Incorporation of L-methionine-methyl-14C into gentamicins. II. Large-scale preparation of methyl-14C-gentamicins.

Methyl-14C-gentamicins were prepared from incubation of Micromonospora purpurea in the presence of added methyl-'4C-L-methionine, and products with high specific radioactivity were isolated in satisfactory yields. In the previous paper1), we have shown in shake flask fermentation a high and selective incorporation of radioactivity from methyl-14C-L-methionine into gentamicins. Consequently, we chose this isotope for the larger scale fermentative preparation and isolation of methyl-14C-gentamicins. Two batches of fermentation, in the presence of the isotope added at different levels, were run in a 30-liter fermenter. Each fermentation broth was worked up independently, and radioactive products were isolated and purified through a Dowex column. The resulting methyl-14C-gentamicin complex showed a high radiochemical purity. Materials and Methods Radioactive Isotope. Methyl-14C-L-methionine of specific activity 0.307 mCi/mg was purchased from New England Nuclear, Inc. Resins. IRC-50, Amberlite IRA-401S and Dowex 1 x 2 (50 ti 100 mesh) were purchased respectively from Rohm and Hass Co., Mallinckrodt Chemical Co., and J. T. Baker Chemical Co. Paper Chromatography. Paper chromatograms were developed on Whatman No. 1 paper in a descending system of the lower phase of chloroform-methanol-17 % ammonia (2: 1: 1 by volume). Measurement of Radioactivity and Bioassay. A liquid scintillation counter (Intertech. Inst. Inc., SL 30) was used for the measurement of radioactivity, a Nuclear Chicago scanner (Model 1002) for the determination of percent radioactivity incorporated into gentamicin components, and Staphylococcus aureus ATCC 6538 P for bioassay. Fermentation. In batch 1, 3.5 ml portion of frozen cells of Micromonospora purpurea (ATCC 15835) was inoculated into a 300-m1 flask containing 70 ml of inoculum medium (3 g beef extract, 5 g tryptose, l g dextrose, 24 g potato starch, 5 g yeast extract, and 2 g CaCO3 in 1 liter tap water). The flask was shaken for 48 hours at 35°C on a rotary shaker at 300 rpm. Twenty-five ml each of the culture broth was inoculated into two 2-liter flasks containing 500 ml of the same inoculum medium, and the flasks were shaken for 48 hours at 28°C at 300 rpm. The inoculum (750 ml), developed in two stages, were added into a 30-liter New Brunswick fermenter (Model MF-128 S) containing 15 liters of fermentation medium (50g potato dextrin, 5 g dextrose, 35 g soybean meal, 7 g CaCO3, 0.0013g Cobalt chloride, and 0.5 ml antifoam (Dow