Studies on the Production of Nucleosides by Microorganisms

Studies on Inosine Fermentation-Production of Inosine by Mutants of Bacillus Subtilis

“…pH was adjusted to 7.0 with KOH followed by autoclaving at 110° for 5 min. KH2P04 was not added, because inosine formation in the cell suspension was fairly repressed by this salt (8,9), and further it was observed that, after 30 hr of the growing culture, phosphate ion in the medium was measured less than 5 mg% as KH2P04. After being cultured for about 30 hr in growing culture medium, at which time the cell growth had reached nearly the stationary phase and inosine was increasing linearly with progress of culture time, CaC03 was separated by centrifugation at 800 rpm for 1 min, and then the cells were harvested by centrifugation at 10,000 rpm for 10 min.…”

“…After incubation for various periods, the reaction was stopped by placing the test tube in a boiling water for 5 min, and the products were quantitatively determined after their separation by paper electrophoresis as described previously (8). In all the cases, hypoxanthine, together with a small amount of inosine was formed, and the deaminating activity was indicated by the total amount of hypoxanthine and inosine.…”

“…After being cultured for 15 hr in various growing culture media, at which time the cells were increasing exponentially, the cells were harvested and washed cells were prepared by the same method as described above. To 5.0 ml of sterilized 0.1 N phosphate buffer (pH 6.5) or the cell suspension medium, both being supplemented with 150 mg% of adenine, 1.0 ml of the washed cells suspended in 0.9% KCi solution was added at about 0.3 of 0D502 (8), and incubation was carried out at 31.5° with or without reciprocal shaking.…”

“…The cells, precultured on nutrient agar slant at 31.5° for 24 hr, were harvested, suspended in the respective basal medium, and seeded in the growing culture medium at about 0.02 as OD562 (8). For non-proliferating washed cell suspensions, the following basal medium was used: 3.5% of glucose, 1.0% of NH4C1, 0.04/ of MgSO4.7H2O, 0.15% of KC1, and 0.001% of both FeSO4.7H2O and MnSO4 • H2O.…”

“…Quantitative determination of inosine was made either by paper chromatographic method (8) or by direct estimation of UV absorption values (OD25o~n~-OD2so~) of the culture medium after removing insoluble materials such as the cells, CaC03, etc., by centrifugation.…”