Nine sulfur-containing acetylenic compounds were isolated and characterized as constituents of Arctium lappa L. The acetylenes named arctinone-a, b (I, II), arctinol-a, b (III, IV), arctinal (V), arctic acid-b, c (VI, VII), methyl arctate-b (VIII) and arctinone-a acetate (IX) were found to be 5'(1-propynyl)-2, 2'-bithienyl-5-yl derivatives on the basis of chemical and spectroscopic evidence.

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A plasmid (pMR226) containing the cDNA coding for the complete amino acid sequence of mouse renin was constructed. The cDNA was reconstructed so as to code for either fused mature renin or preprorenin, and inserted into a plasmid designed to express a protein under the control of the Escherichia coli tryptophan promoter. When introduced into E. coli, the resultant plasmids (pMR304, pMR324) directed the synthesis of proteins that cross-reacted with an antiserum raised against purified mouse renin. The renin precursor produced was readily activated on trypsin treatment and the activity was inhibited by pepstatin and antirenin-antibody.

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The synthesis of lipid oligosaccharide intermediates of a tunicamycin resistant mutant of Chinese hamster ovary cells was investigated both in vivo and in vitro. It was shown that tunicamycin resistant mutant cells had a significantly lower rate of synthesis of lipid oligosaccharide in vivo and their pool of lipid oligosaccharide was smaller.
The formation of lipid oligosaccharides in vitro using microsomes, however, did not show any defect in the enzymes which were involved in this reaction. The rate of transfer of [³H]glucose from UDP-[³H]glucose into oligosaccharide of lipid intermediates was the same in wild type and mutant cells while that into glycoproteins was higher in mutant cells than in wild type cells.

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Pyridoxaminephosphate oxidase (EC. 1.4.3.5: deaminating) is found in many varieties of plants, seedlings, and callus. In plant seedlings, the activity of pyridoxine 5'-phosphate oxidase (PNP oxidase) is greater than that of pyridoxamine 5'-phosphate oxidase (PMP oxidase); the ratios of these activities varied somewhat with the species. Leguminous seedlings had more PMP oxidase activity than seedlings of cereals. Calluses produced from seedlings of soybean, mung bean, and wheat, had these two enzyme activities, but less than those of the original seedlings. The decrease in PNP oxidase activity was especially large.

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We bred potent starch fermenting strains of Saccharomyces diastaticus by means of protoplast fusion. To improve the starch fermentation ability, hybrids carrying unlinked STA genes, which permit fermentation of starch, were introduced by protoplast fusion of haploid strains of identical mating type. Combination of protoplasts prepared from cells of haploid strains with amino acid requirements, cycloheximide resistance or respiration deficiency were fused in the presence of CaCl₂ and polyethyleneglycol 6000. Such fusants thus obtained were selected as regenerated colonies on a minimal medium. The frequencies of fusant formation varied depending upon the strains used and were 7.6 × 10^-6 to 4.1 × 10^-4 for the regenerated protoplasts. Most of the fusants could mate with strains of the opposite mating type, although spore formation by the resultant non-maters was poor. The DNA content and cell volume of the fusants were greater than those of the parental strains. Some fusants produced more ethanol and amylase than the parental strains. Thus, the method we used seemed to be favorable for the breeding of potent starch fermenting strains.

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¹³C NMR spectroscopy was applied to studies on histidine biosynthesis in Corynebacterium glutamicum N-730, a histidine producing mutant. When it was cultured in a medium containing [1-¹³C]glucose as the carbon source, the ¹³C isotopomer was abundantly incorporated into C-6 (28 atom%) and C-1 (21%), and to a lesser extent into C-5 (7%) of the histidine accumulated. The high incorporation into C-6 was explained as being due to that most of the one-carbon unit for histidine biosynthesis is derived from serine. On the bases of this view and the ¹³C population value at C-6, .the contributions of the Embden-Meyerhof and hexose monophosphate pathways to the histidine production were calculated to be 56 and 44%, respectively. The significance of the label incorporation into C-1 and C-5 was discussed.

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The degradation of biotin and dethiobiotin by resting cells of a biotin-degrading bacterium, Mycoplana sp. No. 166, was studied. The metabolites were isolated from reaction mixtures supplemented with biotin or dethiobiotin. By mass and NMR spectral analyses, the hiotin metabolites were identified to be bisnorbiotin and β-hydroxybisnorbiotin, and the dethiobiotin metabolites to be methyl tetranordethiobiotinyl ketone, bisnordethiobiotin, β-hydroxybisnordethiobiotin, and tetranordethiobiotin. The two β-hydroxy metabolites and the keto metabolite were new compounds.

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In order to investigate the relationship between tobacco headspace volatiles and their smoking quality, the volatiles of 44 Japanese flue-cured tobacco samples were trapped by active carbon and analyzed by gas chromatography (GC). The volatiles related to smoking quality were studied by multiple regression analysis (MRA). Prior to MRA, the information on their GC profiles was condensed into the six principal components (PCs) which accounted for 84% of the variance in the 40 GC peaks. The results of MRA using three PCs showed precise predictions for scores of tobacco smoking quality (r = 0.90). The samples were roughly separated into groups according to their variety and stalk position on the plane for the selected PCs. The influences of the variety and stalk position on the smoking quality were also investigated by stepwise discriminant analysis (SDA). As a result of this study, it was found that the volatiles which were partially governed by their variety and stalk position may be used to evaluate the smoking quality of tobacco.

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A specific layer at the outermost surface of mycelia of Glomerella cingulata G 4-1-4 was observed. The substance isolated from this layer was a polysaccharide ([α]²⁰_D +82.6°C) composed of o-mannose (97.6%), protein (2.0%) and a trace amount of phosphate. A high proportion of 3, 4-di-O-methylmannose (38mol%) was found on methylation analysis. Digestion of this mannan with a bacterial α-D-mannosidase released 78% of the D-mannose. In addition, the results of acetolysis, partial acid hydrolysis, and ¹H- and ¹³C-NMR spectroscopy showed that the G. cingulata G 4-1-4 mannan has a backbone joined by α-l, 6-linkages, and α-1, 2- and 1, 3-linked mannose residues are attached to the C-2 position of the main chain segment, giving it a highly branched structure. Although G. cingulata G 4-1-4 mannan has the above properties, its basic structure appears to be similar to that of baker's yeast mannan.

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Colistin was observed to bind to eell wall preparations of colistin-producing Bacillus colistinus 11-4. The binding occurred at 0 and 30°C, and was much suppressed by divalent cations (Mg²⁺ and Ca²⁺), acidic pH and the cationic detergent, cetylpyridinium chloride, but not by the neutral detergent, Triton X-100. When the carboxyl groups in the cell wall preparations were chemically modified to neutralize their electrochemical charges through the formation of glycine ethyl ester, the modified wall showed extremely decreased binding capacity for colistin. The molar ratio of charged carboxyl groups in the wall and maximum binding to colistin was calculated to be 1:0.25. A large excess of the wall preparation protected the protoplasts of colistin-producing organisms from the action of colistin, but the modified wall did not.

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A method of polyethyleneglycol(PEG)-induced protoplast fusion was developed for obtaining better Brevibacterium amino acid producers. By this method, interspecific and intraspecific recombinants among mutants of Brevibacterium sp. were obtained at a frequency of 10^-3-10^-5. In order to improve the threonine productivity, a genetic marker of lysine-requirement was introduced by protoplast fusion into a strain of B. lactofermentum that produced threonine and lysine simultaneously. The resultant lysine-requiring recombinants accumulated only threonine, with an increased yield. On the other hand, protoplast fusion was carried out to improve the glucose metabolizing activity of industrial lysine producers. The low rate of glucose consumption of a lysine producer due to multi-step breeding was improved by protoplast fusion with a strain showing rapid glucose consumption. The rate of glucose consumption of the recombinant was 3 times higher than that of the parental lysine producer, and the lysine production yield of the recombinant was the same as that of the parental strain.

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The distribution of microorganisms in 15 samples of selected spices and the effects of irradiation of them were studied. The total aerobic bacteria in black pepper, white pepper, turmeric, rosemary and basil were determined to be 3 × 10³ to 5 × 10⁷ per gram. Coliforms were also determined in 8 samples to be 2 × 10² to 2 × 10⁶ per gram. The main aerobic-spore-formers were identified as Bacillus pumilus and B. subtilis. Molds were determined in 10 samples to be 1 ×0 10² to 2 × 10⁴ per gram which consisted mainly of the Aspergillus glaucus, A. restrictus, A. flams, A. fumigatus, A. niger groups and Penicillium. A study on the inactivation of microorganisms in spices showed that gamma-irradiation doses of 1.2 to 1.5 Mrad were required to reduce the total aerobic bacteria to below a detectable level, while doses of below 1.0 Mrad were required to decrease the spore-forming bacteria to below 10³ per gram, the Japanese hygenic standard. Coliforms were eliminated with 0.4 to 1.0 Mrad irradiation. In the storage study, at humidity levels higher than 84% at 30 or 35°C, mold counts increased more than 10⁶ per gram in many kinds of powdered spices in polyethylene pouches during 1 to 2 months of storage, while samples subjected to 0.4 Mrad irradiation were free from molds.

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The antifungal antibiotics, neopetins A(1), B(2) and C(3), were found to be cyclic lipopeptides containing unusual amino acids, their structures being elucidated on the basis of chemical and spectroscopic evidence. They inhibited mannoprotein and β-1, 3-glucan synthetases from Saccharomyces cerevisiae. The structure-biological activity relationship is discussed.

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Autoxidation of ascorbic acid (AsA) in the presence of Cu²⁺ generates some oxygen radicals, which induce the cleavage of glucosidic linkages of β-cyclodextrin (β-CD). The oxidative cleavage of β-CD gave several kinds of oligosaccharides and their reducing terminal sugars were mainly D-glucose. D-Erythrose, D-threose, D-arabinose, and D-xylose were also detected as minor reducing terminals by GLC and GC-MS analyses. From the experiments using several radical scavengers, the hydroxyl radical (OH radical) was identified as the main active radical.
During this reaction, 2-thiobarbituric acid reactive substances (TBARS) were produced by the oxidative degradation of β-CD, but their chemical structures and formation mechanism are still unknown.

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Lipase-catalyzed Stereoselective hydrolysis of 2-acyloxy-3-chloropropyl p-toluenesulfonate (1) was investigated. From the screening tests, lipases from Pseudomonas aeruginosa, Aspergillus niger, Mucor species, Rhizopus delemar and Rhizopus japonicus were found to hydrolyze (R, S)-1 stereoselectively to afford (R)-1 and (S)-2-hydroxy-3-chloropropyl p-toluenesulfonate (2). Among these enzymes, the lipase from Pseudomonas aeruginosa was found to possess the highest hydrolytic activity and stereoselectivity in more than 99% e.e.

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The cysteine proteinase of sprouting potato tubers was purified to homogeniety as judged by PAGE or SDS-PAGE for the first time. The molecular weight of this enzyme was 28, 200 daltons by Sephadex G-75, and 27, 500 daltons by SDS-PAGE. This enzyme has an isoelectric point at pH 5.10, an optimum pH for the activity at pH 5.0-6.0, and a tendency to stability between pH 4.5-6.5. SH-compounds such as DTT (dithiothreitol) and 2-mercaptoethanol stimulated the activity of this enzyme. Heavy metal ions and Zn²⁺ strongly inhibited it, but EDTA slightly increased it. SH blocking reagents such as monoiodoacetate, PCMB, and TLCK inhibited this enzyme activity. Chymostatin, antipain, leupeptin and E64 caused complete inhibition.

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Cystathionine γ-lyase from Streptomyces phaeochromogenes catalyzes not only the α., γelimination reaction of L-cystathionine, but also the γ-replacement reaction of L-homoserine in the presence of thiol compounds. Substrates for the enzyme in the γ-replacement reaction were examined. It was found that D-cysteine, L- and D-homocysteine, and 3- and 2-mercaptopropionate served as preferable substrates in the γ-replacement reaction. D-Allocystathionine, ^*1 L- and meso-homolanthionine, S-carboxyethyl-L-homocysteine and S-methylcarboxymethyl-L-homocysteine were enzymatically synthesized from L-homoserine and the corresponding thiol compounds. The thus synthesized S-substituted L-homocysteine derivatives were isolated from large scale reaction mixtures and identified physicochemically.

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The potential of liposomes as vehicles for introducing membrane-impermeable substances into yeast protoplasts was studied using a membrane-impermeable water-soluble fluorescent marker, fluorescein isothiocyanate dextran (FITC-dextran). Simple mixing of FITC-dextran encapsulated in liposomes with protoplasts didn't result in the delivery of liposome-encapsulated FITC-dextran into protoplasts. However, when a liposome-protoplast mixture was treated with glycerol after this incubation, the liposome-encapsulated FITC-dextran was delivered into the protoplasts. Three critical factors affected the efficiency of this delivery: the concentration of the glycerol solution; the concentration of calcium ion in the liposome-protoplast incubation mixture; and the timing of the glycerol treatment. Energy metabolism inhibitors blocked this delivery. Under the best conditions about 70% of yeast protoplasts became fluorescent due to the incorporation of FITC-dextran via liposomes.

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Isocitrate lyase (EC 4.1.3.1) was purified from the pollen of Pinus densiflora Sieb. et Zucc. to apparent homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was 200, 000 by glycerol density gradient analysis and gel filtration on Sepharose 6B, and the subunit molecular weight was 65, 000 by SDS-PAGE. The enzyme was maximumally active at pH 7.6 in the presence of 3 mM MgCl₂ and 0.5 mM EDTA. The enzyme was completely inactivated by heating at 50°C for 5 min, but no activity was lost by the same treatment in the presence-of 3 mM Mg²⁺ and 0.5 mM EDTA. The enzyme was inhibited by PCMB, and the inhibition was reversed by the addition of an equimolar amount of cysteine. ATP, ADP, 3-phosphoglycerate, phosphoenolpyruvate, tartrate, maleate, oxalate, and itaconate inhibited the enzyme. The Km value for DL-isocitrate was 6.6 × 10^-4 M. Itaconate was an uncompetitive inhibitor with respect to isocitrate, and the Ki value for itaconate was 2.8 × 10^-6 M.

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The flexibilities of ovalbumin and lysozyme, detected by the protease-probe method, were monitored during heat-denaturation. The flexibilities of these proteins increased in the heating temperature region where the structural changes were too small to detect by other optical methods, such as surface hydrophobicity measurement. To evaluate the relationship between the structural and functional properties (foaming and emulsifying properties), the thermal denaturation curves for the flexibility and surface hydrophobicity were compared with those for the foaming power and emulsifying activity of ovalbumin and lysozyme. The thermal transition points of the foaming power closely corresponded to those of the flexibility of ovalbumin and lysozyme. On the other hand, the thermal transition points of the emulsifying activity corresponded to those of the surface hydrophobicity of ovalbumin and lysozyme. Thus, it was suggested that the flexibility of proteins may be a main governing factor for the foaming property and that the surface hydrophobicity of proteins may be a main governing factor for the emulsifying property.

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A gram negative bacterium isolated from soil was found to produce a high level of endo-β-N-acetylglucosaminidase in the culture medium. The organism was identified as a Flavobacterium sp. from various bacteriological characteristics. The enzyme from the Flavobacterium sp. was purified to homogeneity from culture broth by fractionation with ammonium sulfate and column chromatographies on DEAE-cellulose, hydroxylapatite, and Sephadex G-150 and G-100. The molecular weight of the enzyme was estimated to be 27, 000 and 30, 000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, and it appeared to consist of a single polypeptide chain. The optimal pH for activity was 5.0 to 6.0 and the stable pH range was 5-7. The Michaelis constant was 0.30 mM with dansyl-Asn-(GlcNAc)₂(Man)₆ as the substrate. The enzyme hydrolyzed oligosaccharides of native ovalbumin, bovine pancreatic ribonuclease B and a yeast invertase.

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We studied the role of surface substances on germinated spores in host-parasite interaction using rabbit antiserum against the substances from germinated spores of sweet potato strain of Ceratocystis fimbriata, compatible with sweet potato. The antiserum agglutinated the germinated spores of this strain and also decreased ethylene production in sweet potato root tissues infected by this strain, which was used here as an index of pathogenicity. On the other hand, the antiserum showed little effect on ethylene production in the root tissues infected by coffee strain, incompatible with sweet potato. The surface substances from germinated spores of sweet potato strain were fractionated into four fractions with ammonium sulfate; precipitates by 0-20%, 20-40%, and 40-60% saturation of ammonium sulfate and the final supernatant. These fractions were assayed for spore agglutination inhibitory activity, ethylene production stimulative activity and antiserum adsorptive activity. All three activities were localized in the same fraction, that precipitated by 0-20% saturation, suggesting the involvement of the same entity or entities in these activities.

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Crude acid phosphatase from wheat germ was separated into two enzyme forms by ion exchange chromatography. One of these, termed P-I, was composed of acidic isozymes with pI's of 4.0-4.7, and the other form, termed P-II, had more neutral ones with pI's of 4.7-7.0. There were no large differences between P-I and P-II in the relative rates of hydrolysis of substrates and optimal pH's. Immunological study, however, revealed that the two enzyme forms have distinct antigenic characteristics.

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The effect of phenol derivatives on cyanide-insensitive respiration of Sp. ruberrimus was studied. Among various phenol derivatives tested, the o-diphenols, pyrocatechol and pyrogallol, were found to be specific inhibitors of the cyanide-insensitive respiration of Sp. ruberrimus but they did not affect its glucose oxidation. m-Diphenols and p-diphenols had no specific inhibitory effects on the cyanide-insensitive respiration. Also, the cyanide-insensitive respiration was inhibited by the addition of salicylhydroxamic acid, which is a specific inhibitor of the cyanide-insensitive terminal oxidase of plant mitochondria.

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Sporobolomyces ruberrimus is insensitive to antimycin A which is a respiratory inhibitor of the cytochrome system, as cyanide is. When this red yeast was cultured in the presence of antimycin A, the growth curve showed the same pattern as that of the normal culture in the absence of it, but the growth mass was only about 70% of that of the normal culture. The antimycin A-insensitive and cyanide-insensitive respiration of Sp. ruberrimus was inhibited by pyrocatechol and salicylhydroxamic acid. Sporobolomyces red yeasts have two characteristic terminal oxidase systems; one is a cytochrome oxidase system and the other is a cyanide- and antimycin A-insensitive oxidase system. The proportions of the two respiratory systems differed among the species and strains of Sporobolomyces red yeasts examined.

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Microorganisms utilizing 1-chlorobutane as a sole carbon and energy source for growth could release halogens under anaerobic conditions, while microorganisms which could utilize 1, 9-dichlorononane released the halogens only under aerobic conditions. A 1-chlorobutane-utilizing bacterium, strain m15-3, converted 1-chlorobutane to butyric acid and 1, 3-dichloropropane to 3-chloropropionic acid under aerobic conditions and 1-chlorobutane to butanol under anaerobic conditions. In the latter case, the participation of halidohydrolase was suggested.
Methane-utilizing bacteria catalyzed the removal of halogens from the terminal positions of short chained chlorinated hydrocarbons. Methane-utilizing bacteria dehalogenated 1, 2-dichloroethane to ¹⁸O-incorporated 2-chloroacetic acid in the presence of ¹⁸O₂ gas.
All of the seven bacterial strains used in this study dehalogenated 3-chlorinated aliphatic acids, but only one strain out of seven could dehalogenate 2-chlorinated aliphatic acids.

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Triacylglycerol lipase activity appeared on the 8th day of embryonic development of the Japanese quail in the egg yolk sac and its contents, and activity was highest on the 12th day, although yolk lipids decreased markedly at the later stage of embryonic development. We extracted the lipase from the defatted yolk sac of 12-day eggs, purified it to a 200-fold specific activity. This purified preparation gave at least 2 bands having hydrolyzing activity against β-naphthylbutyrate upon polyacrylamide gel electrophoresis, with molecular weights of 55, 000 and 57, 000. The enzyme had maximum activity at pH 8.0 and 38°C. Concerning substrate specificity, the enzyme hydrolyzed a variety of triglycerides, but had the highest specificity for tributyrin. This lipase had some heterogenous forms on isoelectric focusing, with pI values of 4.8 to 5.2. The profile of isoelectric focusing was changed by neuraminidase treatment, showing that sialic acid content affects the net charge of the lipase molecules.

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The 4-pyrone and 4-pyridone series were examined as a new type of photosynthetic inhibitor because of a structural resemblance to the co-enzyme plastoquinone, which lies between photosystem II and cytochrome b. These compounds inhibited a Hill reaction at "the plastoquinone pool." From our study of the structure/activity relationships for 4-pyrones and 4-pyridones, it has become clear that these compounds need no strict structural resemblance to the plastoquinone. It is also demonstrated that the brominated 4-pyridone series has remarkably higher inhibitory activities than those of its non brominated analogues.

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Glucoamylase was purified from the culture fluid of the yeast Saccharomyces diastaticus carrying STA1. The molecular weight of the protein was about 250K by both gel-filtration and acrylamide gel electrophoresis. The protein was glycosylated with asparagine-linked glycosides whose molecular weight was 70K. The amino-terminal sequence of the protein began from the 33rd amino acid residue from the first mcthionine of the putative precursor deduced from the DNA sequence of STA1. The amino acid composition of the purified protein matched the predicted amino acid composition. These results confirmed that STA1 codes for a structural gene of glucoamylase. Yeast cells secreted enzymatically active β-lactamase into the culture medium after transformation with hybrid plasmids containing the glucoamylase promoter and the DNA sequence encoding the extended peptide of 32 amino acids fused to a structural gene for Escherichia coli β-lactamase. The result suggested that the amino-terminal peptide of the putative glucoamylase precursor functions as a signal sequence for protein secretion. Taking into consideration the subunit structure of the previously purified glucoamylase [Yamashita et al., Agric. Biol. Chem. 48, 1611 (1984)], we present the molecular structure of the glucoamylase precursor and also a model for its proteolytic processing.

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A lignin-degrading Basidiomycete, Phanerochaete chrysosporium, produced peroxidases other than ligninperoxidase in a low nitrogen culture. One of the peroxidases was purified by DEAE-Sepharose column chromatography. The enzyme (NADH-peroxidase) oxidized NADH besides phenol red and other substrates for peroxidases. The product on NADH oxidation by the enzyme was H₂O₂. The reaction was stimulated by Mn²⁺ and inhibited by heme protein inhibitors. A diarylpropane compound, a lignin model, was degraded in the presence of both the NADH- and lignin-peroxidase with NADH. This reaction showed that NADH-peroxidase played a role as a H₂O₂ donor in the lignin-peroxidase reaction.

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