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A microorganism producing a lytic enzyme preparation that could rapidly lyse bacterial cells such as hiochi bacteria and other lactic acid bacteria was screened. The microorganism was identified as Streptomyces fulvissimus. The enzyme produced by this organism lysed boil-denatured cells quicker than intact cells of hiochi bacteria. A mutant strain of S. fulvissimus producing the enzyme exhibiting high activity against intact cells of hiochi bacteria was screened on plates, containing intact cells inactivated with UV irradiation. The optimal pH for lytic activity against intact cells of the hiochi bacterium Lactobacillus casei S-4 was from 3.5 to 4.0, and the optimum temperature was close to 50 degrees C. This enzyme activity was stable between pH 3.5 and pH 8.0 and up to 60 degrees C. The enzyme exhibits N-acetyl glucosaminidase and muramidase activities. The effects of adjusting the pH and using different inducers for enzyme production were investigated. Chitin was the most effective inducer of enzyme production. Intact DNA was easily isolated from the cells of many lactic acid bacteria following lysis with the enzyme. It is thought that this enzyme will be a good biotechnological tool.

The ATF2 gene, which encodes alcohol acetyltransferase II (AATase II), was cloned from Saccharomyces cerevisiae Kyokai No. 7 (sake yeast). The ATF2 gene coded for a protein of 535 amino acid residues with a calculated molecular mass of 61,909 daltons. The deduced amino acid sequences of the ATF2 showed 36.9% similarity with that of ATF1, which encodes AATase I. The hydrophobicity profiles for the Atf2 protein and Atf1 protein were similar. A transformant carrying multiple copies of the ATF2 gene had 2.5-fold greater AATase activity than the control, and this activity was not significantly inhibited by linoleic acid. A Southern analysis of the yeast genomes in which the ATF2 gene was used as a probe showed that S. cerevisiae and brewery larger yeast have one ATF2 gene, while S. bayanus has no similar gene.

A rapid and sensitive method of detection of the sake spoilage microorganisms hiochi bacteria was developed. The hiochi bacterial common antigen (hb-CA) was identified using a monoclonal antibody (MAb) and its gene was cloned. The nucleotide sequences of the hb-CA gene from four standard strains of hiochi bacteria were then determined. Two polymerase chain reaction (PCR) primers for use in the detection of hiochi bacteria were designed by homology analysis of these sequences. When genomic DNA from the hetero-fermentative true hiochi bacillus (he-T) Lactobacillus fructivorans HI was amplified by PCR using this set of primers and electrophoretically analyzed, a PCR product could be detected when as little as 10 fg of genomic DNA, which corresponds to the amount of DNA in a single cell, had been used. When he-T L. fructivorans H1 was inoculated into 300 ml of pasteurized sake, the cells recovered by filtration, and the DNA analyzed by this method, the detection limit was found to be about 10 cells. This method is much faster than the standard culture method. This PCR technique is a rapid and highly sensitive method of detection of hiochi bacteria which is applicable to quality control of sake. The results of a homology search performed using the deduced amino acid sequence of this common antigen suggest that this antigen is the hiochi bacterial elongation factor Tu (EF-Tu).

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.

We used a plasmid carrying a sequence for autonomous maintenance in Aspergillus (AMA1) and the E. coli uidA gene as a reporter gene to search the A. oryzae and A. niger genomes for DNA fragments having strong promoter activity. Beta-glucuronidase (GUS)-producing A. oryzae transformants containing the No. 8AN derived from A. niger, or the No. 9AO derived from A. oryzae, were constitutive for the expression of the uidA gene when cultivated in the presence of a variety of carbon and nitrogen sources. When the GUS-producing transformants were grown in liquid culture, the No. 8AN showed an increase of approximately 3-fold in GUS activity compared to the amyB (alpha-amylase encoding gene) promoter. There was also a corresponding increase in the amount of GUS gene-specific mRNA. When these transformants were grown as rice-koji, the No. 8AN showed an increase of approximately 6-fold compared to the amyB promoter, and the amount of GUS protein produced also increased. These strong promoter regions might be applicable to the production of other heterologous proteins in Aspergillus species.

Sensory evaluation data obtained from experts were analyzed by numerical methods. The aim of this study is to identify a model that can objectively estimate the sensory evaluation results based on the concentrations of components in sake. To this aim, a learning model in which Dempster-Shafer's measure was learned by genetic algorithm (GA) was constructed. The learning process was performed by discovery of the assignments of basic probabilities according to the decrease in error between the observed and estimated data. When the model was compared with back propagation and multiple regression analysis by cross validation, the predictive faculty of the present model was as good as that of back propagation. The experiential rule by experts for time series data of sensory evaluation could be more sufficiently explained by the present model than by back propagation. The main advantage of this model was that its predictive faculty was compensated by Bayesian probabilities.

Ten standard strains of hiochi bacteria were selected based on the SDS-PAGE patterns of their cellular proteins. We then obtained ten hybridoma systems that secreted highly reactive monoclonal antibodies (MAbs) to the whole cells of each strain. It was apparent that these MAbs were highly reactive and specific for hiochi bacterial whole cells when using an enzyme-linked immunosorbent assay (ELISA). Three MAbs (two against the homo-fermentative hiochi lactobacilli and an MAb against the hetero-fermentative true hiochi bacilli) showed cross-reactivity to some of the other strains of lactobacilli tested. However, the other MAbs did not react with strains other than the immunogen. A sensitive ELISA method for the detection of hiochi bacteria was examined. It was possible to detect the order of 103 cells of the ten standard strains. Using this procedure and a mixture of the ten MAbs, a detection limit of 104 cells or less could be obtained for 98.2% of the hiochi bacteria isolated from sake brewing factories. ...

Optimal control of sensory evaluation estimated from 13 component concentrations on the basis of Dempster-Shafer's measure (DS) was attempted in the fermentation process for mashing Ginjyo-shu (sake). The control system consisted of fuzzy simulators generated by a genetic algorithm (GA) and an optimization procedure based on another GA. The fuzzy simulators simulated the dynamics of the ethanol production rate and sensory evaluation. Decentralized learning of fuzzy rules was also introduced. The fermentation period was divided into 4 phases, with a set of fuzzy rules corresponding to each phase. In order to construct an adaptive system based on the fuzzy simulators, only the set of rules corresponding to the current phase was adaptively identified, with the result that the fuzzy rules adapted to fluctuations in the relationship between the temperature and the ethanol production rate. By optimizing the control in this way, the optimal quality sake was successfully obtained.

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per micrograms of integrative vector and 100 colonies per micrograms of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.

Alcohol acetyltransferase (AATase) catalyzes the esterification of isoamyl alcohol by acetyl coenzyme A. The enzyme was solubilized from the microsomal fraction of Saccharomyces cerevisiae Kyokai No. 7, using Triton X-100 and then purified by a series of chromatographic separations: Poly Buffer Exchanger 94 (PBE94), DEAE Toyopearl, Toyopearl HW60, hydroxyapatite, Octyl-Sepharose CL-4B, and hexanol-affinity column chromatography. When the solubilized enzyme was put on PBE94, two active fractions were obtained. The enzyme obtained after affinity column chromatography had a single band on an SDS polyacrylamide gel, and its molecular mass was estimated to be 60 kDa. The enzyme was most active at pH 8.0 and 25 degrees C. It was stable between pH 7.5 and 8.5, but was unstable at temperatures above 10 degrees C. The activity was markedly inhibited by heavy metal ions such as Cd2+, Cu2+, Zn2+, and Hg2+, and sulfhydryl reagents. The Km for acetyl-CoA was 0.19 mM. The internal peptide sequences were also identified.

Isoamylalcohol and isoamylacetate concentrations were analyzed at various isothermal conditions in sake brewed with a saccharified rice solution. Kinetic models for formation of these components were constructed in which the relation between the isoamylalcohol and ethanol formation rates were linear, and the isoamylacetate formation rate was expressed as the sum of two terms, formation and decomposition. An optimal control strategy for isoamylacetate formation was calculated using dynamic programing. The calculations suggested that the fermentation temperature should be gradually lowered as the fermentation progresses in order to maintain the isoamylacetate formation rate at a high level.

The whole cellular proteins of various hiochi bacteria were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of the electrophoretic patterns, the hiochi bacteria were generally divided into four distinguishable groups, agreeing with the conventional groupings based on physiological and biochemical characters; homo- and hetero-fermentative true hiochi bacilli and homo- and hetero-fermentative hiochi lactobacilli. The SDS-PAGE patterns of the other lactobacilli were clearly different from that of the hiochi bacteria. The SDS-PAGE patterns of SDS-extracted protein from hiochi bacteria can be used as a simple and a useful tool for identification of the microorganisms causing putrefaction of sake.

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.

The nucleotide sequences of alcohol acetyltransferase genes isolated from lager brewing yeast, Saccharomyces carlsbergensis have been determined. S. carlsbergensis has one ATF1 gene and another homologous gene, the Lg-ATF1 gene. There was a high degree of homology between the amino acid sequences deduced for the ATF1 protein and the Lg-ATF1 protein (75.7%), but the N-terminal region has a relatively low degree of homology. Southern analysis and contour-clamped homogeneous electric field analysis of Saccharomyces strains suggest that the ATF1 gene is located on chromosome XV in S. cerevisiae and that the Lg-ATF1 gene might originate from the "non-S. cerevisiae' genome of S. carlsbergensis, which is similar to that of S. bayanus and S. pastorianus.

An autonomously replicating plasmid is expected to increase the frequency of Aspergillus transformation. To construct this type of plasmid, we developed a rapid method of re-isolating autonomously replicating plasmids from Aspergillus transformants. Transformants grown in MM medium under selective pressure for 1-2 days were converted to protoplasts with a cell wall lytic enzyme (e.g. Yatalase). The protoplasts were lysed with phenol/chloroform followed by precipitation with ethanol. The total DNA was treated with RNaseA, re-precipitated with PEG, and then used to transform E. coli. These re-isolated plasmids were mainly the plasmid monomer.

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.

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。

固定化酵母を用いた米糖化液の連続発酵における条件, 特に, 雑菌汚染を防ぐ方法について検討を行った。1. pH 4.0に調整した米糖化液を通常の方法で連続発酵する場合は, 殺菌した原料糖化液を通液する必要がある。2.固定化酵母カラムの前に, 固定化ゲルより溶出した遊離酵母を含む前槽を設置した場合, 前槽液中のアルコール分を平均4%以上に保つことにより, pH 4.0の糖化液を未殺菌のままフィードしても, 長期間にわたり汚染を防止できることが判った。3.さらに前槽を撹拌した場合には, アルコール分を平均6%以上に保持することができ, pH未調整 (pH 5.5), 無殺菌の糖化液をフィードし連続発酵を行ったが, 45日間にわたり雑菌汚染もなく, 安定した酒質のものを得ることができた。尚, 本報告の内容については, 昭和61年度日本農芸化学会大会において発表した。終りに臨み, 本研究の遂行に当り, 終始ご指導を賜りました幣社常務取締役総合研究所長布川弥太郎博士に深く感謝致します。