Bacillus subtilis 1A20 transformed with a hybrid plasmid, pNP150, to which a DNA fragment from Bacillus amyloliquefaciens F was attached, produced a large amount of a neutral protease. To identify the origin of the gene specifying this neutral protease, neutral proteases from B. amyloliquefaciens F, B. subtilis NP58 (a derivative of Marburg 6160), and B. subtilis 1A20 transformed with pNP150 were purified. We investigated their immunological properties and primary structures.
The proteases from these two species were indistinguishable by chromatography, but they were distinguishable from each other by SDS-polyacrylamide gel electrophoresis and double immunodiffusion. Amino acid sequencing of these two proteases by Edman degradation showed that there were four substitutions in the 20-residue amino acid sequence from the N-termini.
Neutral protease from the transformant had the same immunological characteristics and N-terminal amino acid sequence as that from B. amyloliquefaciens. These results meant that the gene in question was derived from a gene specifying the neutral protease in this bacterium.

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By ion exchange chromatography on DEAE-cellulose in the presence of 4 M urea, bovine κ-casein A was fractionated into 9 subcomponents, all of which were identified as κ-casein from immunological analyses. The microheterogeneity of the subcomponents was explained by stepwise increase of their carbohydrate contents (0-4mol/mol of GalNAc, and 0-8mol/mol of NANA). The micelle-stabilizing ability of κ-casein subcomponents increased with the increase of their carbohydrate contents: the carbohydrate rich subcomponent 7 possessed twice the stabilizing ability of the carbohydrate free subcomponent 1. The sensitivity of synthetic casein micelle composed of κ-casein subcomponents and α_S1-casein to the wheat germ lectin-induced aggregation also increased with the increase of their NANA contents.

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The leaves of Ocimum gratissimum (Clocimum) exhibited strong volatile fungitoxicity against betelvine (Piper betle L.) pathogens - Alternaria alternata, Colletotrichum capsici and Sclerotium rolfsii. Fifteen compounds could be identified from the fungitoxic constituents- the essential oil. The oil at its minimum inhibitory concentrations of 50, 250 and 500ppm against S. rolfsii, A. alternata and C. capsici, respectively, was fungistatic, although, fungicidal at higher concentrations. Eugenol was found to be the major fungitoxic principle in the oil. The oil was either equally effective or superior to synthetic commercial fungicides and was non-phytotoxic to the host plants. Thus, the oil can be used as a valuable indigenous and biodegradable agent against fungi that cause losses to the betelvine industry.

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Guinea pig liver transglutaminase is a Ca²⁺ dependent enzyme which catalyzes the formation of inter- and intramolecular ε-(γ-glutamyl)lysyl cross-links between protein molecules. We have found that solutions of several proteins (α_s1-casein, and soybean 11S and 7S globulins) were gelatinized firmly by transglutaminase. The gel formation depended on the protein concentration. In the case of α_s1-casein, a reaction mixture containing below 2% was incapable of gelation. However, above 3%, a firm gel was formed by transglutaminase. As to soybean 11S and 7S globulins, reaction mixtures containing below 5% did not form gels, while, above 8%, firm gels were formed. The protein solutions in the presence of EDTA, an inhibitor of transglutaminase, were not gelatinized on treatment with transglutaminase. Thus, transglutaminase and a higher concentration of a substrate protein are indispensable for firm gel formation. It is supposed that the protein gels are formed through covalent bonds with transglutaminase.

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Toxins in ovaries and livers of the puffer fish, Fugu flavidus, were purified successively on activated charcoal, Bio-Gel P-2 and Bio-Rex 70. Besides tetrodotoxin (TTX), a minor toxin (toxin D) was newly detected in both tissues; toxin D accounted for 3% and 4% of the toll lethal potency in ovaries and livers, respectively. Although there were differences in the specific lethal activity, dose-survival time relation and signs in mice between toxin D and TTX, positive reactions to Weber reagent and 10% KOH, and a negative one to 1% H₂O₂ were observed for both toxins. Moreover, toxin D, like TTX, gave rise to the C₉-base upon alkali-degradation. These results suggested that toxin D is a TTX-like compound having a quinazoline skeleton.

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To investigate the function of proteinases in the fruiting of Basidiomycetes, we purified the neutral proteinase in vegetative mycelium of Lentinus edodes. About 1.6 mg of purified enzyme was obtained from 1.5 kg of mycelium. The purified enzyme was confirmed to be monodispersi ve on disc electrophoresis.
The neutral proteinase was most active around pH 7.5 toward hemoglobin and 7.0 toward casein and was extremely labile with temperature. The enzyme was strongly inhibited by EDTA or Talopeptin (MK-I). The molecular weight and isoelectric point of the enzyme were 45, 000 and pH 5.3, respectively. The enzyme contained no methionine residues. The enzyme hydrolyzed the bonds involving hydrophobic or bulky amino acid residues of oxidized insulin B-chain such as His-Leu (10-11 and 5-6), Leu (17)-Val (18) and Ala (14)-Leu (15).
These characteristics are compared with those of the metal proteinase in the fruit-body of the same fungus, which was purified and characterized at the same time as in vegetative mycelium. We also compare it with proteinases from other microbes.

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The disaccharide, 2, 3-di-O-methyl-4-O-(3, 6-di-O-methyl-β-D-glucopyranosyl)-L-rhamnopyranose, the distal segment of phenolic glycolipid I, that is a specific antigen from Mycobacterium leprae, and some related disaccharides were synthesised as the glycosides of methyl 3-(p-hydroxyphenyl)propionate. The methyl 3-(p-hydroxyphenyl)propionate was coupled with 2, 3, 4-tri-O-acetyl-L-rhamnosyl bromide, deacetylated, acetonated, coupled with 2, 4, 6-tri-O-acetyl-3-O-methyl-D-glucosyl bromide, and converted into a variety of p-(2-methoxycarbonylethyl)phenyl 4-O-(3, 6-di-O-methyl-D-glucopyranosyl)-containing disaccharides that are amenable to ready conjugation with protein carriers, thereby providing neo-glycoconjugates for the specific serodiagnosis of leprosy.

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The oxidation of methionine to its sulfoxide, as a possible cause of decrease in the biological value of red clover during drying with aeration, was examined using various model systems, in the presence or absence of polyphenol oxidase. The effects of catalase were also examined. Results indicated hydrogen peroxide as a possible intermediate that directly oxidizes methionine. The methionine oxidation can be one of the causes of the decrease in biological value of red clover during drying, beside the known damage of lysine in the same process.

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Several compounds having the basic α-ionylideneacetic acid structure were tested in Cercospora rosicola resuspensions. At 100μM, all the compounds inhibited abscisic acid (ABA) biosynthesis. Time studies with unlabelled and deuterated (2Z, 4E)- and (2E, 4E)-α-ionylideneacetic acids showed rapid conversions into both (2Z, 4E)- and (2E, 4E)-4'-keto-α-ionylideneacetic acids as major products. Incorporation of the label into ABA was specific for the 2Z, 4E-isomer. Minor products, identified by GC-MS, were (2Z, 4E)- and (2E, 4E)-4'-hydroxy-α-ionylideneacetic acids and (2Z, 4E)-1'-hydroxy-α-ionylideneacetic acid. The conversion to (2Z, 4E)-1'-hydroxy-α-xionylideneacetic acid has not been previously reported and was specific for the 2Z, 4E-isomer. A time study for the conversion of methyl esters of [²H₃]-(2Z, 4E)- and [²H₃]-(2E, 4E)-4'-keto-α-ionylideneacetates showed a slow introduction of the 1'-hydroxyl group and specificity for 2Z, 4E-isomer. Conversion of the ethyl esters of (2Z, 4E)- and (ZE, 4E)-1'-hydroxy-α-ionylideneacetates into the ethyl esters of both ABA and (2E, 4E)-ABA demonstrated that ABA can be formed by oxidation of the 4'-position after the insertion of the 1'-hydroxy group. The ethyl 1'-hydroxy acids were also isomerized to the corresponding ethyl (2Z, 4E)- and ethyl (2E, 4E)-3'-hydroxy-β-ionylideneacetates. Ethyl (2Z, 4E)-1'-hydroxy acid also gave small amounts of ethyl 1', 4'-trans-diol of ABA. These results suggest that ABA may be formed through a (2Z, 4E)-1'-hydroxy-α-ionylidene-type intermediate in addition to the previously proposed route through (2Z, 4E)-4'-ketoa-α-ionylideneacetic acid.

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A non-phenolic β-O-4 lignin substructure model, 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (I), was metabolized by a ligninolytic culture of Coriolus versicolor. Based on the identification of the metabolic products (II∼XI), the following reactions were found to occur in the culture; a) oxidation (III) and reduction (II) at the benzyl (Cα') position of the substrate (I), b) β-ether cleavage to give arylglycerols (IV, V), and c) Cα-Cβ cleavage of the arylglycerols and/or arylglycerol moiety of the substrate (I). In addition, β-deoxy diol (VI) and γ-formylglycerol (VII) were obtained as degradation products from substrate (I).

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Microorganisms which hydrogenate 2-nitro-1-phenyl-1-propene were screened in type cultures and soil samples. Some actinomycetes belonging to Rhodococcus, Nocardia and Mycobacterium asymmetrically reduced the substrate and gave optically active 2-nitro-1-phenylpropane. Among them, Rhodococcus rhodochrous IFO 3338 gave the best results. The saturated compound was obtained quantitatively, when cultivation was carried out for 3 days at 30°C with a substrate concentration of 0.4%. The optical purity of the product was seriously affected by the pH of the medium. The more acidic the medium, the higher the enantiomeric excess. The results suggested that non-enzymatic racemization of the product takes place even under neutral conditions. Other substrates, such as 2-nitro-1-propene were also converted to optically active 2-nitro-1-substituted propane.

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Equimolar aqueous solutions of D-glucose and glycine were heated at 50°C and 95°C at pH 6.7. The headspace volatiles and the ether extracts from the reaction mixture were analyzed by gas chromatography and gas chromatography-mass spectrometry, using a fused silica capillary column. The major components formed were identified as diacetyl, furfuryl alcohol, two pyrroles, one pyranone and two amides. In order to elucidate the formation mechanisms of the amides formed from amino-carbonyl reactions, two model systems were adopted. N-Butylacetamide and N-butylformamide were formed as major components from diacetyl-butylamine and glyoxalbutylamine systems, respectively. The results obtained suggest that such α-dicarbonyls as 3-deoxyosone, 1-deoxy-D-erythro-2, 3-hexodiulose and diacetyl generated in the amino-carbonyl reaction react with amino compounds, amides then being formed by cleavage of the C-C bond in the α-dicarbonyls.

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The structure of an acidic polysaccharide elaborated by Bacillus polymyxa S-4 was investigated in relation to its physiological activity, particularly, its hypocholesterolemic effect on experimental animals. The polysaccharide is composed of D-glucose, D-mannose, D-galactose, D-glucuronic acid, and D-mannuronic acid (molar ratio 3:3:1:2:1). Methylation and fragmentation analyses, such as Smith degradation and partial acid hydrolysis showed that the polysaccharide has a complicated, highly branched structure, consisting mainly of (1→3)- and (1→4)-D-glycosidic linkages. The backbone chain containing D-glucuronic acid, D-mannose, and D-galactose residues is attached at the C-3, C-4, and C-4 positions, respectively, with side chains of single or a few carbohydrate units, which are terminated with D-glucose or D-mannose residues.

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To investigate the substrate specificity of α-L-rhamnosidase from Aspergillus niger, the following seven substrates were synthesized: methyl 3-O-α-L-rhamnopyranosyl-α-D-mannopyranoside (1), methyl 3-O-α-L-rhamnopyranosyl-α-D-xylopyranoside (2), methyl 3-O-α-L-rhamnopyranosyl-α-L-rhamnopyranoside (3), methyl 4-O-α-L-rhamnopyranosyl-α-D-galactopyranoside (4), methyl 4-O-α-L-rhamnopyranosyl-α-D-mannopyranoside (5), methyl 4-O-α-L-rhamnopyranosyl-α-D-xylopyranoside (6), and 6-O-β-L-rhamnopyranosyl-D-mannopyranose (7).
Compounds 1∼6 were well-hydrolyzed by the crude enzyme, but 7 was unaffected.

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The sulfite reductase of Desulfovibrio vulgaris, strain Miyazaki F (MF), was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Ultrogel AcA34, and hydroxylapatite. The molecular weight was estimated to be 180, 000 by gel filtration. It had a subunit structure of α₂β₂; the molecular weight of the α subunit was 50, 000 and that of β, 39, 000. The absorption spectrum with characteristic peaks at 629 and 409 nm and the amino acid composition resembled those of the sulfite reductase from D. vulgaris, Miyazaki K. The MF enzyme reduced sulfite to trithionate, thiosulfate, and sulfide by hydrogen when coupled with a hy-drogenase-methyl viologen system, like other sulfite reductases from Desulfovibrio.

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The separation of oil from wheat germ by extracting with supercritical carbon dioxide (CO₂) is described. The solubility of wheat germ oil in supercritical CO₂ at 200 atm and 40°C was about 0.35 weight%. The effect of pressure on the extraction process with liquid or supercritical CO₂ was of great significance. On the other hand, the effect of temperature on the extraction process was small. Oil extracted with supercritical CO₂ was lighter in color and contained less phosphorus than that extracted with hexane. The contents of α- and β-tocopherol in the oil extracted with supercritical CO₂ were comparable to those in the hexane-extracted oil

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(S)-2-Hydroxy-β-ionone of 96% e.e. was synthesized from (S)-3-hydroxy-2, 2-dimethyl-cyclohexanone, which was easily obtained by the baker's yeast reduction of 2, 2-dimethylcyclohexane-1, 3-dione.

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Maltopentaose-producing microorganisms were screened from a wide variety of sources. A bacterium which produced maltopentaose as the main product from a culture medium containing soluble starch was isolated from soil and identified as Bacillus cereus from its morphological, physiological, and biochemical properties. The culture conditions for the production of maltopentaose were investigated. The optimum medium consisted of 2.5 to 8% soluble starch, 1.5% peptone, and 0.25% NaCI. Under the optimum conditions of cultivation, the ratio of maltopentaose produced was in the range of 29 to 47% to the residual sugar.

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An obligate methanol-utilizing bacterium, Methylomonas sp. YK 1, was isolated and used as a cytochrome c producer. The strain was mutagenized so as to be resistant to metabolic inhibitors related to the function of cytochrome c. The strain, YK 56, which was derived as a KCN-resistant mutant contained 3 times the cellular level of cytochrome c compared to the parent strain. Optimization of the culture conditions for the mutant to enhance the cytochrome c productivity was performed. Peptone, succinate, L-malate or FeSO₄ • 7H₂O increased the productivity when added to the culture medium. Under the optimal culture conditions, strain YK 56 produced about 60 mg cytochrome c per liter when methanol and peptone were fed to the medium during the cultivation.

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A pepstatin-insensitive carboxyl proteinase of Polyporus tulipiferae (formerly Irpex lacteus) was purified by methods including affinity chromatography with chymostatin as the ligand.
Although the enzyme's maximum proteolytic activity on hemoglobin was at pH 2.8, it was not. affected by carboxyl proteinase inhibitors such as DAN, EPNP, and pepstatin. On the other hand, the enzyme was inhibited by chymostatin competitively and its Ki value was estimated to be 1.6 × 10^-5 M. The enzyme was very heat labile and was inactivated completely at pH 4.6 by heating at 45°C for 15min. Polyporus enzyme and Scytalidium lignicolum enzyme A_l hydrolyzed the same peptide bond of Z-tetrapeptides, but their primary specificities were slightly different. Polyporus enzyme as well as Ganoderma lucidum enzyme contains histidine, and the amino acid compositions of Polyporus enzyme and other pepstatin-insensitive carboxyl proteinases resembled each other.

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Microsomal cytochrome P-450 from tulip bulbs (Tulipa gesneriana L., Balalaika) was purified to an almost electrophoretically homogeneous preparation. The specific content of cytochrome P-450 in the final preparation was 6.68nmol/mg protein, which was 30-fold enriched from that of the solubilized fractions of microsomes. The molecular weight of purified cytochrome P-450 by SDS-gel electrophoresis is 52, 500. The Oxidized form of the purified cytochrome P-450 had absorption peaks at 392, 552, and 645 nm and the absolute reduced CO spectrum peaked at 448 nm. Judged spectrally, the purified cytochrome P-450 is in high spin in the oxidized state. Antiserum against this cytochrome P-450 previously has shown to be highly specific for its antigen but showed a single precipitin line with solubilized microsomal proteins from tulip bulbs of several other cultivars. The physiological role of this cytochrome P-450, however, is unknown in these dormant tulip bulbs.

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A shuttle vector for Gluconobacter suboxydans and Escherichia coli was constructed by ligation of a cryptic plasmid, pMV201, found in G. suboxydans IFO 3130 to E. coli plasmid pACYC177. The chimeric plasmid named pMG101 carries the ampicillin resistance gene derived from pACYC177 and transforms G. suboxydans var. α IFO 3254 as well as E. coli. The transformation conditions for G. suboxydans var. α IFO 3254 were examined using pMG101 DNA. Competent cells were induced efficiently by treatment with LiCl or RbCl. CaCl₂ which induced the competency of Acetobacter was much less effective. Addition of polyethylene glycol enhanced the transformation efficiency significantly. An efficiency of approximately 10² transformants per μg DNA was finally obtained.

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Degradation of phenylmercuric acetate (PMA) and p-chloromercuribenzoic acid (PCMB) by cells of a mercury resistant strain of Bacillus cereus was demonstrated. Degradation of PMA was also demonstrated with the crude extract of the B. cereus cells grown in nutrient broth containing 1 μM PMA or 10 μM HgCl₂. The crude extract seems to split the carbon-mercury bond of PMA and form benzene and the mercuric ion as degradation products. The splitting enzyme was separated from mercuric reductase which catalyzes the reduction of the mercuric ion by gel filtration on Sephadex G 100.

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A synthesis of nitrogen analogues of the natural insect anti-juvenile hormones precocene I and II is reported. The crucial step of this synthesis is thermal cyclization of the N-alkynylaniline 3 into the corresponding 1, 2-dihydroquinoline when promoted by a copper catalyst. Biological testing of the anti-juvenile hormone activity was carried out against Callosobruchus chinensis by observing the inhibition of eclosion. The 7-methoxy analogue showed higher activity than the 6, 7-dimethoxy analogue. However, the former showed lower activity than precocene I.

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The mechanism of purine arabinoside synthesis from uracil arabinoside and purine bases via the bacterial transarabinosylation reaction was investigated. Arabinose-1-phosphate was isolated from the reaction mixture in the form of the barium salt and proved to be the intermediate of the reaction. Two enzyme fractions were obtained from Enterobacter aerogenes by means of heat treatment, ammonium sulfate fractionation and DEAE-cellulose column chromatography. One enzyme split uracil arabinoside into uracil and arabinose-1-phosphate in the presence of inorganic phosphate and the other synthesized hypoxanthine arabinoside from arabinose-1-phosphate and hypoxanthine. The substrate specificity of these enzymes indicated that the former was uridine phosphorylase and the latter was purine nucleoside phosphorylase, respectively. Hypoxanthine arabinoside was synthesized from uracil arabinoside and hypoxanthine only in the presence of both enzymes and inorganic phosphate.

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The effects of dietary casein and adrenal hormone on the dietary induction of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSDase) activity in rat liver and kidneys were examined. Of three levels of casein in the diet examined (7.5%, 15% and 30%), only the feeding of a 30% casein diet to normal rats after three days on a protein-free diet resulted in a significant increase in the activity of ACMSDase in liver and kidneys on the following morning. The degree of the increase in this activity was higher in the liver than the kidneys. Dietary induction of ACMSDase activity disappeared when rats were adrenalectomized six days prior to the start of the experiment. In sham-operated rats, dietary induction of liver ACMSDase activity was as high as that in normal rats, however, that in kidneys disappeared. Prednisolone (synthetic adrenal hormone with glucocorticoid activity) injection of adrenalectomized rats led to the recovery of the dietary induction of liver ACMSDase activity. Blood serum corticosterone levels in normal rats on the last day of the feeding period remained maximum and then decreased gradually until 04: 00, and tended to be higher in rats fed a protein-free diet than in those fed a 30% casein diet. These results suggest that the dietary induction of ACMSDase activity occurs only when a sufficient amount of dietary casein is ingested in the presence of a physiologically significant concentration of blood serum glucocorticoid in rats.

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A new polysaccharide was obtained from the culture nitrate of a bacterium, Alcaligenes latus strain G66A. This polysaccharide induced differentiation of mouse myeloid leukemia cells (M 1), and has been named latosillan.

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The structure of latosillan was elucidated by a degradative study and NMR spectral analysis. This revealed that latosillan is a heteroglycan composed of repeating units of the pentasaccharide, →2)-β-D-Man-(1→2)-{β-D-GlcNAc-(1→4)}-α-L-Rha-(1→4)-α-L-Rha-(1→4)-α-L-Rha-(1→, shown in Fig. 1.

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GA₃ was identified as a major GA in Neurospora crassa by gas chromatography/selected ion monitoring (GC/SIM) and its content was measured at various stages (0-96 hr after inoculation) of conidial germination and mycelial growth. The GA₃ content in the fungus was 190ng/g dry weight at the initial stage and then decreased rapidly; that per liter culture decreased soon after the inoculation, and then increased to 17.6ng 96hr after inoculation. The GA3 concentration in the culture medium was around 10^-11 M throughout the 96-hr incubation. The physiological role of endogenous GA in Neurospora crassa is also discussed.

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