Various kinds of eucaryotic microorganisms were screened as to their tocopherol production by means of high performance liquid chromatography. Non-photosynthetic microorganisms did not produce detectable amounts of the vitamin. In cells of photosynthetic algae grown photoheterotrophically, tocopherols, mainly of the α-type, were detected. Euglena gracilis Z was employed for further study to optimize the culture conditions for α-tocopherol production. The α-tocopherol production was affected by the concentrations of the carbon and nitrogen sources, glucose and peptone. In a constructed medium, the amount of α-tocopherol in cells reached 9.9 mg per liter of culture broth or 1.1 mg per gram of dry cells. The effects of the addition of some compounds to the culture medium on α-tocopherol production were then studied. Homogentisate and L-tyrosine were very effective as additives. Feeding of ethanol and peptone also increased the production. With the feeding of ethanol, peptone, homogentisate and L-tyrosine three-times, the amount of α-tocopherol reached 143.6 mg per liter of culture broth or 5.1 mg per gram of dry cells.

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The p-nitrophenol-releasing activity, from p-nitrophenyl (p-NP) β-D-fucoside, of β-D-glucosidase I from Bifidobacterium breve clb was enhanced by the addition of many kinds of sugars and alcohols, suggesting the occurrence of a β-D-fucosyl transferring reaction. The enhancement on glucose addition was dependent on the reaction pH, and the concentrations of p-NP β-D-fucoside and glucose, and the activity reached 430% when 100 mM glucose was added to the mixture containing 20mM p-NP β-D-fucoside at pH 4.5. A mixture of transfer products was separated from the other constituents by activated charcoal column chromatography. Further purification of the mixture by paper and thinlayer chromatographies gave four oligosaccharides, which were identified as β-D-fucosylglucoses with β1→2, β1→3, β→4 and β1→6, linkages from the absorption spectra obtained with the phenolsulfuric acid method, and the results of acid and enzymatic hydrolyses, and methylation analyses. The mixture of oligosaccharides was well assimilated by many bifidobacteria but not by any other intestinal bacteria tested.

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"Delicious peptide" was reported to be as delicious as beef soup. The peptide and its fragments were synthesized to study its taste active sites. "Delicious peptide" was found to produce a umami and a sour taste. By preparing several di- or tripeptides composed of basic or acidic amino acids, we found that the taste of "delicious peptide" was produced by an interaction between the basic and the acidic fragments in the peptide.

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The crystallization rates of cocoa butter with and without seeding were measured at 25 and 30°C with a viscometer to examine seeding effects on the solidification kinetics of cocoa butter. Three polymorphic forms of cocoa buffer were used as the seed crystals to examine the influence of polymorphism. We found that the crystallization was greatly accelerated by the addition of the seed powders of Forms III, V, and VI; Form VI was most effective. However, the polymorphic form of the crystallized cocoa butter was primarily set by the temperature of crystallization irrespective of the polymorphism of the seed crystal: Forms IV and V at 25°C, and Forms V and VI at 30°C.

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Trypsin inhibitor (JBTI) was extracted and purified from the bran of Job's-tears (Coix lacryma-jobi L. var. Ma-yuen Stapf) seeds. Its molecular weight was estimated as 16500 by SDS-PAGE. Trypsin inhibitory activity of JBTI was enhanced in the presence of divalent cations, such as Ca²⁺ or Mg²⁺, and inhibited by divalent heavy metal ions, such as Cu²⁺ or Zn²⁺ . Its activity was stable against pepsin digestion, while JBTI was denatured by subtilisin BPN' or Actynase E digestions. Its activity was stable against chemical modification of the lysyl residues, while JBTI lost its activity by the modification of the arginyl residues. The thermal unfolding of JBTI was studied in an adiabatic differential scanning calorimeter and it was found that the protein unfolds at 93°C and pH 7.0 in 50 mM phosphate buffer.

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A novel maltose-producing amylase, which strictly hydrolyzes the α-I, 4-glucosidic linkages in starch with maltose units and releases α-maltose, was found in the culture filtrate of a strain of Bacillus megaterium newly isolated from soil. The enzyme was purified to almost homogeneity by means of ammonium suit ate fractionation, DEAE-Sepharose column chromatography and Bio-gel A 0.5m column chromatography. Its optimum pH and temperature were around 7.0 and around 60°C, respectively. The enzyme was inhibited by sulfhydryl reagents such as mercuric chloride and p-chloromercuribenzoate, and the inhibition was recovered by cysteine. Its molecular weight was estimated to be about 60, 000 by the gel filtration method. Its enzymatic properties, including its action pattern, were very similar to those of β-amylase, except for the configuration of the maltose produced.

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Field studies on the control of potato common scab were carried out using a Streptomyces scabies-antagonistic biofertilizer produced from swine feces with a selected strain of Streptomyces albidoflavus CH-33. The field soil, which was supplemented with lime in order to adjust the pH into the agricultural range of 6 to 7, was mixed with 3.8 kg of biofertilizer per subplot (1.0 × 1.2 m) to a depth of 15 cm. The scab severity, in terms of the percent coverage of the total surface area of the potato tubers, was 18.3 % and 23.2 % in the control and the subplot supplemented with barnyard manure, respectively, whereas the lesions were light in the biofertilizer subplot, the scab severity being only 1.7% in terms of percent coverage. The field soil supplemented with the biofertilizer gave the maximum potato production. In the biofertilizer subplot, the viable count of Streptomyces scabies in the field soil had decreased to 2 × 10² per gram soil by 80 days, whereas that of actinomycetes including Streptomyces albidoflavus CH-33 had increased to 4 × 10⁸ per gram soil.

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When added directly to the assay medium under standard conditions, 3-bromopyruvate inhibited wheat dihydrodipicolinate synthase (DHDPS) competitively with respect to pyruvate and uncompetitively with respect to aspartate semialdehyde. When DHDPS was incubated with 3-bromopyruvate at pH 7.5 and 30°C, this reagent irreversibly inactivated the enzyme. The inactivation followed pseudofirst-order kinetics during the early period of inactivation; then the inactivation rate decreased. For the early period of inactivation, the pseudo-first-order rate constant was 0.29 min^-1 and the Ki of 3-bromopyruvate was 1.8mM. Pyruvate protected against inactivation, while aspartate semialdehyde partially did so and lysine did not. The Hill constant for lysine of the enzyme inactivated by 73 % was about half of that of the untreated enzyme.

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A new enzyme, N-acyl-D-hexosamine oxidase, was purified to homogeneity on polyacrylamide gel electrophoresis from Pseudomonas sp. 15-1 by successive procedures involving CM-cellulose fractionation and chromatographies on Phenyl-Sepharose, QAE-Sephadex, SE-Sephadex, and Sephadex G-200, and some properties were investigated. Its molecular weight was 170, 000 on gel filtration and it was composed of one each of four non-identical subunits; 63, 000, 44, 000, 36, 000, and 22, 000. The absorption spectrum of the enzyme was the same as those of many flavoproteins. Its isoelectric point was at 7.9. Characteristically, the enzyme showed its activity throughout a wide pH range and the optimum pH was 8.0. N-Acetyl- and N-glycolyl-D-glucosamine and N-acetyl-D-galactosamine were oxidized as good substrates of the enzyme and their apparent Km were 0.24, 2.6 and 0.10 mM, respectively. But N-acetyl-D-mannosainine was a poor substrate. The anomeric configuration of the substrate required was β-form. The reaction products from 1 mol each of N-acetyl-D-glucosamine and oxygen were found to be 1 mol each of N-acetyl-D-glucosaminic acid and hydrogen peroxide. The enzyme was inhibited specifically by Zn²⁺. A preliminary study suggested that the enzyme might be useful for measurement of N-acetyl-β-D-glucosaminidase.

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In addition to head-to-tail tandem repeats of injected pSV2-gpt-gE1A DNA, head-to-head and tail-to-tail tandem structures were detected in a transgenic mouse, pEl A/3, which showed the existence of inverted repeats. The injected DNA molecules in the founder mouse were segregated into two groups in its progeny. One group had about 80 copies of pSV2-gpt-gE1A and the other had 20 copies per diploid genome. Both the high and the low copy number lines contained inverted repeats. Breeding experiments showed that the inverted repeats in about half of the transgenic progeny were lost during the transmission from generation to generation. Progeny of the mice that lost the inverted repeats carried only head-to-tail tandem repeats.
Besides the inverted sequences, another portion of the transgene in the high copy line was found to be lost during germline transmission but independently.

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The culture of mouse spleen cells with concanavalin A (Con A) or phytohemagglutinin (PHA) increased histidine decarboxylase (HDC) activity as a function of incubation time. The histamine level of the medium increased in parallel with the increase in HDC activity. Lymphocyte proliferation induced by Con A was enhanced by cimetidine, a histamine H₂-antagonist, in the absence of exogenous histamine. Dimaprit, an H₂-agonist, blocked lymphocyte blastogenesis at high doses (>10^-5M). Diphenhydramine, an H1-antagonist, abolished mitogenesis induced by Con A. At low doses (10^-9-10^-7 M), the H₁-agonist 2-pyridylethylamine augumented mitogenesis mediated by Con A. The addition of alphafluoromethylhistidine or histaminase (EC 1.4.3.6), at low doses, to the culture enhanced Con-A-dependent lymphocyte proliferation. At high doses they inhibited the reaction. Histamine itself augmented [³H]thymidine uptake by lymphocytes induced by Con A at the low doses of10^-13-10^-11 M;at higher doses (>10^-5 M), it suppressed this response. These results suggest that mitogen-induced lymphocyte proliferation is regulated by histamine, formed in the system per se through the action of HDC.

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PNP-(GlcNAc)₅, which is available as a substrate of a lysozyme assay, was efficiently synthesized by transfer of the tetra-N-acetyl-chitotetraosyl residue to the 4-position of PNP-GlcNAc using lysozyme in an aqueous solution containing DMSO in a high concentration. The use of an aqueous DMSO system in this reaction not only ensured a sufficient solubility of PNP-GlcNAc substrate, but also resulted in the high yield of PNP-(GlcNAc)₅. PNP-(GlcNAc)₅ insolubilized from the reaction mixture was directly obtained in a high state of purity by washing with aqueous methanol without using any column chromatographic separation.

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Interspecific protoplast fusion between Candida utilis IAM 4277 and Candida lipolytica OUT 6340 was carried out. A stable fusant, No. 1, was selected from among many fusants which were able to grow on both maltose and n-hexadecane. Fusant No. 1 seems to be an intermediate between the parents with respect to morphological and physiological characteristics. Further chemotaxonomic studies on the coenzyme Q type, fatty acid composition and enzyme patterns, and antigenic analysis revealed that fusant No. 1 is a hybrid having both a major part of the characteristics of C. lipolytica OUT 6340 and a minor part of those of C. utilis IAM 4277.

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Based on studies on the acceptor specificity of the transglucosylation with cyclodextringlucosyltransferase (CGT-ase) as well as on the structure-sweetness relationship of steviol glycosides, selective elongation of the 13-O-glucosyl moiety of steviol bisglycosides was done to improve sweetness. The β-D-glucosyl ester group at the 19-carboxylic acid of rubusoside and stevioside was chemically replaced by β-D-galactosyl group as a blocker against the glucosylation and both the galactosyl esters were regioselectively 1, 4-α-glucosylated on the 13-glycosyl moiety with CGT-ase using soluble starch as a donor. Great improvement in sweetness was observed for the products which have three or four glucosyl units at the 13-position, while the products with five or six glucosyl units had less sweetness than the starting materials. It was also revealed that the sweetness of the products of the stevioside series were better than the corresponding isomers of the rubusoside series.

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A screening test was done to isolate microorganisms that produced nucleoside oxidase. The enzyme activity was found in a few strains of Gram-negative bacteria. One strain (LB-86) of these microorganisms showed strong ability to produce the enzyme. The morphological and physiological characteristics of strain LB-86 were studies, and this strain was found to be Pseudomonas maltophilia. From the examinations of culture conditions, suitable conditions for enzyme production were found, and strain LB-86 was aerobically cultured in shaking flasks at 26°C in a medium containing 2.5 % yeast extract, 3% glucose, 0.1% KH₂PO₄, 0.1% KCI, and 0.05% MgSO₄•7H₂O, pH 7.2. The highest activity was obtained after 22-24 hr of cultivation.

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Tyrostatin, a new carboxyl proteinase inhibitor, was discovered in a culture filtrate of an actinomycete. From taxonomical studies, the strain was identified and named Kitasatosporia sp. No. 55. The inhibitor was extracted with ethylacetate, purified by Sephadex LH-20 and silica gel column chromatographies, and crystallized from methanol. The yield was about 100 mg from 18.2 1 of culture filtrate. The structure of tyrostatin was N-isovaleryl-tyrosyl-leucyl-tyrosinal.
Tyrostatin strongly inhibited pepstatin-insensitive carboxyl proteinases originating from Pseudomonas sp. No. 101 and Xanthomonas sp. No. T-22, but not any of the other pepstatininsensitive carboxyl proteinases tested. It inhibited all of the carboxyl proteinases tested of the pepstatin-sensitive group, although the inhibitory activities were not so potent. It also strongly inhibited such cysteine proteinases as papain.

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We investigated the optimal conditions for anthocyanin production in suspension cultures of a Euphorbia millii cell strain obtained by successive cell-aggregate selections. A modified Gamborg's solution containing 5 to 7% sucrose (w/v), a 1:16 ratio of NH₄⁺:NO₃^-, 30 mM of nitrogen source, 10^-6 M 2, 4-D, 10^-8 M BA, and low concentrations of Fe²⁺ and SO^2-₄ greatly increased anthocyanin production. 2, 4-D was superior to NAA in increasing anthocyanin production. In the anthocyanin production medium we established, E. millii cells produced 32 mg anthocyanin per liter per day.

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Three ingenol esters as piscicidal constituents and an ingol ester were isolated from Euphorbia trigona (a popular indoor foliage plant in Japan) and their structures were unambiguously elucidated.

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We have developed a novel transformation system for yeasts carrying no selectable markers. By using a plasmid, pYHH-1, containing an internal region of the CAN1 gene, a wild type yeast strain was endowed with L-canavanine resistance (canl) through integral disruption of the resident CAN1 gene. Diploid strains as well as haploids can be transformed, indicating that L-canavanine resistance can be used as a positive selectable marker for the transformation of industrial yeasts.

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Quinoxaline derivatives derived from D-glucuronic acid (GlcUA) and D-galacturonic acid (GalUA) with o-phenylenediamine (OPD) under deoxygenated and heated conditions in alkaline media were analyzed by gas-liquid chromatography, GC-MS and HPLC. Both hexuronic acids gave the same quinoxaline derivatives as those derived from D-glucose, but the quantity of each quinoxaline formed differed, depending on the material used, with regard to the configuration of the OH group at the C-4 position. The results of quantitative analyses of the quinoxaline derivatives derived from GlcUA and GalUA suggested that carbon-carbon linkages of the uronic acids were split between the C-3 and C-4 positions. No quinoxaline derivatives with carboxylic acid in their structures were formed, so the alkaline degradation probably started after the decarboxylation of GlcUA and GalUA. The patterns of degradation and condensation of the uronic acids and OPD were similar to those of glucose after the decarboxylation of GlcUA and GalUA.

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A major trypsin inhibitor was purified from the extract of the whole grain of foxtail millet, Setaria italica, to an electrophoretically homogeneous protein by conventional methods. This inhibitor (FMTI-II) has a molecular weight of 7500 and contains high levels of basic amino acids, acidic amino acids, prolinc, and half-cystine. FMTI-II inhibited bovine and hog trypsins in a 1:1 (M/M) stoichiometry: the K_i values were 3.0 × 10^-11 M and 2.2 × 10^-10 M, respectively. Bovine α-chymotrypsin, subtilisin BPN', hog pepsin, and papain were not inhibited. The inhibitor was stable in a wide range of pH and was heatresistant at acidic pH. The chemical modification suggested that FMTI-II had a Lys-X bond as a trypsin reactive-site. FMTI-II resembles rice bran and wheat germ trypsin inhibitors, showing that it is a Bowman-Birk type inhibitor.

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PM-toxins are host-specific pathotoxins produced by the corn fungal pathogen Phyllosticta maydis. Three PM-toxin analogues of a new type which have oxygen atoms in the skeletal carbon chain were synthesized as stereoisomeric mixtures. The linear ester PML-777 was about 100-times less active than the potent synthetic analogue PM-777 in an NADH oxidation assay using corn mitochondria. A large cyclic analogue, PMC₃₂-7777, was only about 4- to 10-times less active than the parent linear ester PML-777, but over 500-times more active than a smaller sized analogue, PMC₂₀-711. The striking loss of biological activity in PML-777 clearly shows the significance of a carbonchain skeleton like PM-777 for higher biological activity. The unexpectedly slight decrease of activity from PML-777 to PMC₃₂-7777 may suggest that the active conformation of the linear analogue (PML-777) was similar to that of the cyclic analogue (PMC₃₂-7777).

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Penidllium simplidssimum AK-40, which was isolated from a soil sample, produced novel insecticidal compounds when cultured with okara (the insoluble residue of whole soybean). The metabolites, named okaramine A (1) and B(2), were crystalline products with molecular formulas C₃₂H₃₂N₄O₃ and C₃₃H₃₄N₄O₅, respectively. The structure of 1 was deduced from the structure of acetylokaramine A that was established by single crystal X-ray diffraction. The structure of 2 was determined from the data of UV, IR, NMR (¹H and ¹³C) and mass spectrometry. 1 and 2 showed insecticidal activity against the 3rd instar larvae of silkworm at doses of 3 μ/g of diet and 0.1 μg/g of diet, respectively.

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The structure/activity relationships of phlorophenone derivatives including grandinol and homograndinol, which are potent inhibitors of photosynthetic electron transport (PET) in Eucalyptus grandis, were examined. The results indicate that at least one acyl functionality on a phloroglucinol nucleus was essential for PET inhibition, and that the lipophilicity and/or length of the acyl group was the most prominent factor affecting the activity. In addition, the introduction of a formyl group to monoacylphloroglucinols remarkably enhanced the activity. The structural requirements for PET inhibition in these compounds were found to be very similar but not identical with those for the phenol type of inhibitors proposed by Trebst et al.

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An E. coli mutant (M8328) was isolated by NTG mutagenesis from E. coli KMS3207 which has an inducible N-acetylneuraminate lyase (NPL). The mutant was resistant to catabolite repression and produced NPL constitutively. To improve the productivity of NPL, a hybrid plasmid, pMK6, carrying the npl gene was introduced into the mutant, M8328. The NPL activity level was increased more than 5-fold in the pMK6-harboring mutant compared with that in the mutant, when the cells were grown in inducer (N-acetylneuraminic acid)-free medium. The NPLs produced by the mutant and the pMK6-harboring cells were structurally and immunologically identical with that purified from the parent strain (KMS3207).

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A family of secretory or transmembrane proteins have a unique glycopeptide which contains abundant, clustered threonine and serine residues. Such a sequence is also found in a yeast extracellular glucoamylase encoded by STA1. To investigate the role of the threonine- and serine-rich tract (TS) in protein secretion, I constructed and introduced into yeast a series of internal deletions of STA1 and chimeric genes which code for various amounts of Sta1 amino-terminal peptide and a constant carboxy-terminal peptide of either intracellular glucoamylase (encoded by SGA) or β-glactosidase (encoded by laeZ), and examined the secretory nature of their gene products. I found that the mutant Sta1 proteins without TS were not secreted and that the hybrid β-galactosidase proteins carrying TS were transported to the cell envelope. These results suggest that TS can serve as a signal for intracellular transport of proteins.

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The gene for maltohexaose-forming amylase (G₆-amylase) from alkalophilic Bacillus sp. H-167 was cloned into Escherichia coli HB101 using pBR329 as a cloning vector. Recombinant pi as mid, pSB404, containing a 3.0kb fragment derived from H-167 DNA, was shown to direct the synthesis of G₆-amylase, which was partly secreted into the culture broth on the addition of a certain carbohydrate. The pH- and temperature-activity profiles of the G₆-amylases from HB101 (pSB404) and H-167 were essentially the same. The G₆-amylase produced by HB101 (pSB404) was observed to be multiform as well as H-167; the molecular weights were 90, 000, 73, 000 and 60, 000, respectively. Protease inhibitor experiments showed that the multiple expression was due to intracellular proteolysis of the enzyme.

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Polymyxin acylase, which produces deacylated polymyxins by hydrolyzing only the fattyacyl groups of polymyxin antibiotics without affecting the peptide moiety, was purified from acetone-dried cell powder of Pseudomonas sp. M-6-3. The cell-free enzyme, solubilized by Triton X-100, was further purified on successive DEAE-cellulose, hydroxyapatite, and Sephacryl S-300 columns to a homogeneous state. This purified enzyme (Type I) had a single band with an MW of 62, 000 in SDS-polyacrylamide gel electrophoresis. Gel filtration on a calibrated Sephacryl S-300 column also gave an estimated MW of 62, 000. The isoelectric point of the enzyme was 5.7. Enzyme activity was optimal at pH 9.0 for colistin A (polymyxin E₁) and was stable up to 50°C for 24 hr. The observed V_max and Km values were 1750nmol/min/mg and 3.85 mM, respectively. This enzyme was almost not affected by metal chelators and various thiol-enzyme inhibitors (other than p-chloromercuribenzoate), and showed high tolerance for organic modifiers; for example, half of the activity remained in 50% ethylene glycol buffer. This enzyme deacylated not only polymyxins but also various N-fattyacyl compounds (peptides and amino acids). Among several fattyacyl groups (C₂-C₁₆) of N-acyl DL-methionines, the caprinoyl (C₁₀) group was most easily liberated, and the benzyloxycarbonyl (Z) group was also slightly susceptible. When the enzyme was solubilized in 0.2 M KCl-containing buffer by Triton X-100, the enzyme (Type II) showed a slightly different substrate specificity and an increased activity for some Z-derivatives. With this enzyme, it is possible to remove the Z group from several Z-peptides under mild conditions.

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The chemistry of several of the specific toxins isolated from a culture medium of the phytopathogenic fungus Helminthosporium victoriae, a causal agent of Victoria blight disease of oat, was studied. The structures of the amino acid components of the isolated HV-toxin M were identified, and the absolute configurations of the asymmetric α-carbons were elucidated as (S) (I - V, Fig. 1), (S) for C-3 of 3-OH-leucine (II), (R) for C-3 of 3-OH-lysine (V) and (Z)for 3-chlorodehydroalanine (VII).
The structure of the other toxin simultaneously isolated, HV-toxin H, was found to be identical with victorin C.

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Rice prolamin extracted with 60% (v/v) n-propanol from protein-rich rice bran was fractionated into individual polypeptides by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The prolamin polypeptides of 10, 13, and 16 kDa were acid hydrolized, and the amino acid composition of each polypcptide was analyzed. These prolamin polypeptides were rich in glutamic acid/glutamine and leucine but poor in lysine as reported previously for prolamins of many cereals. The 10 and 16 kDa polypeptides had a markedly high content of sulfur-containing amino acids.

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Five antioxidative phenolic acids were isolated from the leaves of oregano (Origanum vulgare L.). The structure of a new compound (5a) was determined to be 2-caffeoyloxy-3-[2-(4-hydroxybenzyl)-4, 5-dihydroxy]phenylpropionic acid on the basis of spectroscopic evidence. Especially, phenyl glucoside (1) and 5a showed an activity comparable to that of BHA.

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Dinophysistoxin-3 (DTX3, 3), the principal toxin of scallops implicated in diarrhetic shellfish poisoning, is a mixture of 7-0-acyl esters of 35-methylokadaic acid. To investigate the effects of unsaturation in the acyl moieties on biological activities, three demethyl analogs of DTX3, 7-O-palmitoyl, 7-O-linoleoyl, and 7-O-docosahexaenoyl okadaic acid, were prepared and tested for mouse lethality, cytotoxicity, and diarrheagenicity. The activities of okadaic acid were generally decreased by acylation of 7-OH. The decrease was most significant in the mouse lethality, moderate in cytotoxicity, and only slight in the fluid accumulating potency in mouse intestinal loops. Diarrheagenicity measured by suckling mouse assays was affected little by the acylation. Potency of analogs genellaly increased in parallel with the unsaturation in the acyls.

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