Although 95 % of the enterotoxin B produced by Staphylococcus aureus appears during the latter part of the exponential phase of growth, growth per se is not necessary for toxin synthesis. A procedure is described whereby a concentrated suspension (at least 6 x 1010 cells per ml) of a 16-hr culture of S. aureus was found to be capable of producing toxin, without replication, when air and glucose were present. This technique allows the growth requirement to be separated from toxin formation. Although higher (100 ,ug/ml) concentrations of toxin appeared in the medium when nitrogen was present, lower levels (30 ,ug/ml) were produced in the absence of N-Z-amine A. Toxin production proceeded without any net increase in deoxyribonucleic acid, ribonucleic acid, or protein. Chloramphenicol did not inhibit toxin formation in a nitrogen-free medium. The optimal pH for toxin production in a nitrogen-free medium was 8.0 to 8.5; for synthesis in a medium where nitrogen was available, the optimal pH was 7.0 to 7.5. Increasing the rate of aeration increased toxin release during growth, but decreased the amount of toxin subsequently produced when the bacteria were resuspended. These results suggest the presence of a precursor pool in the cells collected after 16 hr of growth. MATERIALS AND METHODS Media. Medium 1, as described by Rosenwald et al. (12), contained 4.0% N-Z-amine type A (Sheffield Chemical, Norwich, N.Y.), 0.4% yeast extract (Difco), and 0.1% K2HPO4 in distilled water. Medium 2 contained 4.0% N-Z-amine type A and 0.1% K2HPO4. Medium 3 contained 0.5% glucose and 3.5% K2HPO4. This medium was considered the nitrogenfree medium. Culture propagation. Stock cultures of S. aureus S-6 were maintained on slants of Trypticase Soy 506
Effect of culture conditions on the production of D-galactose oxidase by Dactylium dendroides. Appl. Microbiol. 13:686-693. 1965.-The effects on enzyme production of inoculum size and age, medium composition, and culture conditions were studied in shake flasks and in a pilot-plant fermentor. Using a medium consisting of glucose, yeast extract, and inorganic salts in deionized water, we found that the addition of Cu++ was essential for the formation of active enzyme. Cultures grown in the absence of added copper produced an inactive enzyme protein which could be activated by 103 M Cu+. Thiamine fulfilled all requirements for exogenous vitamins for growth and enzyme production. Glucose concentrations higher than 1% markedly suppressed enzyme formation. The mycelium inactivated the enzyme on prolonged incubation of the culture. Mycelial autolysates and sonic extracts were found to contain a thermostable and slowly dialyzable galactose oxidase-inactivating factor. The experiments suggest that this factor operates as a chelating agent which forms complexes with the copper of the enzyme. Copper ions (10I8 M) prevented enzyme inactivation and restored activity to samples previously inactivated by this factor.
SummaryA substrain of Bacillus cereus 569/II produced above 10,000 units of penicillinase/ ml. when grown in a pilot-plant fermentor using a medium containing Casamino acids techn. (Difco) or N-Z-Amine type B (Sheffield), and salts. Simplified purification and concentration procedures give an overall yield of 5&65% enzyme. The freeze-dried enzyme preparation had a good storage stability in vacuumsealed ampules kept a t 4, 30, and 37°C. In vials tontaining air in the head space, partial inactivation occurred in two months a t 30 and 37°C. The freezedried preparation showed satisfactory performance in the production of yoghurt fermented milk.
The effects on enzyme production of inoculum size and age, medium composition, and culture conditions were studied in shake flasks and in a pilot-plant fermentor. Using a medium consisting of glucose, yeast extract, and inorganic salts in deionized water, we found that the addition of Cu ++ was essential for the formation of active enzyme. Cultures grown in the absence of added copper produced an inactive enzyme protein which could be activated by 10 -3 M Cu ++ . Thiamine fulfilled all requirements for exogenous vitamins for growth and enzyme production. Glucose concentrations higher than 1% markedly suppressed enzyme formation. The mycelium inactivated the enzyme on prolonged incubation of the culture. Mycelial autolysates and sonic extracts were found to contain a thermostable and slowly dialyzable galactose oxidase-inactivating factor. The experiments suggest that this factor operates as a chelating agent which forms complexes with the copper of the enzyme. Copper ions (10 -3 M) prevented enzyme inactivation and restored activity to samples previously inactivated by this factor.
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