工程にわずかながら影響を及ぼしていることが示唆された。

S.Kubota, I.Takeo, K.Kume, M.Kanai, A.Shitamukai, M.Mizunuma, T.Miyakawa, H.Shimoi, H.Iefuji, D.Hirata.

The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds ofalcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. Thisstudy shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion ofF-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, thenegative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and theamount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes atransient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains inthe complete set of 4847 non-essential gene deletions and identified at least 256 genes that are importantfor cell growth in the presence of ethanol.

H.Obata. H.Ishida, A.Kawato, Y.Abe, T.Akao, O.Akita and E.Ichishima

We have cloned a novel tyrosinase-encoding gene (melB) specifically expressed in solid-state culture of Aspergillus oryzae. A tyrosinase-encoding gene (melO) from A.oryzae was already cloned and the protein structures of its catalytic and copper binding domains were investigated. However, our recent results revealed that the melO gene was highly expressed in submerged culture but not in solid-state culture. Because tyrosinase activity was also detected in solid-state culture, we assumed that another tyrosinase gene other than melO is expressed in solid-state culture. Another tvrosinase gene was screened using the express ed sequence tag (EST) library. One redundant cDNA clone homologous with the tyrosinase gene was found in the collection of wheat bran culture. Northern blot analysis revealed that the gene corresponding to the cDNA clone was specifically expressed in solid-state culture (koji making), but not in submerged culture. Molecular cloning showed that the gene carried six exons interrupted by. five introns and had an open reading frame encoding 616 amino acid residues. This gene was designated as melB . The deduced amino acid sequence of the gene had weak homology (24%-33%) with MelO and other fungal tyrosinase but the sequences of the copper binding domains were highly conserved. When the melB gene was expressed under the control of the glaB promoter in solid-state culture, tyrosinase activity was markedly enhanced and the culture mass was browned with the melanization by MelB tyrosinase. These results indicated that the melB gene encodes a novel tyrosinase associated with melanization in solid-state culture.