A deoxyribonuclease (DNase) was purified approximately 200-fold from 14-day-old chick embryos. The purified DNase did not contain detectable phosphomonoesterase, phosphodiesterase or ribonuclease activity, and it was homogeneous on gel electrofocusing and gave a single band of protein on polyacrylamide gel electrophoresis. The enzyme was activated slightly by Mg²⁺, inhibited completely by Hg²⁺ and moderately by Cu²⁺, had a pH optimum at 5.3, a molecular weight of 37, 000 and an isoelectric point of 6.5. Its mode of action was primarily endonucleolytic, yielding oligonucleotides ending in 5'-phosphates, and a preference was shown for native DNA as substrate.

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In Schizosaccharomyces pombe, inositol-deficiency caused a decrease in Crabtree effect and an enhanced Pasteur effect. A time-course estimation of intracellular glucose, glycolytic intermediates, ATP and other components showed that decreased glycolytic activity possibly occurred before low glucose uptake activity and that low transport activity could hardly have caused of low glycolytic activity. The activities of all glycolytic enzymes tested seemed to be low in vivo, as well as the activities of enzymes in the hexose mono-phosphate shunt. These results suggest that inositol-deficiency caused pleiotropic situation, possibly due to structural abnormality. The possible cause of low aerobic fermentation activity may be due to the difference in Km values between pyruvate dehydrogenase complex and pyruvate decarboxylase. An oxidative phosphorylation-uncoupling mutant was isolated and was used in these investigations.

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The enzyme inactivating showdomycin was extracted from Streptomyces sp. No. 383 and purified 20-fold by ammonium sulfate fractionation followed by DEAE-Sephadex A-50 and Sephadex G-100 column chromatography. The enzyme preparation with a pH optimum of 7.0 effectively converted showdomycin into isoshowdomycin at 35 to 40°C in 30min. The molecular weight was calculated to be approximately 125, 000 by gel filtration. The enzyme activity was not affected by metal ions, except for Hg²⁺ and Cu²⁺.

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Synthetic activity and existence of ppGpp and pppApp in an anthracycline-producing strain Streptomyces galilaeus were determined by radioimmunoassay and ³²P-labeling method during cultivation under both the antibiotic productive and non-productive conditions. The cellular ppGpp(pppGpp)-synthesizing activity was highest at the end of exponential growth, and 3-fold higher in the antibiotic-productive cultivation than in non-productive cultivation. The intracellular level of ppGpp determined by radioimmunoassay was high at the end of exponential growth, and afterwards its level decreased by one fifth. The low level of cellular ppGpp during the period of intense antibiotic production suggests that ppGpp consumption may play an important role in antibiotic production of S. galilaeus. The concentration of pppApp was not determined intracellularly by radioimmunoassay.

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In a continued search for new potential pesticidal agents, two new substituted 1, 2, 4-triazole derivatives were synthesized. These were condensed with various substituted 1, 3, 4-thiadiazole and N-chloroacetyl-N'-aryl urea derivatives. The compounds thus obtained were screened for their insecticidal and bactericidal activities. Most of these have been found to possess significant pesticidal activities. The relation between their pesticidal properties and chemical structure has been studied.

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The effect of the organophosphorus fungicide, edifenphos (O-ethyl S, S-diphenyl phosphoro-dithiolate, EDDP, Hinosan^®), on DNA, RNA, protein, chitin and lipid biosynthesis was investigated in Pyrieularia oryzae. The incorporation of [2-¹⁴C]thymidine, [2-¹⁴C]uridine and L-[U-¹⁴C]amino acid mixture into DNA, RNA and protein, respectively, was not significantly affected in mycelial cells of P. oryzae treated with 10ppm edifenphos. The fungicide scarcely inhibited chitin synthetase of the microsomal fraction at the ED₅₀ concentration value for mycelial growth. On the other hand, addition of edifenphos to the mycelial cell suspension caused a striking decrease in the incorporation of L-[methyl-¹⁴C]methionine and S-adenosyl-L-[methyl-¹⁴C]methionine into phospha-tidylcholine, which is the most abundant phospholipid in P. oryzae, but scarcely inhibited the incorporation of [methyl-¹⁴C]choline into phosphatidylcholine and that of L-[methyl-¹⁴C]methionine into simple lipids. Of enzymes responsible for biosynthesis from glycerol to phosphatidylcholine by way of Greenberg's pathway, phospholipid Nmethyltransferase was specifically inhibited and the ED₅₀ value was 13 ppm. These data suggest that the primary antifungal action of edifenphos is due to interference of phosphatidylcholine biosynthesis by the transmethylation reaction of Sadenosyl-L-methionine. Therefore, the mode of action of edifenphos has a strong resemblance to that of IBP (Kitazin P^®).

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The sugar sequence of the main carbohydrate portion of bovine whole casein was investigated. A major trisaccharide consisting of N-acetylneuraminic acid, D-galactose and N-acetylgalactosaminitol in the molar ratio of 1:1:1 was obtained by alkali treatment of glycopeptide, which was isolated by enzymic digestion of caseinoglycopeptide from chymosin-treated casein. The trisaccharide and the glycopeptide were subjected to methylation and gas chromatographic analysis. On the basis of the analytical data and other data obtained from enzymic examination, the chemical structure of the main sugar portion isolated from bovine whole casein was identified to be N-acetylneuraminyl-α-2→3-galactosyl-β-1→3-N-acetylgalactosaminyl-X (threonine or serine).

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Low concentrations of ionic detergents with 8 or more carbons in the alkyl chain were predicted to shift the native-denatured transition in the lysozyme to denatured state. Fatty acids were found to exert a similar effect and their participation was proposed in proteolysis of alimentary canal digestion and intracellular catabolism.

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About 200 strains of phenol-assimilating yeasts and fungi were isolated from 168 soil and sewage samples. These isolated phenol-assimilating microorganisms were classified into five groups according to morphological properties: typical yeasts, Rhodotorula species, Trichosporon species, Black Yeasts and fungi. There were differences in the groups in ability to assimilate the three isomers of cresol and monohydroxybenzoate. The Rhodotorula species utilized m- and p-hydroxybenzoates, while Trichosporon species did so with m- and p-cresols. “Black Yeasts” were able to metabolize all isomers very well.
A strain in the group of Black Yeasts identified as Aureobasidium pullulans was further tested for growth on cresol. The strain grew at nearly the same rate in media containing different concentrations (90_??_668μg/ml) of o-cresol. Successive feeding of o-cresol at concentrations of less than 200μg/ml resulted in a high yield of intact cells capable of decomposing o-cresol actively.

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Soybeans were roasted at 200°C for 10, 20 and 30min, and their headspace volatiles trapped by Tenax GC were analyzed by gas chromatography and gas chromatography-mass spectrometry and compared with those of raw soybeans.
Fifty compounds were identified, and the quantity of important compounds for beany flavor, such as hexanal and l-hexanol, decreased with longer roasting. However, the decrease was not remarkable, especially between 10 and 20min roasting (in other words between 110 and 150°C), while pyrazines, furans and pyrrole were newly formed in this period and increased with roasting. Judging from the results on sensory evaluation, it is evident that the flavor changed from a beany note to a more desirable note at between 10 and 20min roasting, These three findings suggest that the roasted flavor masked the beany flavor.

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The structure of the extracellular acidic polysaccharide succinoglycan from Alcaligenes jaecalis var. myxogenes 10C3 was elucidated by successive fragmentation of the polysaccharide with extracellular β-D-glycanase (succinoglycan depolymerase) and intracellular endo-β-(1→6)-D-glucanase of Flavobacterium sp. M64 into two tetrasaccharides via its octasaccharide repeating unit, and then methylation analysis and enzymic hydrolysis of the products. The extracellular acidic polysaccharides from Rhizobium meliloti, Agrobacterium radiobacter, Agrobacterium rhizogenes and Agrobacterium tumefaciens were also analyzed by this method and found_??_o be identical with the polysaccharide from Alcaligenesfaecalis var. myxogenes, irrespective of their modes of acylation. The structure was identical with that of extracellular polysaccharide from Rhizobium meliloti presented by Jansson et al. (J. Am. Chem. Soc., 99, 3812 (1977)), except for the occurrence of the succinic acid residue.

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N⁶-(o-Hydroxybenzyl) adenosine-d₄ was synthesized by dehalogenation of 2-chloro-6-(o-hydroxybenzylamino)-9-β-D-ribofuranosylpurine-d₂ with deuterium gas in 0.3N-sodium deuter-oxide solution, which was obtained by condensation of 2, 6-dichloro-9-(2', 3', 5'-tri-O-acetyl-β-D-ribofuranosyl) purine and salicylamine-d₂. The total extent of deuteration of d₄ compound was 86.6%. The d₄ compound was heated in water to yield N⁶-(o-hydroxybenzyl)adenosine-d₃, whose total deuteration was 94.6%.

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Quantitative relationships were investigated between α-tocopherol and polyunsaturated fatty acids (PUFA) or PUFA>18:2 (PUFA with three or more double bonds) in porcine red (M. biceps femoris) and white (M. longissimus thoracis) muscles.Their effects on the development of oxidative rancidity in refrigerated precooked muscles were also investigated. The differences in the molar ratios of PUFA or PUFA>18:2-to-α-tocopherol or in the concentrations of α-tocopherol (pmol) to PUFA or PUFA>18:2 (per gram) were not statistically significant in the two types of muscles, although red muscle had lower molar ratios of PUFA-or PUFA>18:2-to-α-tocopherol and higher levels of α-tocopherol per gram of PUFA or PUFA>18:2 than did the white muscle. 2-Thiobarbituric acid (TBA) values for cooked ground muscles, subsequently refrigerated for 3 days tended to increase with decreasing concentrations of α-tocopherol per gram of PUFA and PUFA>18:2 of total lipids.

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α-Amino acid ester hydrolase, which can synthesize various kinds of semi-synthetic cephalo-sporins from 7-aminocephem compounds and α-amino acid esters, was purified to homogeneity from the cell-free extract of Xanthomonas citri IFO 3835 by ion-exchange chromatography and gel filtration. The purified enzyme migrated as a single band on disc gel electrophoresis and sedimented as a single symmetric peak on ultracentrifugation (s_20.W=11.8S). It showed a UV absorption maximum at 280nm at pH 7.0 (E^1%_1cm=16.4) and had an isoelectric point at pH 7.8. Cysteine and cystine were not found in its molecule. Carbohydrate was not substantially detected. The molecular weight was estimated to be 270, 000_??_280, 000 by ultracentrifugation and gel filtration. The enzyme dissociated into subunits with an identical molecular weight of 72, 000 in the presence of sodium dodecyl sulfate or guanidine. HCl.

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Enzymological properties of the purified α-amino acid ester hydrolase from Xanthomonas citri IFO 3835 were studied. The enzyme catalyzed both hydrolysis of α-amino acid esters (e.g. D-α-phenylglycine methyl ester (D-PG-OMe)) and transfer of the acyl group from the amino acid esters to amine nucleophiles (e.g. 7-aminocephalosporanic acid, 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and 6-aminopenicillanic acid). The substrate specificity of the enzyme in hydrolysis was parallel with that in transfer. The enzyme required a free amino group on the α-carbon of the donor ester, but did not exhibit absolute stereospecificity. α-Amino acid derivatives having an acid-amide bond were hydrolyzed at much lower rates than the corresponding ester derivatives. The optimum pH for both hydrolysis and transfer was 6.4, and the optimum temperature, 35°C. The enzyme was inactivated completely within 15 min at pH 6.0 and 50°C. FeSO₄, CuSO₄, and HgCl₂ inhibited the enzyme. At pH 6.4 and 30&C, the Michaelis constants were estimated to be 8.26mM for D-PG-OMe and 2.99mM for cephalexin. The apparent Michaelis constant for 7-ADCA was estimated to be 5.08 mm, With D-PG-OMe as an acyl donor, the molecular activity (k_o) for hydrolysis was calculated to be 1.11×10⁴ sec^-1.

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Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed D-α-phenylglycine methyl ester (D-PG-OMe) to give equimolar amounts of D-α-phenylglycine and methanol. With D-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the aryl group from D-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (k_o) of the enzyme-catalyzed hydrolysis of D-PG-OMe, whereas it did not alter the Michaelis constant (K_M), and plots of 1/ko against the initial concentration of a nucleophile (n_o) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor.

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D-Aminoacylase was found to be produced not only by S. olivaceus 62-3 isolated from soil but also by three strains of type culture of Streptomyces species. All four of these strains produced D-aminoacylase intracellularly only when an inducer was added to the culture medium. D-Amino acids or N-acetyl-D-amino acids were effective as inducers.
As S. tuirus showed the highest D-aminoacylase activity, the enzyme extract of this strain was subjected to further investigation to determine the optimal conditions for optical resolution of N-acetyl-DL-phenylglycine. Almost all contaminating L-aminoacylase in the enzyme extract could be eliminated by DEAE-Sephadex adsorption. D-Phenylglycine of 99.9% optical purity was obtained after complete hydrolysis of D-isomer with the use of D-aminoacylase solution.

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Euglena gracilis grew with propionate as the sole carbon source under illumination but not in the dark. The metabolic pathway of propionate was determined in E. gracilis growing under illumination. Labeled propionate was first recovered in the amino acid fraction and gradually transferred to protein and paramylon with a lapse in incubation time. In the amino acid fraction aspartic acid was most densely labeled initially, followed by glutamic acid indicating that propionate is metabolized via succinate. The result agreed with the radiorespirometric pattern of specifically labeled propionates: 1-C>2-C=3-C. E. gracilis contained propionyl-CoA carboxylase, but not α-hydroxyglutarate synthase. The reaction product of the enzyme was identified as methylmalonyl-CoA. The enzyme activity increased in the log phase when propionate in growth medium was actively utilized for growth. From these results we have concluded that illuminated Euglena metabo-lizes propionate through propionyl-CoA and methylmalonyl-CoA to tricarboxylic acid cycle intermediates for growth.

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Vitamin B₁₂-dependent ribonucleotide reductase purified from Rhizobium meliloti catalyzes the reduction of 5'-diphosphates of guanosine, adenosine, cytidine and uridine (GDP, ADP, CDP and UDP). The enzyme activities were regulated by Mg²⁺ and deoxyribonucleoside triphosphate effectors as follows: in the presence of Mg²⁺, allosteric effector deoxyguanosine triphosphate (dGTP) had the most stimulatory effect on reduction of ADP and UDP; deoxyadenosine triphosphate (dATP) on reduction of CDP; and thymidine triphosphate (dTTP) on reduction of GDP. These stimulatory effectors were active at a low concentration of 10μM. Other deoxyribonucleotides may be negative or weakly positive effectors. Without effectors, the rate profile of ADP and GDP reduction showed a sigmoidal curve. In the absence of Mg²⁺, the activities of the reductase showed nearly maximal levels, and the addition of effeetors rather decreased the activities, except in the case of UDP reduction which was most strongly stimulated by dGTP. The effect of Mg²⁺ can be replaced by Ca²⁺. Monovalent cations such as Na⁺ and K⁺ had a negligible effect on the activities of ribonucleotide reductase.

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Subsite affinities of soybean β-amylase were evaluated by a new method to elucidate the effect of multiple attack. Product analysis of the reducing end-labeled maltooligosaccharides showed that G^*₆ and G^*₇ were degraded through the multiple attack pathway, in 0.02_M acetate buffer, pH 5.4 at 25°C. The apparent first-order rate constants, (k_o/K_m)e_o, for n-mer substrates (n=3-7) were obtained from the decrease of substrate followed by paper chromatography under the condition of [S]_??_K_m. Using the constants and the cleavage distribution of G^*₃^*, the subsite affinities (A₁, A₄ and A₅) of β-amylase were evaluated. It was revealed that the first subsite of soybean β-amylase had a larger subsite affinity than any subsites of other amylases so far evaluated. Thus the first subsite seems to play an important role for the characteristic action pattern of this enzyme, namely exclusive maltose-forming action. The frequency of multiple attack decreased as either or both pH and temperature become unfavorable for enzymic activity.

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Acetone-dried cells of Arthrobacter simplex having appreciable steroid Δ¹-dehydrogenase activity were immobilized by mixing the cell suspension with water-miscible urethane prepolymers synthesized from toluene diisocyanate and polyether diols. The entrapped cell activity in the transformation of hydrocortisone to prednisolone was affected by the properties of urethane prepolymers, such as the isocyanate group content in prepolymers, the molecular weight of polyether diols and the ethylene oxide content in diols. The addition of 10% of organic solvents, such as methanol and glycols, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and the reaction rate of the immobilized cells. The activity of immobilized cells remained high even in the system containing 30% of methanol, which drastically inhibited the activity of free cells. The presence of an electron acceptor, phenazine methosulfate or 2, 6-dichlorophenolindophenol, significantly stimulated the steroid conversion with entrapped cells, as well as free cells. The stability of the cells over repeated reactions was greatly improved by immobilization.

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4-Guanidinobutyrate amidinohydrolase (EC 3.5.3.7) was purified about 360-fold to apparent homogeneity from Pseudomonas sp. ATCC 14676. The molecular weight of the enzyme was estimated to be 180, 000_??_186, 000 by gel filtration procedure and the method of Hedrick and Smith. The subunit molecular weight was estimated to be 33, 000_??_36, 000 by SDS-polyacrylamide gel electrophoresis. The enzyme was optimally active at pH 10.2 in sodium carbonate buffer. EDTA inactivated the enzyme during incubation at 50°C in phosphate buffer (pH 7.0). Incubation of the inactivated enzyme with Mn²⁺ restored full activity. The enzyme was inactivated with PCMB, and the PCMB-inactivated enzyme was reactivated by incubation with 2-mercaptoethanol. The enzyme specifically acted toward 4-guanidinobutyrate; 5-guanidinovalerate and 6-guanidinocaproate were hydrolyzed at very low rates. The Km value for 4-guanidinobutyrate was 33mM. Propionate and n-butyrate were competitive inhibitors of the enzyme with Ki values of 2.0mM and 0.5mM, respectively. These properties were compared with those of the analogous guanidinoacetate amidinohydrolase from the same organism.

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When Erwinia carotovora Er, a bacteriocinogenic strain, was induced after irradiation by ultraviolet (UV) light or inhibitors of DNA synthesis, such as mitomycin C or nalidixic acid, pectin lyase and bacteriocin (designated carotovoricin) activity appeared in the culture fluid. The optimal dose of each of these agents for producing the enzyme or bacteriocin was identical, and the time courses for both were essentially the same. Therefore, we assumed that the synthesis of the enzyme and bacteriocin was regulated by the same mechanism, in which a repressor inactivated by UV light, mitomycin C or nalidixic acid was involved. The other three bacteriocinogenic strains of E. carotovora also formed pectin lyase, in addition to carotovoricin in the presence of mitomycin C, indicating that simultaneous syntheses of pectin lyase and carotovoricin were widespread pheno-menon in bacteriocinogenic strains of E. carotovora.

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Guanosine triphosphate cyclohydrolase (EC 3.5.4.16) was previously shown to exist in two forms (GTP cyclohydrolase D-I and D-II) in Serratia indica IFO 3759, and they were homogeneously isolated. The present study deals with the characterization of their reaction products. A fluorescent product formed from guanosine triphosphate by GTP cyclohydrolase D-II was identified as 7, 8-dihydroneopterin triphosphate by its absorption spectra, phosphate analysis and gas chromatography-mass spectrometry of the dephosphorylated trimethylsilyl derivative. After oxidation and dephosphorylation, the D-erythro configuration of the side chain was made clear by the elution profile on ECTEOLA-cellulose chromatography, Rf values on thin-layer chromatography and by biological activity to Crithidia fasciculata ATCC 12857. The fluorescent products from GTP cyclohydrolase D-I and D-II were indistinguishable.

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From the mycelial mats of Aspergillus multicolor Sappa, five metabolites were isolated. Two of them were new metabolites; one was 5, 6-dimethoxysterigmatocystin (1) and the other 5, 6-dimethoxydihydrosterigmatocystin (4). The structure of 1 was determined by chemical degradation, NMR techniques of benzene-induced solvent shifts and X-ray structure analysis of its acetate (2). The three metabolites were identified as sterigmatocystin, averufin and versicolorin C, respectively.

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A new antibiotic, toromycin (antibiotic B-21085) was isolated as yellow crystals from the culture broth of Streptomyces collinus subsp. albescens subsp. nov. The antibiotic has the molecular formula C₂₇H₂₆O₉ and is the polycyclic aromatic hydrocarbon group of antibiotics. Toromycin is active against gram-positive bacteria, mycobacteria, mycoplasma, DNA viruses and some bacteriophages.

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The enzyme utilizing metaphosphate for nicotinamide adenine dinucleotide phosphorylation was purified 500-fold from B. ammoniagenes and its properties were studied. The isolated enzyme appeared homogeneous on disc gel electrophoresis; its molecular weight was determined to be 9.0×10⁴ by gel filtration. This enzyme specifically phosphorylated nicotinamide adenine dinuc-leotide at the optimum pH at 6.0. Of phosphoryl donors tested, metaphosphate was most effective for the reaction, and adenosine-5'-triphosphate was less effective. The activity was inhibited by adenosine-5'-monophosphate, adenosine-5'-diphosphate or reduced pyridine nucleotides. The enzyme did not exhibit catalytic activity in the absence of a divalent cation. We concluded that the enzyme phosphorylating nicotinamide adenine dinucleotide in the presence of metaphosphate is distinct from adenosine-5'-triphosphate-dependent nicotinamide adenine dinucleotide kinase, and tentatively designated it metaphosphate-dependent nicotinamide adenine dinucleotide kinase.

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¹³C NMR spectra were measured for 19 pyrethroids and their related compounds including allethrin, tetramethrin, resmethrin, furamethrin, phenothrin and permethrin. Complete assignment of chemical shifts was accomplished by relative spectral pattern, single-frequency off-resonance decoupling, benzene substituent effects, proton selective decoupling and use of shift reagents. The use of shift reagent was found to be especially efficient for assignment of ¹³C resonances. In the case of allethrin, the splittings of some resonance peaks were observed originating from diastereomerism.

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