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Conditions for the production of thermostable proteases (alkaline proteinase and carboxypeptidase) by a thermophilic streptomycete (Streptomyces rectus var. proteolyticus) were investigated in 20-liter volumes. Proteinase production was affected by the concentration of defatted soybean powder, its optimum being 1.2% in medium containing 2.0% soluble starch. Relatively high concentration of phosphate (0.3 to 0.4% K(2)HPO(4)) was needed for the maximum enzyme production. A large inoculum size (5 to 10%) was favorable, but the inoculum age did not significantly influence the production. The yield increase of 20 to 30% was obtained by feeding of medium during fermentation. The optimal temperature for proteinase production was 50 C, at which the maximal rate of production was 66.2 proteinase units per ml per hr, whereas at 40 C it was 9.0. Production at 50 C reached the maximum within 12 to 16 hr. The optimal agitation rate was different for the production of proteinase and carboxypeptidase, 400 rev/min for the former and 500 rev/min for the latter. The optimal aeration for proteinase production was 20 to 30 liters/min at 400 rev/min, whereas carboxypeptidase production was not markedly affected by aeration rate. The possibility that carboxypeptidase production was correlated with the shear of mycelium was discussed.