Abstract: A crystalline alkaline protease was prepared from Bacillus No. 221 isolated from soil. The characteristic point of this microorganism is especially good growth in alkaline media. The enzyme was most active at pH 11.5~12 towards casein and was stable at pH values from 4 to 11 on 10 min incubation at 60°C. Calcium ion was effective to stabilize the enzyme especially at higher temperatures. The enzyme was completely inactivated by DFP and urea, but not affected by sulfhydryl reagent, EDTA, SLS, and DBS. The specific activity of the enzyme towards casein was about 18,000 unit/mg, and the isoelectric point was higher than pH 9.4. The molecular weight and sedimentation constant was approximately 30,000 and 3.5 S respectively, and N-terminal of the enzyme was identified to be alanine. The results indicate that the No. 221 alkaline protease is different from those of alkaline proteases of Bacillus subtilis.

Abstract: A simple purification method which enables us to obtain homogeneous proteinase C from S. cerevisiae was developed. Physical and chemical properties of the purified enzyme were determined. The extinction coefficient at 280 mμ, , of yeast proteinase C was 14.8, and its isoelectric point was pH 3.60. Partial specific volume, intrinsic viscosity and the sedimentation and diffusion coefficients of homogeneous protein were , 0.71 ml/g, [η], 4.83 × 10−2ml/g, , 4.23 S and , w, 6.1 × 10−7 cm2/sec. From these values, molecular weights, M[·],D, MS,D and M[·],S, of 60,000, 59,000 and 58,000, respectively, were obtained. The sedimentation equilibrium experiment gave a molecular weight, Mequil, of 61,000. Yeast proteinase C contained 11.9% nitrogen and was a glycoprotein with 16.7% carbohydrate: The value of β-function, 2.163×l06 or 2.20×l06 indicates that the molecular shape of yeast proteinase C is a plorate with an axial ratio of 4.0, assuming 35% hydration. Furthermore, yeast proteinase C may be a compact, asymmetr...

Abstract: Bacillus No. A–40–2 isolated from soil produced an alkaline amylase in alkaline media. The characteristic point of this microorganism was especially good growth in alkaline media, and no growth was detected in neutral media such as nutrient broth. The alkaline amylase of Bacillus No. A–40–2 was purified by DEAE-cellulose and hydroxyl apatite columns. The amylase was most active at pH 10.5 and stable pH was about 8.5. Calcium ion was effective to stabilize the enzyme especially at high temperatures. The sedimentation constant was about 3.8 S and molecular weight estimated by the Sephadex gel-filtration method was about 70,000. The enzyme was inactivated by urea, sodium laurylsulfate and sodium dodecylbenzene sulfonate. EDTA, PCMB and DFP did not show inhibitory effect. The enzyme hydrolyzed about 70% of starch and yielded glucose, maltose and maltotriose. If the enzyme is a single entity, this alkaline amylase is a type of saccharifying α-amylase.

Abstract: (1) Chitin-UDP acetylglucosaminyltransferase (E.C. 2.4.1.16., chitin synthetase) in the cell-free system from phytopathogenic fungus Piricularia oryzae, and effects of various polyoxins and related compounds on the enzyme activity were studied. Polyoxins A~M, polyoxin A derivatives, polyoxin C derivatives, 5′-amino-5′-deoxyuridine, uridine and thymidine inhibited equally the incorporation of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) into chitin.(2) Competition between the above inhibitors and UDP-GlcNAc was observed by kinetic studies. The Km for UDP-GlcNAc was determined to be 3.3 × 10−3 m and the Ki values for polyoxins A~M, except polyoxin C, were found to be in the range of 3.3 × 10−5 m to 3.4 × 10−6 m. For polyoxin C, 5′-amino-5′-deoxyuridine and uridine, the Ki values of 2.7 × 10−3 m, 8.0 × 10−3 m and 3.0 × 10−3 m were given, respectively. The inhibitor constants for other related compounds were also calculated.(3) The values of binding affinity, −ΔG, for formation of su...

Abstract: The structure-activity relationships of 3-phenyloxazolidine-2, 4-diones, 3-phenyl-4-imino-oxazolidine-2-ones and n-phenylcarbamates were investigated on Sclerotinia sclerotiorum by the agar medium dilution method. In addition, antimicrobial spectra of several compounds against other 15 pathogenic microbes were investigated by the same method. In each series, 3, 5-dihalo-substituents on benzene ring are essential to high antifungal activity against Sclerotinia sclerotiorum and in the case of n-phenylcarbamates, it is necessary that the α-position of alcohol moiety is substituted by such a group as cyano group, ethoxy-carbonyl group or carbamoyl group. α-Cyanoisopropyl N-(3, 5-dichlorophenyl) carbamate, 3-(3′, 5′-dichlorophenyl)-5, 5-dimethyl-4-iminooxazolidine-2-one and 3-(3′, 5′-dichlorophenyl)-5, 5-dimethyloxazolidine-2,4-dione were the most effective and completely inhibited the mycelial growth of Sclerotinia sclerotiorum at 3.2 γm (about l.0 ppm). In general, 3-(3′, 5′-dichlorophenyl) oxazolidine-2, 4-...

Abstract: Sir: In 1961 Kodaira\" reported that extracts from the deproteinized haemolimph of silkworm larvae (Bombyx mori L.) killed by Metarrhizium anisopliae (Metch.) Sorok., a pathogenic fungus for lepidopterous insects, showed toxicity to healthy silkworm larvae by intrahemocoelic injection. More recently, Roberts2) demonst rated that the blood of the diseased silkworm larvae, even after heat treatment, held toxicity to larvae of the greater wax moth (Galleria mellonella L.) and of the silkworm. These facts suggest the production of some kinds of toxins by the fungus in the blood of infected larvae. On the other hand, Kodaira\" isolated, in 1961, two insecticidal metabolites named destruxins A and B (I and II) from culture broth of M. anisopliae. Quite recently, we succeeded in isolating three new destruxins, destruxins C and D and desmethyldestruxin B

Abstract: A radio-resistant Pseudomonas has been isolated from samples of normal unpolished and commercial rice grains. This species could be classified in chromogenic group of genus Pseudomonas. It’s taxonomic characteristics were found to be sufficiently different from all the described species in this genus to warrant it’s description as a new species and was named as Pseudomonas radiora nov. sp.The radio-resistance of this species was 10 to 40 times higher than that of ordinary species in the genus Pseudomonas such as Ps. fluorescens. The dose at D10 value of the strain No. O-l was ca. 0.14 Mrad, which is similar to that of the Micrococcus radiodurans, and that of the strain No. RP-C was ca. 0.06 Mrad in m/15 phosphate buffer.

Abstract: Relation between sulfhydryl groups in soybean proteins and the physical properties of tofu was studied. Changes in the amount of sulfhydryl groups by heating or treatment with urea were more rapid in 11S protein as compared with 7S protein. Moreover, by changing the amount of sulfhydryl groups in proteins by N-ethylmaleimide, 2-mercapto-ethanol and dithiothreitol, the physical properties of tofu from 11S protein were more significantly effected than that from 7S protein. Namely, tofu-gel from 11S. protein got harder and stronger as the amount of sulfhydryl groups increased.The results may suggest that tofu prepared from IIS protein has more disulfide bonds in its gel than that from 7S protein.

Abstract: Two selection methods of non-foaming mutants of sake yeasts (a kind of cell wall mutants lacking the ability to form froth head in sake mash) are described. The mutants, being different in both the affinity to gas bubble and in the agglutinability from the parent, were concentrated, by removing the wild type cells with froth in froth flotation method and by removing them by agglutination caused by lactobacillus cells in cell agglutination method. Spontaneous non-foaming mutants of Kyokai No. 7 strain were isolated from the concentrates after 9 successive trials of each selection procedure at the rates of 50% by the former and 81% by the latter. The UV-induced mutants were also isolated from the concentrates after the 7 successions at the rates of 80% and 100%, respectively, by the former and by the latter. There were two types among the non-foaming mutants with respect to the agglutinability; the one was non- or almost non-agglutinable type (type 1) and the other was weakly-agglutinable one (type 2). The spontaneous mutants isolated by the froth flotation method were all of type 2, while 54% of those isolated by the cell agglutination method was of type I and the rest was type 2. On the other hand, only type I was found with the UV-induced mutants. It is inferred from the concentration rate determined by the froth flotation method in a model experiment that the mutation would occur spontaneously at a rate of 10-8 and be stimulated about 100 fold by the UV-irradiation in Kyokai No. 7 strain. The usefulness of the non-foaming mutants are discussed from a practical point of view for sake production.

Abstract: product from pullulan was isopanose (6-ƒ¿ maltosylglucose). The mold was cultivated in solid culture on wheat bran, or by shaking culture in rice koji media at 30°C for 3 days. Cells obtaine_??_ from the media were ground with alumina extracted with 0.005 M phosphate buffet (pH 6.0), and crude enzyme was obtained b} adding an equal volume of cold acetone to the extract. The crude enzyme showed maltase activity together with pullulanhydrolyzing activity. The pullulan-hydrolyz ing activity was estimated by determining reducing sugar after the incubation with M

Abstract: Protein fraction, prepared from fresh fruiting bodies of shiitake mushroom, is found capable of converting lentinic acid to lenthionine, the principal aroma-bearing substance of the mushroom. A simple Michaelis-Menten kinetics is also valid for the reaction. The reaction products identified, besides lenthionine and unidentified compounds, are glutamic acid, pyrubic acid, acetaldehyde, ammonia, and formaldehyde. Two different kinds of enzyme are envisaged to participate in the reaction; γ-glutamyl transpeptidase removing glutamyl moiety from lentinic acid, and pyridoxal phosphate dependent s-alkyl-l-cysteine sulfoxide lyase acting next on the resultant intermediate to produce an unstable intermediate which is then converted spontaneously to polythiepanes. A reaction pathway is proposed for lenthionine evolution from lentinic acid based on the experimental evidences obtained in this and other papers.

Abstract: (1) Chitin-UDP acetylglucosaminyltransferase (E.C. 2.4.1.16., chitin synthetase) in the cell-free system from phytopathogenic fungus Piricularia oryzae, and effects of various polyoxins and related compounds on the enzyme activity were studied. Polyoxins A~M, polyoxin A derivatives, polyoxin C derivatives, 5′-amino-5′-deoxyuridine, uridine and thymidine inhibited equally the incorporation of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) into chitin.(2) Competition between the above inhibitors and UDP-GlcNAc was observed by kinetic studies. The Km for UDP-GlcNAc was determined to be 3.3 × 10−3 m and the Ki values for polyoxins A~M, except polyoxin C, were found to be in the range of 3.3 × 10−5 m to 3.4 × 10−6 m. For polyoxin C, 5′-amino-5′-deoxyuridine and uridine, the Ki values of 2.7 × 10−3 m, 8.0 × 10−3 m and 3.0 × 10−3 m were given, respectively. The inhibitor constants for other related compounds were also calculated.(3) The values of binding affinity, −ΔG, for formation of su...

Abstract: The volatile compounds from beef fats heated under the cooking condition-145°C, l0 min-were isolated, and nonacidic compounds separated from them were further fractionated into five fractions by silicic acid column chromatography. The odor of nonacidic compounds significantly resembled the heated flavor of beef fats. Several carbonyl compounds, hydrocarbons, alcohols, lactones and pyrazine compounds in the fractionated compounds were identified with the techniques of gas chromatography and gas chromatography-mass spectrometry. Their possible contribution to the heated beef fat flavor was discussed. The typical heated flavor could probably be ascribed to a proper combination of aldehydes, ketones, esters and sulfur-containing compounds.

Abstract: Strand scissions in DNA of M, radiodurans after in vivo irradiation with 60Co gamma rays were investigated by the sedimentation analysis using neutral sucrose gradients Double-strand scission in DNA was estimated to occur at the rate of one double cut per 800 eV This rate is in a good agreement of the value reported for mammalian cells The rejoining of these double-strand scissions was observed during the repair process of the post-irradiation incubation and the mean rejoining time, ie, the time reducing the remaining fraction of the double-strand scission to 037, was found to be 52 min This rejoining repair was inhibited by adding chloramphenicol, tetracycline or actinomycin D to the postirradiation incubation medium It is suggested that the high resistance character of M radiodurans to gamma rays may be due to the efficient capacity of this rejoining repair

Abstract: A sulfur-containing peptide, acting as an enzymatic precursor of the aroma-bearing substance, was isolated in crystalline form from fruiting bodies of the shiitake mushroom. The isolation procedure consists of methanol extraction, repeated ion-exchange chromatographies, and crystallization from methanol-water. The common name “lentinic acid” is given for the isolated peptide. Amino acid analysis, performed after acid hydrolysis of the oxidized or desulfurized peptide, indicates involvement of a glutamic acid and S-substituted cysteine sulfoxide in the molecule. Results of enzymatic assays led to the designation of l-configuration for the component amino acids. The presence of a γ-glutamyl peptide bond is shown, since glutamic acid constitutes the N-terminal amino acid and a free amino group is adjacent to a free carboxyl group. The structure proposed for lentinic acid is, The precise structure for the portion in brackets is unidentified.

Abstract: Microorganisms which produced n-alkane ω,ω′-dicarboxylic acid (DC) from n-alkane were selected from natural sources. It was found that the best three producers thus obtained belonged to yeast. All of the stock cultures which are able to assimilate n-alkane and are belonged to genus Candida and Pichia were also found to produce DC from n-alkane.Candida cloacae 310, a representative strain selected from natural source, was able to produce DCs having 5 to 16 carbon atoms from various n-alkanes. Among them, DCs with 5 to 9 carbon atoms were more heavily accumulated than those with more than 9, except those with the same number of carbon atoms as the substrates which were the main products from the substrates with less than 15 carbon atoms. It was also clearly demonstrated that DCs with odd carbons alone were produced from n-alkanes with odd carbons, while DCs with even carbons alone from n-alkanes with even carbons.Then, cultural conditions of Candida cloacae 310 were studied for the production of DC-12 from ...

Abstract: The viscosity change of myosin A concentrated solution with or without other com-ponents was measured as the incubation time elapsed at 30°C. The viscosity of myosin A solution increased, but that of F-actin solution did not. The shear stress at 0.04 sec-1 was not increased to 1.0 dyne/cm2 in the former, but in the latter was below 0.5 dyne/cm2. The viscosity of myosin B solution increased slightly, but that of native tropomyosin-free myosin B solution decreased remarkably. In both the shear stress at 0.04 sec-1 was greater than or equal to 15 dynes/cm2. The speed of the viscosity increase in the presence of 3mM pyrophosphate and 3mM MgCl2 was higher in concentrated solution of myosin B than in that of native tropomysin-free myosin B. The shear stress at 0.04 sec-1 after 6 hr at 30°C was 11.5 and 8.2 dynes/cm2, respectively. The effect of native tropomyosin and actin on the viscosity change was discussed.

Abstract: The effect of concentration of each substrate in the reaction catalyzed by sucrose synthetase isolated from sweet potato roots was determined. For the sucrose synthesizing reaction, UDP-glucose(ADP-glucose)+fructose→sucrose+UDP(ADP), the substrate saturation curves for UDP-glucose, ADP-glucose and fructose were hyperbolic in shape and the reaction was strongly inhibited by UDP competitively. On the other hand, the substrates for the reversal of sucrose synthetase reaction, sucrose+UDP(ADP)→UDP-glucose(ADP-glucose)+fructose, exhibited a sigmoidal shaped saturation curve which was deviated from the Michaelis-Menten equation. The plot of data according to the empirical Hill equation gives a values greater than 1.0 for every substrate examined in the latter case. In view of these experimental data, the major role of sucrose synthetase is postulated in that this enzyme is involved in the breakdown of sucrose in sweet potato root tissues instead of the sucrose synthesizing reaction. The molecular weight of the ...

Abstract: The structure of fluorescein chromophore was studied with infrared and nuclear magnetic resonance spectrometries. Fluorescein chromophore formed acid salt with strong acid and this salt was decomposed with water. Fluorescein derivatives were allowed to crystallize into trifluoroacetic acid salts. The new compounds, trifluoroacetic acid salts of fluorescein thiohydantoin (FTH)* amino acids, were synthesized and they were studied with ultraviolet, visible, fluorescence and infrared spectrometries, as well as optical rotatory dispersion and nuclear magnetic resonance. The trifluoroacetic acid salts of FTH-amino acids were superior to trifluoroacetic acid-free form as the standard materials of N-terminal analysis.

Abstract: Many strains of yeast which can utilize n-alkanes as the sole source of carbon were isolated from flowers and fruits. Among them, a strain, OH23, identified as Candida tropicalis, formed acidic substances from n-alkanes. The principal products from n-alkanes with odd and even numbers of carbons were identified as glutaric and adipic acids, respectively. The culture conditions for their formation were investigated. n-Pentadecane and n-hexadecane were the best substrates for the formation of glutaric and adipic acids, respectively. Yields of 170 mg of glutaric and 64 mg of adipic acid were obtained from 100 ml of media containing 4% (v/v) n-pentadecane and n-hexadecane, respectively, and 0.5% casamino acids.

Abstract: A gluconate-utilizing strain of Corynebacterium was found to be capable of utilizing aldopentoses and producing corresponding pentitols when pentoses were added to the medium containing gluconate as a carbon source during the cultivation of the organism.Pentitols produced from d-xylose, l-arabinose, and d-ribose were isolated from the cultured medium and identified as xylitol, l-arabitol, and ribitol, respectively.The pentitol production was significantly influenced by the concentration of gluconate in the initial medium and that of pentose added to the medium during the cultivation.The amount of xylitol, l-arabitol, and ribitol reached 69 mg/ml, 60 mg/ml, and 32 mg/ml, respectively, after 14 days of incubation when pentoses were added to the medium containing 9.6% potassium gluconate to give a final concentration of 150 mg/ml.

Abstract: (1971). Inactivation of Bacteriophages by Ascorbic Acid. Agricultural and Biological Chemistry: Vol. 35, No. 2, pp. 294-296.

Abstract: Seven gibberellin glucosides (F-I_??_F-VII) were isolated from immature seeds of Japanese morning-glory (Pharbitis nil). The structures were elucidated as 2-O-β-glucosyl-gibberellin A3 (F-I), 2-O-β-glucosyl-gibberellenic acid (F-II), 2-O-β-glucosyl-isogibberellin A3 (F-III), 3-O-β-glucosyl-gibberellin A26 (F-IV), 3-O-β-glucosyl-gibberellin A27 (F-VI) and 3-O-β-glucosyl-gibberellin A29 (F-VII). The one of the glucosides (F-V) was identified as 3-O-β-glucosyl-gibberellin As isolated from Phaseolus multiflorus.

Abstract: The crude enzyme preparation obtained by isopropanol-precipitation of a water-extract of wheat bran culture of Trichoderma viride was first fractionated with ammonium sulfate precipitation, followed by DEAE-Sephadex A-25 column chromatography. Two active fractions, F-1 and F-4, were obtained. Fraction F-4 was futher purified by column chromatography with CM-Sephadex C-50 and was finally crystallized by the addition of ammoniun sulfate to 35% saturation. This purified xylanase A was homogeneous as judged by ultracentrifugation and discelectrophoresis. Maximum conditions for the activity of crystalline xylanase were pH 3.5 and 50°C. The enzyme was stable in a concentrated solution below 40°C and between pH 2 and 7, but at low enzyme concentration, i. e. 9.7μg per ml, it was easily inactivated under these same conditions.

Abstract: The enzymatic decomposition of phenyl mercuric acetate (PMA) to metallic mercury by a mercury-resistant Pseudomonas has been studied. The formation of the system involved in the decomposition was found to be inducible. Glucose dehydrogenase (D-glucose: NAD oxidoreductase), arabinose dehydrogenase (L-arabinose: NADP oxidoreductase), cytochrome c and the “decomposing enzyme” which catalyzes the splitting of the C-Hg linkage, were separated from cell free extract by gel filtration on a column of Sephadex G-150. The cytochrome fraction was further separated into two types, c-I and c-II, by chromatography on a column of CM-Sephadex. Each of these cytochromes showed absorption peaks at 416, 519 and 547mμ fe in the reduced form, but they were different in the molecular weight; cytochrome c-I was estimated to be about 26000, and cytochrome c-II about 14000. The reconstruction of these enzymes demonstrated that a reduced NAD(P) generating system, glucose dehydrogenase or arabinose dehydrogenase, cytochrome c-I and the “decomposing enzyme” were required for the decomposition of PMA. A hypothetical scheme for the decomposition of PMA was proposed and the decomposition mechanism was discussed.

Abstract: A 7S protein in soybean globulins consisted of at least nine polypeptide chains (subunits). Complete dissociation into subunits, having a sedimentation coefficient of 1.1~1.4S and a molecular weight of 22,000~24,000, occurred in the presence of 8m urea and 4m guanidine hydrochloride. However, it was found by sedimentation, ultraviolet spectrophotometry and optical rotatory dispersion that the dissociation with various concentrations of urea accompanied simultaneously with the destruction of the internal structure of the protein. No disulfide bond appeared to participate in the binding between subunits from the results of sedimentation and disc electrophoresis. These results suggested that the subunits were very compactly and complicatedly folded on the formation of the gross structure, and hydrophobic bond with hydrogen bond also participated in the interaction between subunits.The dissociation was interfered with the increase of ionic strength using sodium chloride, particularly in low concentration of u...

Abstract: In order to search for microorganisms able to synthesize D(−)-α~aminobenzylpenicillin (ampicillin) from 6-aminopenicillanic acid (6-APA), experiments were undertaken to screen out organisms possessing potent penicillin acylase (EC 3.5.1.11). From numerous bacteria, Actinomycetes, Yeasts and Basidiomycetes, we selected as acylase producers Pseudomonas crusiviae, Kluyvera citrophila, Streptomyces ambofaciens, and Nocardia globerula. The latter also could form the enzyme on a n-paraffin medium. Among these, K. citrophila KY 3641 was a most promising organism for ampicillin biosynthesis from 6-APA. Approximately 10 mg/ml of 6-APA were formed from penicillin G in about 90% yield by use of its intact cells.