Phthalthrin labelled with ¹⁴C in the alcoholic moiety was synthesized via an intermediate Δ¹-tetrahydrophthalic anhydride-¹⁴C. A preparative pathway leading to the intermediate was investigated in order to shorten the synthetic steps. Thus the intermediate was prepared in two steps starting with potassium cyanide-¹⁴C. Position of double bond introduced into the molecule was confirmed from IR spectrum and melting point. Chemical and radiochemical purities of intermediates and the final product were ascertained on scanning the radio-thin-layer chromatograms. A brief description on the self-decomposition of phthalthrin-¹⁴C and an intermediate is also given.

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An intracellular nuclease inhibitor was 1270 times purified from a heat treated cell free extract of fresh mycelia of Aspergillus oryzae, by ammonium sulfate fractionation and chromatographies using DEAE-cellulose and Sephadex G-75. The purified sample of the inhibitor showed a UV absorption curve typical for protein, and it was inactivated by proteases such as chymotrypsin. The inhibitor stoichiometrically inactivated nuclease O (an intracellular nuclease of Asp. oryzae), forming an enzyme-inhibitor complex. But, it did not affect nuclease S₁, RNase T₁, RNase T₂ or pancreatic RNase. The inhibitor was insensitive to 10-⁵M p-chloromercuribenzoate or 10-⁴M Pb²⁺. Molecular weights estimated by the method of Andrews were 23, 000 for the inhibitor, 47, 000 for nuclease O, and 82, 000 for the enzyme-inhibitor complex. The nuclease activity was recovered from the inactive complex by the action of chymotrypsin.

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Nuclease O of Asp. oryzae was purified and crystallized from 113.5kg of wet mycelia and 2kl of culture filtrate, by salting out with ammonium sulfate and by chromatographies on CM-Sephadex C-50 and Sephadex G-100. The purified nuclease showed α single peak with apparent sedimentation constant 2.9S in an ultracentrifuge. The molecular weight measured by short column method was 64, 000. The nuclease was completely inhibited by the specific nuclease inhibitor obtained from Asp. oryzae. The nuclease was activated by 0.1mM Mg²⁺ and Mn²⁺, and completely inhibited by 1mM EDTA. Optimum pH for activity was 7.6 for RNA and 7.4 for DNA. The nuclease degraded polyadenylic acid, polyuridylic acid and polycytidylic acid without forming detectable amount of mono-nucleotides. And, the main product from RNA was oligonucleotides. The enzyme showed no nonspecific phosphodiesterase activity.

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From the cells of two strains of Bacillus subtilis, one producing liquefying α-amylase and the other, saccharifying α-amylase, were isolated each two kinds, four in total, of aminopeptidases. Their stabilities and activities were different from one another under various conditions, although all of them required either manganese or cobalt ions for the enzyme actions. They were also distinguished from each other by their specificities towards peptide linkages as tested by synthetic peptides. They showed different activities according to the kinds of amino acids of the N-terminus. The experiment also indicated that the hydrolytic activity of the peptidases was influenced by the kind and sequence of amino acids adjacent to the N-terminal amino acid.

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Intracellular protease (IPLB) of Streptococcus cremoris was extracted from the cells, which were cultivated in liquid media, by momentarily disrupting between two disks by high pressure.
The hydrolyzing modes of α₈-, crude k-, β-, and whole casein by IPLB of Str. cremoris or rennet were observed through the released amounts of tyrosine, sialic acid, NPN, and calcium insensitive substance. Relative specific turbidity of casein solution and dissymmetry coefficient of casein were measured. Particle weight and UV absorption spectrum of each high molecular hydrolyzate of whole casein were also determined.
Among four kinds of casein fractions, α₃- or crude k-casein was most easily hydrolyzed by IPLB of Str. cremoris or rennet. Relative specific turbidity of crude k-casein solution was remarkably, but those of α₈-, β-, and whole casein slightly increased by the action of IPLB of Str. cremoris or of rennet. Changes of dissymmetry coefficients were negligibly induced by these two enzymes. Absorption spectrum of IPLB-Str. cremoris-casein showed some conformational change.

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It was recognized that intracellular protease (IPLB) of L. bulgaricus, L. helveticus or Str. lactis, all together, more easily hydrolyzed α₈-casein than crude k-, β-, and whole casein. By the actions of three IPLBs, relative specific turbidity of crude k-casein solution remarkably but those of α₈-, β-, and whole casein slightly increased, and dissymmetry coefficients of these casein fractions changed negligibly.
Particle weight of whole casein hydrolyzed by each IPLB for five days was larger than that of control casein. UV absorption of each whole casein hydrolyzed by a IPLB increased at the wave length range of 280_??_250 mμ.

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1. Nutritional requirement of Candida lipolytica AJ 5004 were examined, and it was apparent that this yeast required only thiamine as growth factor either in n-paraffins or sugar medium.
2. Even if Candida lipolytica was cultured in synthetic medium containing adequate amounts of thiamine, it was able to produce so high yield of α-KGA as in the medium containing 0.02% of corn steep liquor.
3. Relationship between concentration of n-paraffins and yield of α-KGA in the corn steep liquor containing medium was studied.
It was also shown that the rate of conversion of n-paraffin to α-ketoglutarate gradually increased as the concentration of n-paraffins was decreased or as the incubation time was prolonged. A very high rate of conversion, 71%, was obtained after prolonged culture, for 5 days, with a culture medium containing 8% of n-paraffins.
4. It was observed that in the glucose medium the yeast accumulated large quantity of PA, and the optimal concentration of thiamine which afforded the highest productivity of α-KGA was rather higher in glucose medium than in n-paraffin medium.
5. Productivity of α-KGA from C₉ to C₂₀ n-alkanes was demonstrated, and it was found that higher carbon alkanes in the range of C₁₅_??_C₁₉, especially C₁₇_??_C₁₉, were preferable for α-KGA production.

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In a preceding paper we reported that Rhodotorula flava 194 effectively converted biotin to biotinamide. In a present paper the metabolism of desthiobiotin by R. flava 194 was studied under the same condition as in the conversion of biotin to biotinamide. Two desthiobiotin derivatives (Vitamer I and II) were isolated. Vitamer II (crystalline) was identified as bisnordesthiobiotin and Vitamer I was chromatographically determined as desthiobiotinamide.

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Screening for p-hydroxybenzoate-utilizing pseudomonads was carried out. Species belonging to Fluorescent group could grow at the expense of p-hydroxybenzoate as sole carbon source. Two species in Chromogenic group and three species in Achromogenic group could grow in the same manner. Specic and total activities of crude extracts differed from each other strain under cultural conditions employed. Ps. desmolytica IAM 1123 and Ps. desmolytica 4-B were selected as superior strains for the sources of p-hydroxybenzoate hydroxylase. The type and the activity of protocatechuate oxygenase were also investigated. Protocatechuate 3, 4-oxygenase was found in the cells belonging to Fluorescent and Chromogenic groups whereas 4, 5-oxygenase was found in Achromogenic group.

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In the course of phytopathological studies, it has been observed that phyllosticta sp. produces a toxic compound causing wilting and simultaneous dark coloration of the clover leaf. A toxin, herein refered to as phyllosinol (mp 76_??_77°C) was isolated in a crystalline state from the pure culture and it was proved to be the principal toxic metabolite of the fungus. Although the melting point of phyllosinol differed significantly from that reported for epoxydon (40_??_45°C), the structural evidence deduced from spectrometric and chemical methods indicates that phyllosinol is substantially identical with epoxydon even in stereo-isomeric considerations. In the red clover leaf test 10ppm phyllosinol showed the wilting effect.

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An attempt has been made to isolate the bacteria capable of accumulating amino acids during the growth at low temperature from various natural sources. A psychrophilic strain P145 forming glutamic acid at 5°C was obtained and identified as a Brevibacterium sp. The bacterium grew in the range of 0° to 37°C and exhibited the optimum growth at 15°C. The bacterium was defined as a facultative psychrophile.
The strain strictly required methionine only at above 28°C; below this temperature it grew normally without the amino acid. When methionine was added thiamine and biotin stimulated the growth of this strain at 28°C.

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With the Brevibacterium sp. P145 isolated from soil, the effect of incubation temperature on the extracellular amino acid accumulation has been examined from cultural and enzymological points of view. The strain was found to accumulate L-glutamic acid up to 5.88 mg/ml and L-alanine 0.38 mg/ml at 5°C, whereas it formed 0.21 mg/ml of L-glutamic acid and 2.54 mg/ml of L-alanine at 28°C.
The accumulation of L-alanine in the medium at 28°C seemed to be related to the thiamine requirement of the strain. In the case of thiamine deficiency, L-alanine was the main product in the culture at 28°C. When the incubation temperature was abruptly shifted from 28°C to 5°C or from 5°Cto 28°C, the amino acid accumulation was also changed to that of the final temperature. L-Alanine dehydrogenase existed even in the cells grown at 5°C but was not active at this low temperature. These results were in accord with the informations obtained from cultural experiments.

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Maleate cis-trans isomerase in Alcaligenes faecalis IB-14 was induced by malonate and purified about 100-fold over the crude cell-free extract by treatments of ammonium sulfate fractionation, Sephadex G-100 gel filtration, DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. The preparation was shown to be monodisperse on ultracentri-fugal analysisand Svedberg value was found to be 3.84S.
The enzyme was most active at pH value around 8.3 and was stable over the range of pH 5.0 to 7.0 in the presence of dithiothreitol (DTT) for a few weeks, but in the absence of it, the enzyme activity was markedly decreased, especially in the alkaline region. The enzyme activity was inhibited by various sulfhydryl reagents and oxidizing agents, whereas it was not affected by metal chelating agents. The inhibition by Hg²⁺ and PCMB was overcome by the addition of sulfhydryl compounds such as DTT, 2-mercaptoethanol, L-cysteine and glutathione. It was observed that the enzyme did not require co-factor for its function.
Kinetic studies showed that Michaelis constant for maleate was 2.8×10^-3 M and the enzyme did not catalyze the reverse reaction.

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Intracellular lipase of a strain of Rhizopus fungus which is effective for producing a milk flavor was purified and fractionated into two components, I and II, by DEAE Sephadex A-50 column chromatography. They both proved homogeneous by electrophoresis and ultracentrifugal analysis. The sedimentation coefficient was respectively calculated to be 5.8×10^-13 for lipase I, and to be 2.2×l0^-13 for lipase II. From substrate specificity, it was found that lipase I was an ordinary lipase hydrolyzing olive oil and tributyrin favourably, while, II, rather, a special lipase having a high affinity towards tricaprylin. They, also, respectively had an apparent phospholipase activity on soy-lecithin and, clearing activity on chylomicron prepared from olive oil and human serum. Their mode of action, and the effect of metals and emulsifying agents on their activity are also presented.

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Experiments were made to investigate the regulation of PRPP amidotransferase (EC 2.4.2.14) and of IMP- and of IMP- and GMP-pyrophosphorylases (EC 2.4.2.8) in Brevibacterium ammoniagenes. The PRPP amidotransferase was repressed 61 to 64% by more than 50 μg/ml of adenine in medium and was inhibited 70 to 100% by more than 10^-3M of ATP, ADP, AMP and GMP. The IMP-pyrophosphorylase was susceptible to a feedback inhibition by GTP and, to a much lesser extent, by ATP. The inhibition was enhanced by the simultaneous presence of GTP and ATP. Lower levels of ATP rather stimulated activities of the IMP enzyme. The GMP-pyrophosphorylase was also subject to GTP inhibition, but the extent of inhibition was less than that of the IMP-pyrophosphorylase. Repressions of the two enzymes were not observed.

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Methyl N, N'-diacetyl-α-kasugaminide and its C₄ epimer were synthesized starting from D-glucose, and their configurations were discussed.

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It was found that when Rhodotorula rubra IFO 0911 was grown in a phenylalanine medium, benzoic acid and p-hydroxybenzoic acid besides cinnamic acid were formed in the cultured both. The conversions of cinnamic acid into benzoic acid and of benzoic acid into p-hydroxybenzoic acid, and the degradation of p-hydroxybenzoic acid were demonstrated in intact cells of Rhodotorula rubra. These activities were observed in the cells grown on various media, including the medium containing no phenylalanine, and were found to be distributed widely in Rhodotorula. The cells of Rhodotorula rubra were also able to degrade p-coumaric acid, 3, 4-dihydroxybenzoic acid (protocatechuic acid), p-hydroxyphenyl-acetic acid, 3-methoxy-4-hydroxycinnamic acid (ferulic acid) and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). From these results, the metabolic pathways for phenylalanine and tyrosine in Rhodotorula were discussed.

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Structure of a sugar lipid produced by an oleic acid-requiring mutant of Brevibacterium thiogenitalis was studied and established as (I).

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Relation between biotin and oleic acid was studied using a biotin-requiring organism accumulating L-glutamic acid and its blocked mutants lacking the biosynthetic system of biotin or/and oleic acid. The results support the following considerations. Biotin is not formed from oleic acid and does not substantially affect the growth of L-glutamic acidaccumulating bacteria and their productivity of L-glutamic acid.
Consequently, biotin serves only for the synthesis of fatty acids in the present organisms. The essential factor for their growth and metabolism is an unsaturated fatty acid like oleic acid and not biotin. And also, saturated fatty acids have substantially no relation with their growth and metabolism like accumulation of L-glutamic acid.

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Lysine-lipid (siolipin A) and ornithine-lipid (siolipin B) were found at the same time in Streptomyces sioyaensis. They were also found in mutant strains (Lys-, Met-, Try-, His-) of Streptomyces sioyaensis. Ratio of siolipin A to siolipin B differed, depending on the culture conditions. The young mycelium contained siolipin A predominantly, while the aged mycelium did much more siolipin B. They also varied according to pH of the broth. As the whole, the effect was more conspicuous in the mycelium from jar fermenter than that from Sakaguchi flasks.

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The sterilization activity of the 3-substituted-phenyl-l-methyl-l-nitrosoureas was examined by a feeding test on house fly adults. This series of compounds exhibited toxicity as well as sterility, but, for some compounds, the toxicity was reduced to minimum still retaining the high sterility at lower dose levels in bait. It was suggested that the sterilization activity is dependent on physicochemical properties such as chemical reactivity and solubility in the insect body.

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