平成15年度 研究成果

荒巻功、菊永雪絵、吉井美華、奥田将生、小関卓也、小川雅広、熊丸敏博、佐藤光、橋爪克己

心白を持つ胚乳変異体米16系統を含む米粒の心白の形状、胚乳細胞の配列構造、アミロプラストの構造を実体顕微鏡又は走査型電子顕微鏡により観察した。また、これら試料の酒造適性に関連する項目の分析を行った。7つのタイプの心白の形状、2つのタイプの胚乳細胞の配列構造、6つのタイプのアミロプラスト構造が観察された。心白の中心部が細長く伸張した胚乳細胞から構成される試料は、放射状に胚乳細胞が構成される試料と比較して有意に多くの水を吸収した。アミロプラスト構造のタイプと消化性（Brix）の間には弱い相関が認められた。心白の面積の小さい試料は比較的低いα?アミラーゼ活性を示した。米粒試料の粗タンパク、無機成分、又はアミロース含量と心白の形状、アミロプラスト構造との間には目立った関連は見られなかった。リン含有量は吸水率（20分； r=-0.667、120分；r=-0.635）又は粗タンパク含量（r=0.811）と有意な相関を示した。アミロース含量は吸水率（20分； r=-0.745、120分；r=-0.725、蒸米；r=-0.681）と有意な相関を示した。

荒巻功、神田涼子、菊永雪絵、吉井美華、岩田博、奥田将生、小関卓也、小川雅広、熊丸敏博、佐藤光、橋爪克己

心白を持つ胚乳変異体米16系統を含む米粒の粘度及び糊化特性をラピッド・ビスコ・アナライザー（RVA）及び示差走査熱量計（DSC）によって分析した。清酒醸造適性も分析した。心白部分にシュガリー様の胚乳を持つ試料の糊化エンタルピーはやや小さい値を示した。その他RVA及びDSCパラメーターと心白の形状又は胚乳細胞構造との間には明瞭な関係は見いだせなかった。RVA及びDSCパラメーターは、いくつかの酒造適性分析項目及びアミロース含量と高い相関を示した。6試料の清酒醸造試験において、溶解し易い心白の米粒中における位置が初期の発酵速度に影響していることが見いだされた。シュガリー様の構造を示す胚乳を持つ試料は清酒もろみにおいて高い溶解性を示した。試験した変異体試料のうちのいくつかは親品種又は山田錦よりも優れた酒造適性を示した。

K. Miyashita, K. Sakamoto, H. Kitagaki, K. Iwashita, K. Ito and H. Shimoi

Almost all sake yeasts form a thick foam layer on sake mash during fermentation. To reduce the amount of foam, nonfoaming mutants were bred from foam-forming sake yeasts. To elucidate the mechanism of this foam formation, we have cloned a gene from a foam-forming sake yeast that confers foam-forming ability to a nonfoaming mutant. This gene, named AWA1, encodes a glycosylphosphatidylinositol(GPI) anchor protein that is localized to the cell wall and is required for cell surface hydrophobicity. In this paper, we describe the genomic analysis of the AWA1 gene in a nonfoaming mutant strain K701 derived from a foam-forming sake yeast strain K7. K701-AWA1 was cloned in a cosmid and its sequence was compared with that of K7-AWA1. Although the 5' half of K701-AWA1 was identical to that of K7-AWA1, the 3' half of K701-AWA1 was different from that of K7-AWA1, resulting in a loss of the C-terminal hydrophobic sequence of Awa1p. Since this sequence is considered to be required for the anchoring of Awa1p to the cell wall, K7-Awa1p could not confer both cell surface hydrophobicity and foam-forming ability to strain K701 cells. Since the change found in K701-AWA1 was not a point mutation but a larger scale event, we analyzed chromosome rearrangement by pulsed-field gel electrophoresis Southern blot analyses. The results suggest that the left subtelomeric region of chromosome IX in strain K7 was translocated to the AWA1 gene in chromosome XV by a nonreciprocal recombination.

K.Nagamine, K.Murashima, T.Kato, H.Shimoi, and K.Ito

Aspergillus kawachii produces two kinds of α-amylase, one is an acid-unstable α-amylase and the other is an acid-stable α-amylase. Because the quality of the shochu depends strongly on the activities of the α-amylases, the culture conditions under which these α-amylases are produced were examined. In liquid culture, acid-unstable α-amylase was produced abundantly, but, acid-stable α-amylase was not produced. The acid-unstable α-amylase was produced significantly when glycerol or glucose was used as a carbon source, similarly to the use of inducers such as starch or maltose. In liquid culture, A. kawachii assimilated starch at pH 3.0, but no α-amylase activity was recognized in the medium. Instead, the α-amylase was found to be trapped in the cell wall. The trapped form was identified as acid-unstable α-amylase. Usually, acid-unstable α-amylase is unstable at pH 3.0, so its stability appeared to be due to its immobilization in the cell wall. In solid-state culture, both kinds of α-amylase were produced. The production of acid-stableα-amylase seems to be solid-state culture-specific and was affected by the moisture content in the solid medium.

S.Yano, T.Asano, N.Kurose, J. Hiramatsu, H.Shimoi, and K.Ito

A yeast with high organic acid productivity was isolated from α-ketoglutarate-resistant mutants obtained by mutagenizing a sake yeast, Kyokai no. 701(K-701). The new strain, 2OG-R39, produces about twice as much malate and succinate as the parental strain. DNA microarray analyses revealed that the transcriptional levels of genes involved in the TCA cycle, oxidative phosphorylation,and respiration were higher in strain 2OG-R39 than in strain K-701. Expression of these genes is regulated by the Hap2/3/4/5p complex, and especially by expression of the HAP4 gene. In a Northern blot analysis,the transcriptional level of the HAP4 gene was higher in strain 2OG-R39 than in strain K-701. We constructed a plasmid that expresses the HAP4 gene constitutively and introduced it into strain K-701. The HAP4-overexpression-strain produced more malate and succinate than strain K-701 both in YPD medium and in a sake brewing test. These results indicate that strain 2OG-R39 produces more organic acids than strain K-701 because strain 2OG-R39 has a higher level of expression of the HAP4 gene than strain K-701.

岩田 博、磯谷敦子、宇都宮仁、猪谷富雄、西尾尚道

米α-グルコシダーゼのデンプン蓄積との関連を解明するため、日本晴と山田錦を試料とし、登熟中のα-グルコシダーゼ、α-アミラーゼ、結合型デンプン合成酵素（GBSS）、枝切り酵素（プルラナーゼ）各活性の変化、及び試料のアミロース、アミロペクチン含量等の変化について検討した。

その結果、α-グルコシダーゼ活性は、米重量（日本晴0.9801、山田錦0.9661）、GBSS活性（日本晴0.9684、山田錦0.9504）、アミロース含量（日本晴0.9772、山田錦0.9958）といずれも5%の危険率で高い相関があった。また、精米歩合の異なる米の酵素活性の分布から、α-グルコシダーゼは、米内部で活性が高く、GBSSやプルラナーゼの分布に類似していた。さらに、山田錦から精製したα-グルコシダーゼを生デンプンに作用させたところ、主にグルコースを生成した。以上の結果から、登熟中の米α-グルコシダーゼは、デンプン蓄積と何らかの関連のあることが示唆された。

水野昭博、天野仁、向井伸彦、佐藤和夫、高橋利郎

α-グルコシダーゼをピッチング時に高濃度麦汁へ直接添加し、並行複発酵を行うことにより、マルトース資化性は弱いがアルコール発酵能の高い清酒酵母、ワイン酵母を用いて高濃度醸造を行うことが可能であった。α-グルコシダーゼを原麦汁エキス分20%Platoの高濃度麦汁に添加して清酒酵母K-14を用いて100Lパイロットプラント試験を行ったところ、ビール酵母を用いた酵素剤無添加の仕込みより主発酵は速く完了し、製成酒はエステル香の高い清酒用香味の吟醸酒を想起させる酒質のものとなった。この製造方法においては、使用する酵母のマルトース資化性に関わりなく、清酒酵母及びワイン酵母等を用いることが可能であり、品質の多様化に有効であると考えられた。

Y. Hara, Y. Hinoki , H. Simoi, K. Ito

Three endoglucanase genes (cel5A, cel5B, and cel61A) were cloned from an industrial fungus, Aspergillus kawachii. Yeasts transformed with these cDNAs showed endoglucanase activity in medium. Cel5A and Cel61A contained a type 1 cellulose-binding domain (CBD1) at the C-terminus of the enzyme. The putative catalytic regions of Cel5A and Cel5B showed homology with various endoglucanases belonging glycosyl hydrolase family 5 (GH5). Cel5B showed high homology with Cel5A in catalytic region, but it lacked CBD1 and linker. The cel5A contained four introns, whereas cel5B contained five introns. The putative catalytic region of Cel61A showed homology with enzymes belonging to GH61. The cel61A contained no introns.

N. Tomishige, Y. Noda, H. Adachi, H. Shimoi, A. Takatsuki, K. Yoda

The GAS1-related genes of fungi encode GPI-anchored proteins with β-1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1Δ allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1Δ cells confirmed the important role of β-1,6-glucan in cells that are defective in the processing of β-1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1Δ defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1Δ mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2Δ mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1Δ cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.

T. Ban, M. Ishimaru, S. Kobayashi, S. Shiozaki, N. Goto-Yamamoto and S.Horiuchi

The effects of abscisic acid (ABA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on the expression of seven anthocyanin biosynthetic pathway genes in 'Kyoho' grape berries were investigated. In untreated berries, the expression of the UDP-glucose-flavonoid: 3-O-glucosyltransferase (UFGT) gene was detected only at 42 d after full bloom (DAB), whereas the phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) and leucoanthocyaidin dioxygenase (LDOX) genes were expressed throughout the growing period. ABA increased anthocyanin content in the skin and the expression of PAL, CHS, CHI, DFR and UFGT genes at 7 d after treatment. In contrast, 2,4-D inhibited the accumulation of anthocyanin and the expression of all the genes examined. The results clearly show that the anthocyanin levels resulting from the application of ABA and 2,4-D were correlated with the expression of anthocyanin biosynthetic pathway genes.

Takuya Koseki, Masaki Okuda, Shigetoshi Sudo, Yasuzo Kizaki, Kimio Iwano, Isao Aramaki, and Hiroshi Matsuzawa

Two different α-L-arabinofuranosidases from Aspergillus kawachii were purified and characterized. The two enzymes acted synergically with xylanase in the degradation of arabinoxylan and resulted in an increase in the amount of ferulic acid release by feruloyl esterase. Both enzymes were acidophilic and acid stable enzymes which had an optimum pH of 4.0 and were stable at pH 3.0-7.0. The general properties of the enzymes including pH optima and pH stability were similar to those of Aspergillus awamori. These results suggest that the α-L-arabinofuranosidases contribute to an increase in cereal utilization and formation of aroma in shochu brewing. Two different genes encoding α-L-arabinofuranosidases from A. kawachii, designated as AkabfA and AkabfB, and those from A. awamori, designated as AwabfA and AwabfB, were also cloned and characterized. The difference between the sequences of AkabfA and AwabfA was only one nucleotide, resulting in an amino acid difference in the sequence, and the enzymes were assigned to family 51 of glycoside hydrolases. On the other hand, the differences between the sequences of AkabfB and AwabfB and between their encoding proteins were two nucleotides and one amino acid residue, respectively, and the enzymes were assigned to family 54 of glycoside hydrolases. On comparison of the abfA and abfB genes among A. kawachii, A. awamori, and A. niger, the relationship between the two genes for A. kawachii and A. awamori was much closer than those between A. niger and the others. Northern analyses showed that transcription of AkabfB was greater than that of AkabfA in the presence of L-arabitol and L-arabinose, and that transcriptions of both genes were not induced in the presence of sucrose and glucose.

T. Ohnuki, F. Akamoto, N. Kozai, T. Ozaki, I. Narumi, Arokiasamy J. Francis, H. Iefuji, T. Sakai, T. Kamiya, T. Satoh, M. Oikawa

We examined the accumulation and distribution of Cs, and the presence of other elements in yeast (Saccharomyces cerevisiae) cells by the micro-PIXE (particle induced X-ray emission) system developed at the TIARA facility, JAERI, and by energy dispersive spectroscopy (EDS) coupled to a scanning electron microscope. The effects of Cs on yeast growth were determined by measuring the optical density at 600 nm. Addition of 1 mM Cs did not have any effect on the growth of the yeast. Micro-PIXE analysis of cells grown in the presence of Cs showed that Cs was uniformly distributed in the cells. Using PIXE, Cs,P,K and Fe can be detected, whereas only P and S can be determined by the EDS. Cells exposed to Cs showed an increase in Cs peak intensity, and decrease in P,K and Fe with time. These results suggest that micro-PIXE is a useful technique to detect low concentration of toxic elements in icroorganisms as well as to monitor their changes as function of growth.

Hirokazu Tsuboi, Yasushi Wakisaka, Masato Hirotsune, Takeshi Akao, Osamu Yamada, Osamu Akita

We isolated mutants of S. cerevisiae in which expression of the JEN1 gene encoding a pyruvate transporter was insensitive to glucose repression. The isolated mutant GDR19 expressed JEN1 and absorbed pyruvate in the presence of glucose. In a DNA microarray analysis, GDR19 highly expressed many more genes, including JEN1, in the presence of glucose compared with the parental strain B29. Some of these genes are under the control of the transcription factor Mig1p and are normally repressed in the presence of glucose. The concentrations of organic acids in sake made with GDR19 were different from those in sake made with B29. Changes in the pyruvate concentration in the sake mash made with GDR19 were not very different from those in sake mash made with B29, and both GDR19 and B29 expressed JEN1 during fermentation. When the ethanol concentration was over 2%, JEN1 expression in B29 was similar in the presence and absence of glucose. The expression of JEN1 in sake mash in spite of the presence of glucose appeared to be caused by the coexistence of ethanol.

Jin Fujita, Yu-ichi Yamane, Hisashi Fukuda, Yasuzo Kizaki, Saburo Wakabayashi, Seiko Shigeta, Osamu Suzuki and Kazuhisa Ono

We identified three types of acid phosphatase (ACP-I, ACP-II, and ACP-III) produced by Aspergillus oryzae in a submerged culture using only phytic acid as the phosphorous substrate. The optimum pH for the activities of the three enzymes was in the range of 4.5 to 5.5. Analysis of the substrate specificities of these enzymes revealed that ACP-I and ACP-III were acid phosphatases, and ACP-II was a phytase. These enzymes were produced during different periods of mycelial growth: ACP-II was produced during the early phase of cultivation (around 24 h), and ACP-I was produced between 24 to 72 h. ACP-III was detected after the production of ACP-I and ACP-II had ceased. The release of phosphate from phytic acid was expected to be due to the cooperative hydrolysis of these enzymes.

水野昭博、天野仁、向井伸彦、佐藤和夫、高橋利郎

ビールの高濃度醸造における発酵遅延・発酵度の低下、酢酸生成量の増加という課題の解決を目的として、α-グルコシダーゼを酵母添加と同時に麦汁に添加し、糖組成の変換と発酵を同時進行させる並行複発酵型式のビール製造方法について検討した。その結果、α-グルコシダーゼの糖転移反応により、麦汁中のグルコース濃度を過度に上昇させることなく高濃度麦汁の発酵を行うことができ、ビール中の酢酸濃度の低減に効果があった。また、α-グルコシダーゼの加水分解反応により、麦汁中のオリゴ糖は分解され、ビールの発酵度を高めることができた。

Jin Fujita, Seiko Shigeta, Yu-ichi Yamane, Hisashi Fukuda, Yasuzo Kizaki, Saburo Wakabayashi and Kazuhisa Ono

In our previous study, it was determined that phytase produced by Aspergillus oryzae plays an imrportant role in supplying phosphate to yeast in the process of making sake. During koji making, two types of phytase (Phy-I and Phy-II) are produced. The purified phytases have high thermal and pH stability, in comparison to phytase purified from a submrerged culture (ACP-II). In the present study, Phy-I and Phy-II retained their activities for 45 h. The NH2-terminal sequence of Phy-I, which is eight amino acids in length, was identical to that of ACP-II, but the molecular weights of these two forms, as estimated by SDS-PAGE, were quite different from each other (Phy- I, 120 kDa; ACP-II, 58 kDa). From the NH2-terminal amino acid sequence analysis of the predominant phytase (Phy-II), a molecular weight of 116 kDa was expected to reflect a new type of phytase produced only in koji culture. The substrate specificity of Phy-II was sufficiently broad that it hydrolyzed not only phytic acid and p-nitro phenyl phosphate, but also glucose 6-phosphate and glycerol 1-phosphate. In the process of making koji, Phy-I was produced at an early stage, followed by Phy-II; with both phytases being thought to function to hydrolyze phytic acid cooperatively.

露無顕、家藤治幸、岩下和裕、尾崎則篤、福島武彦

1. 蒸留廃液を最もろ過性を向上させる培養実験の条件選定を行ったところ、菌株はA.oryzae RIB128を用い、培養前蒸留廃液の希釈倍率を芋焼酎蒸留廃液、麦焼酎蒸留廃液に関しては原廃液を2倍に希釈した場合に、米焼酎蒸留廃液に関しては1.5倍希釈にした場合にろ過性が最も向上した。その時の濃縮倍率はそれぞれ7.3、10.8、6.4倍であった。
2. 酎蒸留廃液に麹菌を増殖させることで廃液中のpHが4以下から中性に変化した。これは麹菌の増殖のための炭素源として主に有機酸が使用され、ほぼ消費され尽くすことによるものと考えられた。焼酎蒸留廃液は「廃酸」として扱われており、肥料としての散布量の制限などがなされていたが、麹菌処理により肥料その他資源化の視野を広げるものと考える。
3. 焼酎蒸留廃液に麹菌を増殖させてろ過することで、SSを93～97%、CODMn、DOC及びDTN NO3-N、NH4-N、org-Nについてはそれぞれ40～80%除去できることが示された。さらにリン量においては80～90%の除去が認められた。
4. 焼酎蒸留廃液のろ過性向上に影響与えている要因は、麹菌の増殖であることが示された。これは、麹菌が増殖する際、菌糸が旺盛に伸長する過程で、焼酎蒸留廃液中の固形分に絡みながら大きなフロックを形成するためと思われる。
5. 麹菌を利用しろ過した後の固形物は、粗タンパク質、可溶性無窒素物の成分が高く栄養価に富んでいることが認められた。また、焼酎蒸留廃液は有害物を含まないものであり、ろ過処理に用いた麹菌が安全性が確認されているため、畜産飼料としての資源化の可能性が示された。

野村佳司、木曽邦明

チオバルビツール酸反応は、魚油、ダイズ油及び肝臓の細胞ホモジネートなどの脂質の過酸化の程度を測定法として広く使われている。著者らはこのTBA反応によって清酒の劣化の測定を試みた。

高温で貯蔵した清酒とTBA液との反応液の可視スペクトルは低温で貯蔵した清酒の反応液より濃く着色した。HPLCをもちいて、高温で貯蔵した清酒とTBA液との反応液から、マロンアルデヒドとTBAとの反応により生成される赤色色素と同一と推測される色素が分離された。反応液の530nmの吸光度を測定することで、赤色色素の量が求められることがわかった。赤色色素は清酒の貯蔵時間が長いほど、貯蔵温度が高いほど多くなった。このことはTBA反応により清酒の劣化の測定が可能であることを示唆している。