Akihiro Kanda

Alcohol dehydrogenase I of sake yeast Saccharomyces cerevisiae Kyokai no. 7

Abstract The sake yeast Saccharomyces cerevisiae Kyokai no. 7 (K-7) is capable of producing more than 20% (v/v) ethanol in culture. However, the activity of its alcohol dehydrogenase (ADH), which is a key enzyme in alcohol production, is less than that of the laboratory yeast S. cerevisiae X2180-1A. The ADH activities of brewery yeasts which were isolated and bred for the purpose of ethanol production were compared with those of laboratory yeasts. The ADH activities of a number of the brewery yeasts were found to be less than those determined for the laboratory yeasts. The level of ADH activity of each clone derived from a brewery yeast was also lower than that of each clone derived from laboratory yeasts when expressed in Escherichia coli cells. In the ADH1 gene of K-7, the region between the TATA ☐ and ATG start codon and the region of polyadenylation in this nucleotide sequence were consistent with previous data. The ratio of intracellular ADH activity of K-7 to that of X2180-1A was about 1 : 2. On the other hand, the ratio of ADH activity of the clones derived from K-7 and X2180-1A, expressed under the control of the same promoter and in the same host, was about 1 : 1.3. The amino acid sequence of K-7 ADH1 was compared with previous data. The Glu 128 of K-7 ADH1 was unique among yeast ADH1s. Site-directed mutagenesis was carried out using ADH1 DNAs isolated from the sake yeast K-7 and the laboratory yeast X2180-1A, in order to determine amino acid alterations which affect activity. No single amino acid substitutions resulted in a change in the level of ADH activity. However, two double substitutions E128Q and E148Q or E128Q and V152I increased ADH activity to levels similar to that of X2180-1A Adh1. The substitutions of X2180-1A ADH1 to Q128E and Q148E or Q128E and I152V reduced ADH1 activity to the levels observed for K-7 Adh1.

Plasmids of hiochi-bacteria

Abstract Hiochi-bacterial plasmids were screened. Many hiochi-lactobacilli had plasmids of several molecular sizes, whereas only a few true hiochi-bacilli had just one or two plasmids. A 4.7 kb cryptic plasmid in hiochi-lactobacilli homo-fermenter A-1 strain was cloned into pBR322 vector for Escherichia coli ; it has three unique cleavage sites for Hpa I, Bgl II and Mlu I.

Brewing of Sake Containing Glutathione.

現在の清酒に比べグルタチオン含有量の高い清酒を開発するため,液胞生理機能欠損変異酵母を用い検討を行った. (1) 液胞生理機能欠損変異酵母において,グルタチオンを菌体外に漏出することが認められた. (2) 液胞生理機能欠損変異酵母に,醸造適性を付与することによりアルコール16%,グルタチオン20ppm以上の清酒を製造することが可能となった. (3) さらに,酵素剤を併用し仕込方法を改良した結果,香り,酒質など品質面において,従来の製品と同等の清酒が製造可能となった. 終わりに臨み,液胞生理機能欠損変異酵母を分譲していただいた東京大学大隅良典教授,国税庁醸造試験所北本勝ひこ博士,ならびに終始ご指導を賜りました弊社常務取締役総合研究所所長布川弥太郎博士に深く感謝いたします.

Brewing of Sake with Low Urea Contents by the Addition of Inorganic Salts to Moromi Mash

Several laboratory scale brewings were carried out to obtain sake with low urea contents,by adding some inorganic salts to moromi mash. Results are as follows: 1) The addition of KH2PO4 and MgSO4 to moromi mash was effective to reduce urea contents in sake. 2) In case of using both KH2PO4 and MgSO4 in combination,MgSO4 was effective only under low concentration of KH2PO4(1mM). 3) All of the sake brewed under the addition of 4 mM KH2PO4 by 6 strains of yeasts had lower urea contents as compared with the control brewed without addition. 4) Urea contents in sake brewed with 4•`8 mM KH2PO4 were reduced by 24•`64% as compared with the control. Although several sake components were changed,their sensory evaluation was meaningless.

Studies on aseptic filling of Sake. Part IV. Aseptic filling of Sake into glass bottles.

瓶容器での清酒の常温無菌充填について検討し, 次の結果を得た。1. 空中からの火落菌の混入する確率は極めて低いが, 充填機周辺はクリーンルーム乃至クリーンブースの採用が適当と考えられる。2. 熱水 (90℃, 15分間) CIP条件で充填ラインの無菌化が可能であったが, 洗浄面から熱アルカリ工程を入れる必要があった。3.大量充填試験により, 本装置で常温無菌充填が可能であることが分った。4. 熱交換法の代わりに超精密炉過法を行いることにより, 生酒の無菌充填が可能であることが確められた。

Studies on the preparation of aseptic Sake. Studies on aseptic filling of Sake. Part III.

1.プレート式熱交換殺菌法, 限外炉過法, 超精密枦過法のいずれの方法でも殺菌ないし除菌が可能で, 常温の無菌清酒の製造が可能であった。2.熱交換殺菌法は, 三方法のなかで最も容易に, また確実に常温の無菌清酒が製造できた。3.限外炉過法は, 枦過中に一般成分の変化がみられるが, 酵素の除去が可能であることが大きな特徴であった。4.超精密炉過法は, 除菌だけを目的とした場合, 炉過中に一般成分の変化がなく, 又膜のリークテスト・修理が容易など限外炉過法より優れた特徴を持っていた。5.三方法を単独使用または併用することにより, 目的に応じた常温の無菌清酒の製造が可能となった。