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Ten insect-pathogenic species of Entomophthora showed wide variation in their ability to produce alkaline protease in surface culture. E. coronata, the most active producer, was selected for studies in submerged culture together with E. virulenta. All media tested appeared suitable for mycelial growth of these two organisms, but a liver medium was superior for the production of protease. The effect of the constituents of the liver medium upon yield was investigated. The lag between growth and the production of protease was 24 to 40 hr, and only very small amounts of protease were obtained from sonically treated mycelium. The pH values during growth rose from ranges of 4.5 to 7.5 in the initial medium to 7.2 to 7.9, and did not affect the final yields. The optimal temperature for the production of protease by E. coronata was 24 to 32 C, and good growth was observed at temperatures as low as 16 C. The process with E. coronata was scaled up to fermentors without a decrease in yield; 5 enzyme units/liter were obtained after approximately 33 hr. This corresponds to a maximal productivity of 0.45 enzyme unit per liter per hr during the protease-producing phase. The process was insensitive to changes in aeration rate. The liver in the medium was replaced by various agricultural by-products, meat scrap, rapeseed oil meal, cottonseed nutrients, milk powder, and meat hydrolysate, with approximately the same or higher yields of protease.

Protease Production by Species of Entomophthora