Production of Proteases by Psychrotrophic Microorganisms

¹ Purdue University, West Lafayette, IN 47907

Six milk-derived psychrotrophic microbial cultures were screened for the ability to grow at refrigerated temperatures and produce proteases in reconstituted skim milk. Of these, two cultures, Pseudomonas fluorescens M3/6 and Pseudomonas fragi K122, produced extracellular protease(s) beginning 7 d postinoculation when the cultures had entered late log or early stationary phases of growth. Further work with these two cultures showed that intracellular proteases were present after only 20-h incubation, before detection of the extracellular proteases. Using H-D-valyl-(L-leucyl-L-lysyl-4-nitroanilide (S-2251), a sensitive substrate for plasmin activity, P. fluorescens was shown to have greater intracellular proteolytic activity than extracellular activity at 20 h of incubation. The intracellular enzyme activity remained constant while the extracellular and periplasmic activities increased over the remaining 6-d incubation period. The proteases in crude extracellular extracts from both cultures were characterized and were heat stable with broad temperature (7 to 52°C) and pH (pH 5.5 to 8.5) ranges for activity and were inhibited by the metal chelator, EDTA, indicating that they were metalloproteases.

Key Words: psychrotroph • enzyme • protease

Submitted on November 8, 1990
Accepted on May 20, 1991

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