Myo-inositol phosphate intermediates formed during dephosphorylation of phytic acid (by rice bran acid phosphatase with phytase activity F2) were isolated by ion-exchange chromatography. Hydrolysis of phytic acid proceeded via L-myo-inositoI 1, 2, 3, 4, 5 and D-myo-inositol 1, 2, 4, 5, 6-pentakisphosphate, D-myo-inositol 1, 2, 5, 6 and L-myo-inositol 1, 2, 3, 4-tetrakisphosphate, D-myoinositol 1, 2, 6 and myo-inositol 1, 2, 3-trisphosphate, D-myo-inositol 1, 2-bisphosphate and finally myoinositol monophosphate (myo-inositol 2, 93%; D-myo-inositol 1, 7%). Six percent of myo-inositol and 85% of Pi were found in the reaction mixture when the hydrolysis of phytic acid by the enzyme did not proceed further. The initial points of dephosphorylation of phytic acid by the phytase took place at L-myo-inositol 6 (85%), D-myo-inositol 3 (7%), myo-inositol 2 (5%), and myo-inositol 5 (3%) at the positions of the myo-inositol ring.

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Abietic acid, a tricyclic diterpenoidal resin acid, inhibited both (Na⁺+K⁺)- and (H⁺+K⁺)-ATPases, typical membrane-bound enzymes, at a concentration as low as 25μg/ml. Abietic acid also inhibited gastric acid secretion caused by (H⁺ +K⁺)-ATPase. Non-specific inhibition by abietic acid against ATPases suggests that the acid, as well as other biologically active terpenoids, would primarily induce disorganization of the cell membrane constitution.

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Simple and sensitive spectrophotometric methods were developed for the selective determination of L-lysine. The assay methods were based on the spectrophotometric determination of NADH formed in the reaction catalyzed by L-lysine ε-dehydrogenase (Procedure A) or the formazan produced on coupling of the dehydrogenase reaction with the reduction of a tetrazolium salt (Procedure B). Procedure B was applicable to the direct determination of L-lysine in rabbit serum.

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Using bioreactors with yeasts, Zygosaccharomyces rouxii that undergoes ethanol fermentation and produces 2-phenyl ethanol, and Candida versatilis that produces 4-ethyl guaiacol, adsorbed-immobilized on a ceramic carrier, the total time required for production of soy sauce was shortened to 8 days without affecting the product's quality.

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Several endo-1, 4-β-glucanases [1, 4-β-D-glucan glucanohydrolase (EC 3.2.1.4)] from a fungus, Y-94, were purified to homogeneity by ammonium sulfate fractionation, column chromatography with two kinds of ion exchangers (Amberlite CG-50 and PBE 94), gel filtration with Toyopearl HM-55 and polyacrylamide gel electrophoresis, and their physicochemical and chemical properties were investigated. They differed in molecular weight (13, 000-174, 000), sugar content (around 16-20% and 0%), pI value (6.66-<3.8) and so on.

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The enzymatic properties of several purified endo-1, 4-β-glucanase components [1, 4-β-D-glucan glucanohydrolase (EC 3.2.1.4)] from a fungus, Y-94, were investigated. The pH and temperature optima of the enzymes were from 4.5-5.5 and 50-70°C and their stabilities for various pHs and temperatures were observed over a wide range of 3.0-8.5 and 50-80°C, respectively. All the endo-1, 4-β-glucanase components were strongly inactivated by Hg²⁺ and Mn²⁺ and several of them were activated by the addition of sodium dodecyl sulfate (SDS) when carboxymethyl cellulose (CMC) was used as a substrate. Most purified endo-1, 4-β-glucanase components could hydrolyze CMC, hydroxyethyl-cellulose (HEC), phosphoric acid swollen-cellulose (PSC), and xyloglucan. Their Michaelis constants (Km) for CMC [average degree of polymerization (CP)=1050] were from 0.06-0.28% and for the cellooligosaccharides cellotetraose and cellopentaose, from 1.20-6.75 mM and 0.48-1.09mM, respectively. The synergistic effect, generated by a combination of the endo-1, 4-βglucanase components, avicelases, and β-glucosidase, resulted in a 1.3-2.4-fold increase of Avicel-, CMC-, and cellulose powder-hydrolyzing activities, compared to the sum of the activities of the individual enzymes regarding these substrates. No synergism was observed within the endo-1, 4-βglucanase components. Increasing the molecular weight of cellooligosaccharide that was used as a substrate tended to increase the hydrolyzing activities for them of the enzyme components.

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A NADPH-dependent 2-oxoaldehyde reductase, which catalyzes the reduction of 3-deoxyglucosone to 3-deo¥¥ fructose and reduction of methylglyoxal to acetol, was isolated and purified from parsley leaves. 2-Oxoaldehyde compounds were found to be specially good substrates and monocarbonyl compounds were poor substrates for this reductase. The optimum pH of the enzyme activity was 7.0-7.5. The Km for 3-deoxyglucosone and methylglyoxal were 15 mM and 13 mM, respectively. The molecular weight of the enzyme was estimated to be 67, 000 and the enzyme is proposed to be a dimer composed of identical subunits (MW = 35, 000). The enzyme activity was completely inhibited by p-chloromercuribenzoate. The reaction catalyzed by the enzyme was virtually irreversible. The changes in methylglyoxal and NADPH by the enzyme were stoichiometrically equivalent.

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The relative molecular mass (M_r) of p-hydroxybenzoate hydroxylase from Pseudomonas desmolytica was measured by SDS-PAGE and gel filtration. The enzyme was a dimer of identical subunits with M_r of 44, 000 and with one mole of FAD per subunit. The isoelectric point was 4.9. The amino acid composition was also identified. The enzyme contained 6 sulfhydryl groups per FAD. Modification of the groups with p-chloromercuribenzoate accompanied inactivation which obeyed a biphasic kinetics. The faster reaction corresponded to 80% of the total inactivation. p-Hydroxybenzoate and chloride ion (an inhibitor with respect to NADPH) increased the rate of the faster inactivation reaction, but NADPH had a protective effect. p-Hydroxybenzoate markedly restricted the amount of sulfhydryl groups reactive to p-chloromercuribenzoate. The fast phase inactivation could be ascribed to modification of a single sulfhydryl group. The results are highly indicative of the existence of a sulfhydryl group in the NADPH-binding site.

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Nine distinct fragments of Clostridium stercorarium strain F-9 (which was isolated and identified in this laboratory) DNA have been cloned in Escherichia coli and shown to express enzymatic activities related to xylan hydrolysis. Two of the 9 E. coli clones showed xylanase activity, one showed β-xylosidase activity as well as xylanase activity, and the other 6 showed β-xylosidase activity. On comparing the restriction maps of the xylanase and xylosidase genes cloned in this study with those reported so far, it was concluded that all the 9 clones carried hitherto unidentified genes. All the enzymes expressed in the E. coli clones were thermophilic.

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A mutant, AS2-1, was isolated by UV irradiation from Clostridium saccharoperbutylacetonicum Nl-4 ATCC 13564 producing hyper butanol at the molar ratio of acetone-butanol-ethanol (3.3:9.5:1). The mutant proved to be deficient in the production of acetone, butanol, and acetic acid, but enhanced the ethanol (17.7 times increase) and newly produced isovaleric acid at a molar ratio of 1:1. The maximum production of ethanol and isovaleric acid reached 10.6 g/l (0.23 M) and 19.8 g/l (0.20 M) from glucose at 77.7 g/l (0.43 M) in TYA medium. As the equimolar ratio of ethanol and isovaleric acid was also produced in an amino acid-free minimal medium containing ammonium acetate as a nitrogen source, isovaleric acid was not produced from amino acids, but de novo synthesized from the ammonium and glucose.

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Mutant AS2-1, derived from Clostridium saccharoperbutylacetonicum N1-4 ATCC 13564, did not produce acetone, butanol, and acetic acid, but had enhanced ethanol production and newly produced isovaleric acid at the molar ratio of 1:1. The mutant was defective in thiolase (acetyl-CoA acetyltransferase) activity, while the parent N1-4 showed high thiolase activity (103.5 units/g protein). Addition of allyl alcohol into the mutant culture significantly decreased the production of both ethanol and isovaleric acid. AS2-1 mutant, therefore, had NAD⁺ - and NAD(P)⁺-dependent aldehyde dehydrogenase on the pathway to ethanol and isovaleric acid, respectively. It was suggested that an excess of electrons accumulated as the result of the deficiency in thiolase was consumed for the reductive formation of isovalerate from pyruvate. The equation of the novel ethanol-isovaleric acid fermentation was concluded as 1.5mol glucose = 1mol ethanol +1 mol isovaleric acid+ 2 mol CO₂ + 2 mol H₂O +1 mol H₂.

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Legs from freshly caught snow crabs (Chionoecetes opilio) were kept in ice for 28 days. The pH value decreased from around 7 at the start of storage to 6.6 on day 3, and then increased to 8 on day 20. The concentration of free proline, glycine and arginine increased until day 3, and then decreased. The arginine completely disappeared on day 28. In contrast, ornithine, urea and NH₃ continued to increase during storage in ice. The activities of some peptidases, especially of tripeptidase, were clearly detected in a muscle extract. The arginase [EC 3.5.3.1] activity of the muscle extract tended to increase in the alkaline pH range, accounting for the changes in the arginine, ornithine and urea concentrations. Based on these results, it is proposed to use the concentration of urea or ornithine as a parameter for the freshness of snow crab.

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The tryptophan residue located at the low affinity saccharide-binding site (LA-site) of ricin E was identified using two derivatives of ricin E, in which tryptophan residues were oxidized with N-bromosuccinimide (NBS) in the absence and presence of lactose. From the lysyl endopeptidase and tryptic digests of the B-chains of the respective derivatives, peptides containing oxidized tryptophan residue(s) were isolated by gel filtration on Bio Gel P-2 and HPLC. Analysis of the peptides containing oxidized tryptophan showed that tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative, in which the saccharide-binding ability at the LA-site was abrogated by modification of tryptophan residues with NBS in the absence of lactose. On the other hand, two tryptophan residues at positions 93 and 160 in the B-chain were found to be oxidized in the active derivative that retained the saccharide-binding ability at the LA-site after NBS-oxidation in the presence of lactose, indicating that the tryptophan residue at position 37 is protected from NBS-oxidation in the complex with lactose. The results suggest that the loss of the saccharide-binding ability of the LA-site of ricin E by NBS-oxidation is principally due to the oxidation of the tryptophan residue at position 37 on the B-chain.

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The effect was studied of such mutagens as 3-amino-1-dimethyl-5H-pyridol[4, 3-b]indole (Trp-P2), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF2), 5-hydroxymethyI-2-furfural (HMF) and pyrrole on the viability and activity of some enzymes of a human histiocytic lymphoma cell line, U-937, cultured in a serum-free medium. Incubation of U-937 with the mutagens for 12 hr at 37°C in the serum-free medium reduced the viability of the U-937 cells. Especially, Trp-P-2 and MNNG reduced it markedly. Trp-P-2, MNNG, AF-2 and HMF were also found to decrease the formation of formazan in the U-937 cells. AH the mutagens slightly increased the activity of a drug-metabolizing enzyme, NADPH-cytochrome c reductase, in the cells. They also caused a variation of such enzyme activities in the cells as glutamic oxaloacetic transaminase (GOT), catalase and alkaline phosphatase. Leakage of the activities of NADPH-cytochrome c reductase and GOT from the U-937 cells into the culture medium was also observed, especially when the cells were treated with high concentration of Trp-P-2, MNNG and AF-2. No marked effect of Trp-P-2 and MNNG on the activity of β-gaIactosidase was observed.

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The acid ureases from 37 strains belonging to the genera Lactobacillus and Streptococcus were partially purified and characterized. The 37 acid ureases were classified into 6 different types according to their electrophoretic mobilities. The 6 different types of acid ureases, from 6 representative strains (4 species of Lactobacillus and 2 species of Streptococcus), were further characterized. The molecular sizes of the enzymes ranged from 140, 000 for Streptococcus salivarius to 350, 000 for Lactobacillus animalis. The isoelectric points were found to be 4.6 (Streptococcus mitior), 4.7 (Lactobacillus reuteri, Lactobacillus ruminis and Streptococcus salivarius) and 4.8 (Lactobacillus animalis and Lactobacillus fermentum). The optimum pHs ranged from 2.0 for Lactobacillus fermentum and Lactobacillus reuteri to 5.0 for Lactobacillus ruminis. The optimum temperatures ranged from 55°C for Lactobacillus animalis and Lactobacillus ruminis to 65°C for Lactobacillus fermentum, Lactobacillus reuteri and Streptococcus mitior. All 6 different acid ureases eliminated urea in alcoholic beverages that are generally acidic.

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A viscous fish-flesh sol, shio-surimi, prepared from frozen Alaska pollack surimi, water, sodium chloride and starch was cooked at 80°C for 30 min. The cooked product remained viscous if the starch had been pregelatinized. The most viscous type of paste with a consistency of mayonnaise or tomato ketchup was obtained from the shio-surimi containing 4.5-9% of pregelatinized tapioca starch or potato starch and 150 or 300% of water. The viscosity of the paste was increased when salad oil or butter was further added to the shio-surimi. Furthermore, a type of paste with the consistency of French dressing was obtained by grinding the resulting paste with rice vinegar and salad oil or butter. Cooked fish pastes prepared in this way may be utilized as a new form of marine food.

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The influence of the fluidity of water within food hydrogels on their visco-elasticity was investigated by using three hydrogel models of a closed system from which water did not flow out upon their compression. In all the cases, the instantaneous elastic modulus of the models was somewhat increased by the suppression of the fluidity of water, although the viscosity was not always increased. From the results, it is assumed that, upon the deformation of a food hydrogel, water flowed within it, and that its visco-elasticity was also influenced by the fluidity of the water.

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The protein quality of hybridized high-grain yielding varieties, between japonica and indie a type rices, Chugoku 91, Nanjing 11 and Milyang 30, was examined. The contents of protein and amino acids of Nanjing 11, especially lysine, were highest among these three varieties. The food intake, weight gain and feed efficiency of rats given a Nanjing 11 diet also showed the highest values among these three varieties. The supplementation of lysine and threonine to a Milyang 30 diet brought about increases of 11, 44 and 30% in the food intake, weight gain and feed efficiency, together with 20 and 25% increases in the biological value and net protein utilization, as compared with those in rats that received the Milyang 30 alone as the protein source. These results suggested that it is possible to select a variety for concomitant increases in grain yield and protein with good quality, and showed that the supplementation of lysine and threonine to this variety can bring about economically significant improvement of the biological quality.

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A twin-screw extruder was regarded as a pump and its pumping ability was assessed by measuring the throughput of the twin-screw extruder. Materials with high viscosity were extruded under conditions of different screw pitch and screw speed. Characteristic curves were obtained to evaluate quantitatively the contribution of drag and pressure to the throughput. Both pressure flow and drag flow were larger with longer-pitch screws, which indicated that the throughput with shorter-pitch screws was greatest under high back pressure. The characteristic curves give the minimum required screw speed for a given viscosity of material at the inlet, or the minimum required viscosity of a material at the inlet for a given screw speed.

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Erythrina variegata lectin was purified by affinity chromatography with lactose-Sepharose 6B. The purified lectin was homogenous on poly crylamide gel electrophoresis, immunoelectrophoresis, and Sephadex G-150 gel filtration. The molecular weight of the lectin was 60, 000 by the gel filtration, and SDS-polyacrylamide gel electrophoresis gave a single band at a position corresponding to the molecular weight of 29, 000, indicating that the lectin is a dimer composed of two identical subunits. The lectin was a glycoprotein with its neutral sugar content being 9%. It contained high amounts of acidic and hydroxy amino acids, a low amount of methionine, and no cysteine. This lectin agglutinated human erythrocytes of O type at a concentration of 4 μg/ml, which was inhibited most potently by p-nitrophenyl-β-D-galactopyranoside among the sugars tested, suggesting the importance of the hydrophobic contribution of the aromatic region in the sugar binding-activity. Galactose and its related sugars were also inhibitory but to lesser extents.

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By digestion with an enzyme preparation derived from a culture filtrate of Cellulomonas sp., an unsaturated disaccharide was produced accompanied by the release of mannose and β1 → 2mannobiose from the acidic oligomer mixture that was obtained from the acidic polysaccharide of Fusarium sp. M7-1 by acetolysis. The unsaturated disaccharide produced was isolated and identified as 4-deoxy-L-threo-hex-4-enopyranouronosyl α(1 → 2)-D-galactose. The same unsaturated sugar linked to the polysaccharide was also produced accompanied by the release of mannose and β → 2mannobiose from the acidic polysaccharide by the enzyme digestion.

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A degenerated strain of Clostridium saccharoperbutylacetonicum N1-4 ATCC13564 was isolated after 30 subcultures and was designated as DGN32. The product yields of DGN32 drastically decreased to below one-third of those of N1-4, but its acetone productivity remained the same as that of N1-4 when the culture pH was at 5.1. DGN32 with or without pH control evolved almost equimolar amounts of CO₂ and H₂ throughout the fermentation. The amount of NADH consumed for the acid and solvent production of DGN32 corresponded to that of NADH generated in the EMP pathway. These results suggested that the electron flow coupled with pyruvate metabolism to acetyl-CoA in DGN32 would be kept for H₂ evolution, but not for NADH generation. However DGN32 cultivated with a redox dye (benzyl viologen) preferentially produced butanol though the butanol production rate was comparatively low. In DGN32 there was suppression of the NADH generation system coupled with pyruvate metabolism to acetyl-CoA, which could be essential for the metabolic transition from the acidogenic phase to the solventogenic phase.

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A high copy number plasmid pCS86 (3.0kb) was isolated from Clostridium acetobutylicum strain No. 86. For developing the vector plasmid for gene cloning of saccharolytic clostridia, two hybrid plasmids were constructed by joining pCS86 to the E. coli promoter probe vector pKK232-8 carrying the promoterless gene of chloramphenicol acetyltransferase (CAT). One of the hybrid plasmids designated as pTY10 replicated and expressed the CAT gene in E. coli, and transformed a plasmid-free strain, C. acetobutylicum strain No. 220, as a shuttle vector. This is the first shuttle vector constructed from C. acetobutylicum and E. coli plasmids. Reciprocal transformation was possible between E. coli and C. acetobutylicum, though deletion occurred in the sequence originated from pKK232-8. A deleted plasmid pTY101 only replicated in Clostridium, though it showed a high copy number.

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We evaluated electroporation as an alternative system for genetic exchange for one of the coryneform bacteria, Brevibacterium flavum MJ233.
The maximum number of transformants, 6 × 10⁴ cells, was obtained when cells were cultured with Penicillin G (1 U/ml) and harvested at the middle-log phase. Electroporation was done using 12.5kV/cm of pulse field strength, 1 × 10¹⁰ cells, and 1 μg of plasmid DNA.
Other coryneform bacteria, Brevibacterium lactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 31830, and B. stationis IFO 12144 were also transformed by electroporation.
Electroporation has the advantage that intact cells can be used as host cells without the need for protoplast formation and regeneration. Moreover, minimal medium can be used, so auxotrophic complementation of the transformants is possible.

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Rice-straw treated with 5% ammonia water was hydrolyzed with the β-xylanase of Streptomyces sp. E-86, and four kinds of hetero-oligosaccharides, two arabinoxylo- and two glucuronoxylo-oligosaccharides, were isolated from the hydrolysate by charcoal column, gel filtration, ion-exchange, and aminopropyl silica column chromatographies. The structures of the oligosaccharides were identified as 3²-α-L-arabinofuranosyl-xylobiose, 3²-α-L-arabinofuranosylxylotriose, 2³-4-O-methyl-α-D-glucuronosylxylotriose, and 2³-α-D-glucuronosylxylotriose by the analysis of component sugar, partial acid hydrolysis, methylation analysis etc. Based on the structures of above oligosaccharides, the structure of rice-straw arabinoglucuronoxylan and the specificity of the xylanase toward the xylan are discussed.

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N, N'-Dibromo-5, 5-dimethylhydantoin and N-bromosuccinimide were found to be versatile and commercially available synthetic reagents for generating singlet molecular oxygen in organic solvents under mild conditions. Detection was by an ultra-sensitive near-IR chemiluminescence spectrometer designed and constructed in our laboratory.

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To study the relationship between citrate synthase and 2-phosphinomethylmalic acid (PMM) synthase, which catalyzes a very similar reaction comparable to citrate formation in the biosynthesis of a herbicide, bialaphos, citrate synthase was purified from the mycelium of Streptomyces hygroscopicus SF-1293, a bialaphos-producing organism. The overall purification was 440-fold with a yield of 4.4% from cell-free extract. Based on comparison with PMM synthase, it has been concluded that citrate synthase of S. hygroscopicus is quite different from PMM synthase in several aspects such as enzymatic properties, amino acid composition, N-terminal amino acid sequence, and stereo-chemical reaction mechanism.

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Rice leaf POX (EC 1.11.1.7) and LOX (EC 1.13.11.12) activities were induced by press-injured application of ETP, an ethylene generating chemical, ACC and Glu. A higher concentration of Glu was required to induce rice leaf POX when compared with the induction of LOX and the previously reported case of PAL. Both the POX and LOX activities were induced by press-injured inoculation with incompatible or compatible blast fungus conidia, or by press-injured application of its RIF. The induction of POX by press-injured inoculation of incompatible conidia, compatible conidia or RIF was inhibited by a preapplication of TR, AOA, CY or ABA, while the induction of LOX was similarly inhibited by the former three, but not at all, or only weakly inhibited by ABA. The activity itself of induced POX or LOX was not inhibited by the inhibitors used in this experiment. The enzyme induction spectra either by the incompatible or compatible combination in terms of tm (the time to the maximum rate of induction after inoculation) showed good orderliness (O^-₂ FES<PAL< LOX<POX) with the relaxation in compatible combination. A higher outbreak of rice blast was observed when rice seedlings were pretreated with TR, AOA or ABA 4 hr before the spray inoculation.

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The active principle of EP3, a fraction from an extract of the culture medium of Lentinus edodes mycelia (LEM), which activates murine macrophages, causes proliferation of bone marrow cells, and inhibit the replication of Human Immunodeficiency Virus in vitro, was characterized as a water-solubilized lignin. The detailed structural feature of this water-solubilized lignin was investigated and shown to be a highly condensed and polycarboxylated lignin which is denatured and solubilized by Lentinus edodes from bagasse. The water-solubilized lignin itself was confirmed to have both immunological activities and the antiviral activity.

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Enol esters of 2-substituted-3-oxoalkanoates of (Z)-configuration were reduced by bakers' yeast to chiral 2-substituted-3-hydroxyalkanoates. The syn-selectivities of this reaction increased compared with those of the reduction of the corresponding racemic keto esters. The reaction may proceed via an asymmetric hydrolysis of the enol esters, followed by the reduction of the resulting carbonyl group.

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The ligand structure of chelated copper in recombinant human Cu₂Zn₂SOD (r-hSOD) and its metal-substituted SODs was examined. In r-hSOD the configuration of the copper binding site was axially symmetrical and that of metal-substituted SODs had a tendency to change towards a tetrahedral type compared with r-hSOD except for Cu₂(VO)₂SOD. Cu₂(VO)₂SOD was not tetrahedral and in this case the ligand atom of copper was inclined to convert from nitrogen towards oxygen. In Cu₂E₂SOD, Ag₂Cu₂SOD, and Cu₂(VO)₂SOD the specific activities were lower but in Cu₂Cu₂SOD and Cu₂Co₂SOD they were nearly identical with that of r-hSOD. These results suggested the ligand structure of chelated copper was not related to the specific activity of SOD. However, these SODs were less stable than r-hSOD against heat treatment and denaturing reagent. Further they were less stable against attack by an inactivator (hydrogen peroxide) except for Cu₂(VO)₂SOD. In this case the decreased stability of these SODs was associated with the change of the ligand structure of copper from that of r-hSOD. These results suggested that the presence of zinc contributed highly to the stable formation of the ligand structure of copper and the enzymic stability.

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A highly sensitive fluorometric method for determining sulfite in wine is reported. N-(9-Aciridinyl)maleimide (NAM) reacted with the sulfite in wine quantitatively and gave strong fluorescent derivatives. The determinations were conducted by batch and high-performance liquid chromatographic methods. Sulfite contents in wine by NAM methods and the conventional methods showed good accordance. The sample size could be reduced to less than 1/10, 000 by the NAM methods.

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A strain of the yeast Saccharomyces cerevisiae coding for KHR on the chromosome secreted a toxin that kills sensitive yeasts. The transformants of multicopy vectors carrying the KHR gene could secrete 3-4-fold the killer toxin of the donor strain. This toxic substance was purified 80-fold in specific activity from the culture filtrate by gel filtration and hydrophobic column chromatography. The purified toxin gave a single protein band with molecular mass of 20 kDa on SDS-PAGE and had an isoelectric point of pH 5.3. The toxin had novel killer activity against Candida glabrata and S. cerevisiae, but did not affect bacteria, fungi, or other yeasts.

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We have reported that a 37-kDa protein from a soybean isolate shows an affinity for bile acids and modulates insulin action on fat decomposition in vitro [Makino et al., Agric. Biol. Chem., 52, 803 (1988)]. In this study, the major components of the protein were identified as the acidic A_1a and A₂ subunits of glycinin, via amino-terminal sequence analyses of purified proteins and examination of their effects on fat decomposition in rat adipose cells. The most hydrophobic region of the subunits was found to be responsible for the bile acid-binding ability, and the binding region probably does not contribute to the insulin-modulating activity. These bile acid-binding and insulin-modulating properties were also noted in a 40-kDa protein from pea seeds, probably acidic subunits of legumin, suggesting that these characteristics may be common to legume proteins.

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³²P-End-labeled restriction fragments derived from pBR322 and pUC9 DN As were reacted with D-glucosamine or D-glucosamine-6-phosphate in the presence of Cu²⁺, and, after being heated at 90°C in aqueous piperidine, the DNA products were analyzed on poly aery lamide gels for the sequence-specificity of alkali-labile cleavaged sites. The intensity of oligonucleotide bands of cleavaged sites was directly proportional to the concentration of aminosugars, indicating that the DNA cleavage was caused by the action of aminosugars themselves. The preferred DNA cleavage sites induced by these aminosugars were identical, both at pyrimidine-purine (5'→3') sequences, especially at thymineguanine ones, and to some extent at pyrimidine-pyrimidine (5' → 3') sequences. The 6-phosphate moiety of D-glucosamine did not affect the specificity of DNA cleavage.

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1'-Epi-stegobinone [(2S, 3R, 1'S)-2, 3-dihydro-2, 3, 5-trimethyl-6-(1'-methyl-2/-oxobutyl)-4H-pyran-4-one], an inhibitor of stegobinone, which is the sex pheromone of drugstore beetle (Stegobium paniceum L.), was synthesized by stereocontrol at C-2 and C-1' starting from ethyl (R)-3-hydroxybutanoate and methyl (R)-3-hydroxypentanoate.

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