平成13年度 研究成果

下飯仁

清酒酵母協会7号は35℃でパントテン酸要求性を示し、この性質は他の酵母との識別のために利用されている。我々は、 実験室酵母のECM31が協会7号のパントテン酸要求性を相補することを見出した。ECM31は、協会7号とその変異株の形質転換における選択マーカーとして利用可能である。清酒酵母は、発酵が盛んな時期に高泡を形成することが知られている。我々は、高泡形成能を持つ清酒酵母から泡なし変異株に高泡形成能を付与する遺伝子をクローニングし、AWA1と名づけた。解析の結果、AWA1はGPIアンカー型の細胞壁タンパク質をコードしていることがわかった。高泡形成酵母のAWA1を遺伝子破壊したところ、泡なしとなったことから、AWA1は高泡の形成に直接関与しているものと考えられた。清酒酵母の変異株として高濃度のエタノールの存在下でも死滅しにくいエタノール耐性酵母が育種されている。我々は、エタノール耐性酵母のエタノール耐性の原因を解明するために、DNAマイクロアレイを用いて遺伝子発現プロファイルを解析した。その結果、エタノール耐性酵母では、熱やエタノールなどのストレス存在下で発現する遺伝子群が高発現することによってエタノール耐性となっていることが明らかとなった。清酒酵母が含まれているSaccharomyces cerevisiaeは他の微生物に比べて強いエタノール耐性を持っているが、その詳しいメカニズムについては不明な点が多い。我々は、染色体へのトランスポソンの挿入によってエタノール感受性となる変異株を検索した結果、BEM2、ROM2、VPS34、PAT1、ADA2の変異がエタノール感受性をもたらすことを明らかにした。

Y. Yamane, J. Fujita, R. Shimizu, A. Hiyoshi, H. Fukuda, Y. Kizaki, and S. Wakabayashi

The production of cellulose-(CEL), xylan-(XYL), and pectin-degrading enzymes (PEC) by a koji mold, Aspergillus oryzae, was studied, and their contribution to the maceration of the rice endosperm cell wall were investigated with regard to the utilization of available rice in the sake mash. The sake koji mold showed higher CEL and XYL productivities, whereas the miso and soy sauce koji molds showed higher PEC productivity. Statistical analyses indicated that CEL and XYL contribute predominantly and synergistically to the maceration of the rice endosperm cell wall. A. oryzae produced at least three kinds of CEL (Cel-1,2,3) and two kinds of XYL (Xyl-1,2) when cultured in a wheat bran medium. In the solid-state culture, the production of Cel-3 and Xyl-2 was markedly stimulated by decreasing the moisture content of the solid substrate, although the production levels of Cel-1 and Xyl-1 were almost the same. These data suggest that the production of Cel-3 and Xyl-2 is strongly influenced by culture conditions, and that water activity is one of the dominant factors in the regulation of their production.

水野昭博、岩淵正文、木曽邦明、佐藤和夫、高橋利郎

協会14号の2-デオキシグルコース耐性株からリンゴ酸生産能の高い自然変異株を得た。得られた変異株を用いて100kgのパイロットスケールの清酒醸造試験を行い、高いリンゴ酸生成能を確認した。また、製成酒の官能評価において、2-デオキシグルコース耐性株による製成酒は味がなめらかとの指摘が多く、対照の親株を用いた仕込みより総合品質は高かった。

H. Iwata, T. Suzuki, K. Takahashi, and I. Aramaki

In our previous study, we proposed a new alcoholic beverage called nuka-sake, which is made from uncooked rice polish without any enzyme source such as koji. In nuka-sake brewing, the uncooked rice polish serves not only as the fermentation material but also as the enzyme source. In the present study, the results of both laboratory-scale nuka-sake brewing runs with various grades of rice polish and analysis of amylolitic enzyme distribution in rice polish suggested that α-glucosidase (EC 3.2.1.20) is a key enzyme of parallel fermentation in nuka-sake brewing. Miglitol, a specific inhibitor of α-glucosidase, strongly inhibited glucose production from rice polish. To obtain further evidence regarding the contribution of α-glucosidase, this enzyme was purified from rice grain kernels (Yamadanishiki cultivar) and supplied for both rice polish saccharification and nuka-sake brewing. The purified α-glucosidase promoted both glucose production from rice polish and alcohol fermentation in nuka-sake brewing. Based on these results, it was considered that the α-glucosidase contained in rice polish plays an important role in glucose production. This role may be a rate-limiting factor for parallel fermentation in nuka-sake brewing. Moreover, oligosaccarides accumulated during the saccharification of uncooked rice polish, implying the contribution of not only α-glucosidase but also α-amylase (EC 3.2.1.1). Through this result, it can be speculated that the starch contained in rice polish will be decomposed to glucose as a result of the action of α-glucosidase and α-amylase contained in rice polish.

H. Yoshii, T. Furuta, M. Ikeda, T. Ito, H. Iefuji, and P. Linko

The cellulose-binding ability of Geotrichum sp. M111 cells was investigated by the micro-tube method which gives an indication of the binding ability of M111 cells. The optimum pH value and temperature were 3-7 and below 50℃, respectively, from measurement of the aggregation height for a mixture of cellulose powder and M111 cells. The binding constant of 0.3% for M111 cells to cellulose powder was obtained in a 20 mM citrate buffer of pH 5.0 at 30℃. Aggregation was inhibited by such surfactants as sodium dodecylsulfate. The binding ability of M111 cells to cellulose fiber disappeared after a treatment with Driselase or Pronase E. This suggests that the binding ability might be related to the cell surface proteins. The dehydration rate of the distilled waste of sweet potato shouchu was accelerated by a linear viscoelastic model suggests that the acceleration effect might have been due to the space increase between cellulose fibers with the cell addition.

K. Hashizume, K. Tozawa, Y. Hiraga, and I. Aramaki

An S-adenosyl-L-methionine-dependent O-methyltransferase capable of methylating 2-hydroxy-3-alkylpyrazine (HP) was purified 7,300-fold to apparent homogeneity with an 8.2% overall recovery from Vitis vinifera L. (cv. Cabernet Sauvignon) through a purification procedure including column chromatography on DEAE-Sepharose FF, Ether-5PW, hydroxyapatite, G2000SWXL, and DEAE-5PW. The relative molecular mass of the native enzyme estimated on gel permeation chromatography was 85 kDa, and the subunit molecular mass was estimated to be 41 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme also methylates caffeic acid. The Vmax for IBHP and caffeic acid were 0.73 and 175 pkatals/mg, respectively, and the respective Km for IBHP and caffeic acid were 0.30 and 0.032 mM. The optimum pH for IBHP (8.5) was different from that for caffeic acid (7.5).

N. R. Kamini and H. Iefuji

A number of factors affecting the methanolysis of vegetable oils in aqueous medium by Cryptococcus spp. S-2 lipase were investigated. The crude lipase from the yeast efficiently catalyzed the methanolysis of vegetable oils (oil/methanol molar ratio of 1:1) in the presence of 40 wt.% water. The methyl ester content was high with rice bran oil at 30℃ for 96 h and further optimization studies were carried out with varying amount of enzyme, water or methanol. The enzyme was not inactivated by shaking in a mixture containing 4 Meq of methanol against the oil and 100 wt.% water by weight of the substrate and the methyl ester contents increased with increasing molar equivalents of methanol and water contents from 60 to 100 wt.%. The optimal methanolysis conditions were an oil/methanol molar ratio of 1:4, a water content of 80 wt.% by weight of the substrate containing 2000 U of crude lipase with shaking at 160 rpm for 120 h at 30℃. Thus, the reaction was conducted in a single step to avoid the stepwise addition of methanol and the methyl ester contents reached 80.2 wt.% at 120 h. These same conditions were applied for alcoholysis of rice bran oil and primary alcohols to their respective alkyl ester derivatives which are excellent substitutes for diesel fuel.

H. Fukuda, Y. Kizaki, T. Tsukihashi and S. Wakabayashi

An antibiotic-resistant strain of Saccharomyces cerevisiae was isolated from shochu yeast. Three mutants were used for shochu brewing and gave higher ethanol productivities than the parent. The mutants were resistant to cycloheximide, cerulenin, trichothecin and other organic compounds such as lauric acid. In the presence of 20% (v/v) ethanol, the viability of the mutants was 87-96%, but that of the parent was 77%. Zymolyase treatment for 3 h, decreased the viability of the parent by 44% but that of the mutants only by 11-32%. Thus the higher ethanol productivity of these mutants is related to their high ethanol tolerance and resistance to various organic compounds.

岩田博、岩瀬新吾、高浜圭誠、松浦宏行、猪谷富雄、荒巻功

米のα-グルコシダーゼ活性と食味と関連のある理化学的性質との関係を明らかにするため、1997年産の24種類の米のα-グルコシダーゼ活性と他2種の糖化関連酵素活性及び理化学的性質の測定を行った。α-グルコシダーゼ活性は、コシヒカリとあきたこまち（現代品種）が、赤米（対馬）や神力（旧品種）より高かった。一方、α-アミラーゼ活性は、赤毛（旧品種）と赤米（種子島）がコシヒカリより高かった。α-グルコシダーゼ活性は、炊飯特性試験のヨード呈色度、溶出固形分、膨張容積及びアミロース含量と負の相関（r=-0.542, r=-0.570, r=-0.460, r=-0.487）があった。また、α-グルコシダーゼ活性はRVAを用いた粘度特性試験の最高粘度、ブレークダウンと正の相関（r=0.557, r=0.633）があった。これらから、間接的ではあるがα-グルコシダーゼ活性と食味は相関があると推定された。主成分分析を行った結果、第1主成分はアミロースに関する成分を、第2主成分は還元糖に関する成分を示すことが分かった。これらの2つの主成分を軸として主成分得点をプロットすると、第1主成分はアミロースを示すとともに米の品種の年代をも示しているように思われた。更に、この第1主成分に対し、α-グルコシダーゼが0.778という高い主成分負荷量を持ったので、α-グルコシダーゼのアミロペクチン合成への何らかの関与が示唆された。

福田央、日吉智、砂川英之、田中健太郎、藤田仁、山根雄一、若林三郎

清酒もろみの原料利用率の向上を目的とし、セルロース、キシラン、及びペクチン等の、いわゆる植物細胞壁溶解酵素が、清酒もろみの並行複発酵に及ぼす影響を検討した。セルロース、キシラン、及びペクチン溶解酵素を有する市販酵素剤の添加により、アルコール収得量も増加し、原料利用率の向上が得られた。更に、麹菌の培養上清液よりアミラーゼ・プロテアーゼを除き、セルロース、キシラン、及びペクチン溶解活性の高い粗酵素液を調整し、もろみ発酵試験に供した。この結果、もろみ発酵・原料利用率ともに顕著に向上したことから、これらの植物細胞壁溶解酵素が、清酒もろみにおいて並行複発酵の促進効果を有することが強く示唆された。

M. Azumi and N. Goto-Yamamoto

Using nine primer pairs, amplified fragment length polymorphism (AFLP) analysis was conducted to characterize industrial, laboratory, and type strains of Saccharomyces sensu stricto. S. cerevisiae, S. bayanus, S. carlsbergensis, and S. paradoxus had species-specific AFLP profiles with some variations among the strains. Nineteen wine, ale, bakery, whisky, and laboratory strains of S. cerevisiae were differentiated by two primer pairs, while, out of 19 strains of sake yeast, two groups consisting of two and eight strains were not differentiated using nine primer pairs. A phenogram of 41 strains of S. cerevisiae, two strains of S. bayanus, the type strain of S. pastorianus, three strains of S. carlsbergensis, one hybrid strain of S. cerevisiae and S. bayanus, and the type strain of S. paradoxus was obtained by the unweighted pair group method using arithmetic averages (UPGMA) based on the percentage of shared AFLP fragments of each sample pair. This phenogram demonstrated clear separations of S. cerevisiae, S. bayanus, S. carlsbergensis, and S. paradoxus . However, S. pastorianus ATCC 12752T showed highest percentages of shared fragments with the strains of S. bayanus, and formed a cluster with them. Except for the type strain of S. pastorianus, the percentages of shared fragments showed a similar tendency with reported data of DNA relatedness. The cluster of S. cerevisiae separated into three subclusters: a cluster consisting of sake and shochu strains, and a whisky strain; a cluster consisting of bakery, wine, ale, and whisky strains; and a cluster consisting of laboratory strains.

T. Takahashi, H. Simoi, K. Ito

The yeast Saccharomyces cerevisiae exhibits high ethanol tolerance compared with other microorganisms. The mechanism of ethanol tolerance in yeast is thought to be regulated by many genes. To identify some of these genes, we screened for ethanol-sensitive S. cerevisiae strains
among a collection of mutants obtained using transposon mutagenesis. Five ethanol-sensitive (ets) mutants were isolated from approximately 7,000 mutants created by transforming yeast cells with a transposon (mTn-lacZ/LEU2)-mutagenized genomic library. Although these mutants
grew normally in a rich medium, they could not grow in the same medium containing 6% ethanol. Sequence analysis of the ets mutants revealed that the transposon was inserted in the coding regions of BEM2, PAT1, ROM2, VPS34 and ADA2. We constructed deletion mutants for these genes by a PCR-directed disruption method and confirmed that the disruptants, like the ets mutants, were ethanol sensitive. Thus, these five genes are indeed required for growth under ethanol stress. These mutants were also more sensitive than normal cells to Calcofluor white, a drug that affects cell wall architecture, and Zymolyase, a yeast lytic enzyme containing mainly beta-1,3- glucanase, indicating that the integrity of the cell wall plays an important role in ethanol tolerance in S. cerevisiae.

H. Fukuda, Y. Kizaki, T. Ishikawa and K. Takahashi

Several new types of cerulenin-resistant mutants of sake yeast were isolated. These mutants showed respiratory deficiency and could grow on media containing a higher concentration of antibiotics than could the parents. Sakes brewed by the mutants produced less succinate than by both the parent yeast and the mutants with respiratory deficiency induced by ethidium bromide. In addition, the acidity of these mutants was decreased. Since low acidity is favorable in both sake and wine, these mutants might be applicable for both sake and wine brewing.

J. Fujita, H. Fukuda, Y. Yamane, Y. Kizaki, S. Shigeta, K. Ono, O. Suzuki, and S. Wakabayashi

The high phytase producing mutant of Aspergillus oryzae (KL-38) previously isolated was employed for koji making, and the produced koji rice then supplied for sake brewing. The alcohol fermentation was improved compared to that with the parent strain (A. oryzae BP-1). The effect of two phytase isozymes (Phy I and Phy II) produced by A. oryzae on yeast growth and inorganic phosphate liberation were investigated using a synthetic medium containing by the combination of Phy I and Phy II at a ratio of 1 to 3, which was compatible with the production ratio in KL-38. Based on these results, phytase plays important role in sake brewing, and that the maximum inorganic phosphate liberation from phytic acid can be obtained by a suitable combination of Phy I and Phy II.

JEONG. Seok. Tae、後藤（山本）奈美

1. 市販ワイン酵母25株の種を同定したところ、S. cerevisiaeとS. bayanusの交雑株と報告されているS6U以外の供試菌株は、S. bayanusと表示されている3株を含め、すべてS. cerevisiaeであった。
2. 市販ワイン酵母を用いて小仕込み試験を行ったところ、赤ワインのアントシアニン量の指標であるA520 at pH 0.25と、A420、総フェノール及びフラボノイド・フェノール濃度の間に有意な正の相関が見出され、酵母による果皮成分の抽出効率の違いが示唆された。また、生成酒の亜硫酸濃度とアセトアルデヒド濃度の間に正の相関が認められた。酵母の沈降速度の指標とした懸濁9日後の表面から6cmのA660と比重及びグリセロール濃度の間に有意な正の相関が見出され、凝集性のない酵母の沈降速度の違いは、球形粒子の沈降速度と同様、ワインの比重と粘性率によって説明されると考えられた。

K. Hashizume, K. Tozawa, M. Endo, and I. Aramaki

The final step of 2-methoxy-3-alkylpyrazine (MP) biosynthesis has been presumed to involve O-methylation of 2-hydroxy-3-alkylpyarzine (HP), although this reaction has never been demonstrated in organisms. We detected 2-hydroxy-3-isobutylpyrazine (IBHP) and 2-hydroxy-3-isopropylpyrazine (IPHP) in unripe grapes, and S-adenosyl-L-methionine-dependent O-methyltransferase (OMT) activity toward HP in crude extracts from young shoots and unripe grapes that accumulate MP at different levels. The levels of HP in the berries and stems were estimated by using 2-hydroxy-3-sec-butylpyrazine as an internal standard. The OMT activity observed in the crude extracts from young shoot and berries was extremely low, but no MP-decomposing activity was detected in the solutions. The levels of HP and OMT activity were closely related with the level of MP in the grapes. These results indicate that the predicted final step of MP biosynthesis exists in wine grapes.

N. Mukai, C. Nishimori, I. F. Wilson, A. Mizuno, T. Takahashi, and K. Sato

Beer brewing using a fusant between a sake yeast (a lysine auxotrophic mutant of sake yeast K-14) and a brewer's yeast (a respiratory-deficient mutant of the top fermentation yeast NCYC1333) was performed to take advantage of the beneficial characteristics of sake yeasts, i.e., the high productivity of esters, high tolerance to ethanol, and high osmotolerance. The fusant (F-32) obtained was different from the parental yeasts regarding, for example, the assimilation of carbon sources and tolerance to ethanol. A brewing trial with the fusant was carried out using a 100-l pilot-scale plant. The fusant fermented wort more rapidly than the parental brewer's yeast. However, the sedimentation capacity of the fusant was relatively low. The beer brewed using the fusant contained more ethanol and esters compared to that brewed using the parental brewer's yeast. The fusant also obtained osmotolerance in the fermentation of maltose and fermented high-gravity wort well.

橋本忠明、森下めぐみ、中田佳幸、辻安信、伊藤清

醤油麹菌Aspergillus oryzae HL15 株は、醤油麹において、少なくとも3種類のキシロシダーゼを生産した。それらを精製したところ、醤油諸味中でも機能し得る耐塩性の酵素であった。また、キシロシダーゼは液体培養すると菌体結合型酵素として生産されていた。キシロシダーゼと同時にキシラナーゼを作用させると、効率よくキシランを分解した。