Unique response to heat of extracellular protease of Pseudomonas fluorescens M5.

Eight proteases selected from 38 cultures of psychrotrophic bacteria were subjected to heat stability tests. Cell-free filtrates of the broth in which the cultures were grown individually were heated to 40, 50, 60, and 70 degree C for 0, 15, 30, and 60 min. Six of the filtrates were less than 50% inactivated by heating at 40 degree C for 60 min, whereas the enzyme of Pseudomonas fluorescens M5 was inactivated completely by this treatment. Of the five most proteolytic cultures tested, including P. fluorescens M5, losses in protease activity ranged from 9 to 34% on heating at 70 degree C for 60 min. Purified M5 protease retained at least 75% of its activity over the pH range of 4.5 to 7.5. Electrophoresis of active M5 protease from four chromatographed fractions revealed two bands in each with approximate molecular weights of 35,000 and 45,000. Heating at 40 degree C did not alter mobility of either band. Reasons for lability at 40 degree C but stability at 50, 60, and 70 degree C are discussed. Complexation with casein, observed with another protease, was not a possible explanation for stability at 40 degree C.