Separation and purification of proteolytic enzymes of Streptomyces griseus ATCC 3463 were undertaken by fractional precipitation with ethanol and ammonium sulfate followed by dialysis and finally applying zone electrophoresis on starch. In the experiment, peptidase free from proteinase and the latter enzyme free from the former were obtained. The purity was increased approximately 90-fold with the peptidase and 8-fold with the proteinase.

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A peptidase was separated from the culture filtrate of Streptomyces griseus ATCC 3463. The enzyme acts best at pH 8.0 for leucyl peptides and is most stable in the pH range from 6.0 to 9.0. The enzyme is unstable against dialysis, but stable on storage in higher concentrations.
The enzyme has a characteristic substrate specificity different from that of kidney leucine aminopeptidase. It hydrolyzes rapidly DL-leucylglycylglycylglycine and DL-leucylglycylglycine but slowly DL-leucylglycine and L-leucine amide. In contrast with kidney leucine aminopeptidase, the activity of the enzyme is not effected by the addition of certain metal ions.

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The proteinase of Streptomyces griseus ATCC 3463 shows some characteristic properties different from other known proteolytic enzymes from genus Streptomyces. The optimal pH for reaction of this enzyme is 10.0 and the enzyme is stable around pH2.5 but labile at pHs from 3.5 to 7.5. The enzyme hydrolyzes various synthetic substrates for papain, trypsin and chymotrypsin, whereas it acts neither on carbobenzoxy-L-glutamyl-L-tyrosine, a synthetic substrate for pepsin, nor on twenty-four kinds of peptides and amino acid amides splitted by peptidase. The enzyme activity is not inhibited by metal chelating agents, and not activated by metal ion, indicating that it is not a metalloenzyme.

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Adenosine was produced from adenine by means of fermentation using Bacillus subtilis Marburg 160-88 (str, try^-, pur^-). For the study on adenosine production, experiments concerning with pre-culture age, inoculum size, fermentation period, concentration of adenine, carbon source, nitrogen source and supplement of vitamins were carried out by test tube shaker. Furthermore the time course of fermentation was observed using jar fermentor. And it was proved that adenosine was produced about 1mg/ml during the first 40hrs of fermentation in the glucose mineral medium containing 1-2mg/ml of adenine. This fermentation procedure seems to be one of economical methods for adenosine production.

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Adenosine obtained from fermented broth was phosphorylated with fresh cells of Sacch. carlsbergensis IAM 4727 cultured in malt extract, to afford adenylic acid (Na-salt), which was then deaminated chemically to inosinic acid (Na-salt, IMP-Na). Thus, yield of IMP-Na obtained from malononitrile throughout the reactions was calculated, and as the result, it has been cleared that ca. 6g of IMP-Na was produced from 10g of malononitrile (yield ca. 10%). On the other hand, some reaction conditions of adenosine phosphorylation with yeast cells, i.e., concentration of adenosine and yeast cells in reaction mixture, substituting agent for toluene and direct phosphorylation of adenosine without isolation from fermented broth, were examined. Enzymic activities of various molds for deaminating AMP were also examined.

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Each strain of 210 microorganisms (75 bacteria, 38 yeasts, 48 molds and 49 actinomycetes) and many bacteria newly isolated was cultured on a test tube shaker and adenosine formation from adenine in culture fluids was examined by means of paper chromatography. As the results, none of known strains accumulated adenosine obviously. However, two strains of bacteria newly isolated, each of which was identified with Brevibacterium chromogenes nov. sp. and Bacillus licheniformis respectively, accumulated adenosine obviously from adenine in the culture fluids.

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In the present report, the relationships among three genetic markers, purine, tryptophan dependence and strpetomycin resistance, and the activities of adenosine formation from adenine by Bacillus subtilis Marburg are described. Purine or tryptophan independent strain was obtained by means of transformation and streptomycin sensitive strains by back mutation from B. subtilis Marburg 160-88 which is able to produce adenosine from adenine. It is pointed out that the higher adenosine formation is related only to purine dependent maker and not to tryptophan dependent and streptomycin resistant markers.

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It was found that the intercellular space of centrifuged sake yeast cultured in anaerobic medium is 0.263ml/g, which is larger than that of bakers' yeast. On the preparation of the calibration curve, this centrifuged sake yeast was used as standard substance.
The diameters of the majority of cells were included in a range of 3-8μ during the whole course of cultural development. A similar distribution was found on the cells cultured in an abnormal medium. From these results, clearly, the turbidimetric method for the evaluation of the cell amount of yeast cultured anaerobically is unaffected by the cultural conditions.

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The zinc dust distillates of rubrofusarin, nor-rubrofusarin, and methylxanthones (1-, 2-, 3-, and 4-methylxanthones and 1-methyl-3, 6, 8-trihydroxyxanthone) have been investigated.
On zinc dust distillation, rubrofusarin and nor-rubrofusarin afforded naphthalene and anthracene in low yield, but did not give methylxanthene expected from the formerly proposed structure of methylxanthone.
Methylxanthones are considered to give the corresponding methylxanthenes in the distillates; and it is true for 2-, 3-, and 4-methylxanthones. However, in the 1-methyl derivatives the main product was not 1-methylxanthene but anthraccnc. It has been found also that on that condition all methylxanthones examined did not give naphthalene that was a main zinc dust distillate in rubrofusarin.
Thus, we conclude that it is appropriate to assign the structure of naphthalene derivative for rubrofusarin instead of the so far proposed methylxanthone structure.

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Each of reduction-polarograms of vitamin A-alcohol, -palmitate and β-carotene showed three steps in 60 vol% benzene-acetonitrile mixture containing 0.1M-tetrabutylammonium iodide. The first half-wave potentials were determined at-1.41V vs. Hg-pool for the alcohol, -1.23V for the palmitate and-1.02V for β-carotene. The second half-wave poten-tials were determined at -1.65V identically for first two vitamins and-1.67V for β-caro-tene. The third waves were raised at ca.-2.0V but were quite ill-defined. All of the first and the second waves were controlled by diffusion and those wave-heights were proportional to concentrations of the vitamins and β-carotene in the range from 5×10^-5M (ca. 50 IUA·ml^-1) to 10^-3M (ca. 1000 IUA·ml^-1).
Calculations with Ilkovic Eqn. and Perrin Eqn. gave n≈4 for the first and the second waves of β-carotene, and n≈2 for those of vitamin A and its palmitate.
A preliminary study on vitamin A-acetate was also made under the same experimental conditions. The first and the second half-wave potentials of vitamin A-acetate were -1.26V and -1.66V vs. Hg, respectively, and the proportionalities between wave-heights and concentration were held in the same concentration range as above.

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Polarographic and spectrophotometric examinations on reduction-products obtained by electrolyses at controlled potentials led to the following interpretations in electrode-reductions of vitamin A and its related compounds in acetonitrile benzene mixture. Each of vitamin Aalcohol and palmitate is reduced to a structure of conjugated four doublebond system at the first step, and then, at the second step to a conjugated three doublebond system. The first reduction-products of β-carotene are not uniform and their structures cannot be clarified, but the second reduction-products show absorption spectra of conjugated three double-bond system, consequently, products by the third reduction-step would have a structure of conju-gated two double-bond.

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