Production of a tumor-inhibitory asparaginase by submerged fermentation with Serratia marcescens ATCC 60 was studied to ascertain optimal nutritional conditions for large-scale production leading to enzyme purification studies. Five strains of S. marcescens were screened in shake-flask studies and were found to produce 0.8 to 3.7 IU/ml 48 hr after inoculation. The requirements for asparaginase production with S. marcescens ATCC 60, the high producing strain, included the following: 4% autolyzed yeast extract medium (initial pH 5.0), an incubation temperature of 26 C, and limited aeration for a zero level of dissolved oxygen during the fermentation. Addition of various carbohydrates to the fermentation medium did not enhance yields. The peak cell population in the fermentation medium and the maximal asparaginase yields occurred simultaneously. Highest enzyme yields were found when the pH of the fermentation cycle rose to approximately 8.5. Yields of 4 IU of asparaginase/ml of cell suspension have been obtained consistently in 40 to 42 hr from 10-liter volumes (500 ml/4-liter bottle) produced on a reciprocating shaker. Scale-up to a 60-liter fermentor yielded 3.1 IU/ml in 35 hr.

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.

HEINEMANN, BERNARD (Bristol Laboratories, Syracuse, N.Y.), AND ALMA J. HOWARD. Induction of lambda-bacteriophage in Escherichia coli as a screening test for potential antitumor agents. Appl. Microbiol. 12:234-239. 1964.-A simple, rapid, quantitative test procedure to measure induction of phage production in lysogenic Escherichia coli K-12 (X) was described. This test was used in a study of 209 substances, including antibiotics, pyrimirines, purines, alkylating agents, thiols, amino acids, vitamins, and miscellaneous compounds. Minimal inducing concentrations for the 26 (12.5% of total tested) substances found to be effective inducing agents, as well as a listing of the inactive compounds, are presented. Since 21 of the 26 active agents reportedly have antineoplastic activity in rodent tumor systems, it was concluded that the induction test may provide a useful screen for the detection of potentially useful antitumor compounds.

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in p H and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.

A procedure has been developed whereby paper chromatograms of agents which induce X bacteriophage in Escherichia coli can be developed using bioautographs with a lysogenic test system. Well-defined plaque-forming zones are produced indicating the area on the paper chromatogram where the active inducing material can be located. A mixture of the bacteriophage-inducing antibiotic, mitomycin C, and the noninducing antibiotic, paromomycin, was resolved into its components on paper strips with an ethyl acetate-methanol solvent system. The location of both antibiotics could thus be readily observed. Antibacterial and inducing activities were found to be identical with a crude fermentation solid, NSC-B-158,791. The use of this procedure for resolution of multicomponent inducing activities in antibiotic beers and for characterization of active components which may be potential antitumor antibiotics is indicated.