反应详情

A suitable medium consists of cerelose (10.0 g), corn starch (5.0 g) , corn steep liquor (5.0 g) , N-Z Amine YTT (5.0 g), (Registered Trademark for enzymatic digest of casein, Humko Sheffield Chemical Co. Inc.), and cobalt chloride (0,002 g) which is suspended in one liter of water, pH adjusted to 7.0 with sodium hydroxide and dispensed (800 ml) to a Fernbach flask. After sterilization by autoclaving, flasks are inoculated with a slant growth suspension or frozen vegetative mycelia, then incubated with agitation on a shaker at about 200 rev/min and a temperature of 28° C. for seven to eight days. A 50 ml aliquot is then removed and the mycelia homogenized by a Teflon pestle tissue grinder followed by ultrasonic fragmentation. The fragmented mycelia then were centrifuged, washed free of medium, resuspended in 50 ml of fresh medium in a 300 ml Erlenmeyer flask, and incubated by shaking at 32° C. for two hours. Then, the cells were again centrifuged, washed free of medium and suspended in 50 ml of tris(hydroxymethyl)-aminomethane-malate buffer, pH 9.0. Aliquots of this suspension were treated with NTG at concentrations of 750 mcg/ml to 1500 mcg/ml for one hour on a rotary water bath shaker at 250 to 300 rev/min. and a temperature of 34° C. After treatment, the cells were centrifuged, washed free of mutagen and aliquots were serially diluted, plated onto a solid nutrient medium and the plates incubated at 28° C. until the colony forming units were of sufficient size for transferring to slants. A suitable medium for plates and slants is ATCC Medium No. 172 with N-Z Amine Type A (Humko Sheffield Chemical Co. Inc.) decreased to 1.0 g/l The inoculated slants were allowed to grow at 28° C. for 10 to 14 days after which time they were ready for testing. This was done by inoculating 300 ml Erlenmeyer flasks containing 25 ml of a suitable medium (one such medium contains cerelose, 45.0 g; soy flour 10.0 g; corn steep liquor, 15.0 g; MnSO4.H2O, 0.1 g; MgSO4.7H2O, 0.1 g; Cobalt chloride, 0.002 g; and calcium carbonate, 3.0 q; one liter of water and the pH of the medium is adjusted to 7.0). After sterilization by autoclaving for 30 minutes at 121° C., the flasks were inoculated with individual slant growth suspensions and incubated by shaking at 28° C. on a rotary shaker for 7 days. Mutant cultures were detected by examining methylisobutyl ketone extracts of harvested whole broths after spraying developed thin-layer chromatographic plates (silica gel) with vanillin reagent and heating at 100° C. for five minutes. The developing system was composed of 9 parts chloroform to 1 part methanol which gave Rf values of about 0.2 for CP-91,243, about 0.4 for CP-91,244 and about 0.7 for UK-58,852. Two mutant cultures were thus obtained which produce CP-91,243, CP-91,244 and UK-58,852. Those mutants are identified in the culture collection of Pfizer Inc. as FD 28455 and FD 28518. The morphological and cultural characteristics of the thus-obtained mutants are substantially those described for A. roseorufa ATCC 53666. The distinguishing characteristic of these mutants is their ability to produce a mixture of CP-91,243 and CP-91,244.

1. customTwo mutant cultures were thus obtained which