Characterization of a heat-stable protease of Pseudomonas fluorescens P26.
A heat-stable, extracellular proteolytic enzyme was isolated from Pseudomonas fluorescens P26. Brain heart infusion broth (Fisher Scientific Co.), pH 7.5, and incubation at 21 C provided the optimal conditions for bacterial growth for enzyme production. The organism had a D value of 2.6 min at 62.8 C (145 F). The enzyme, however, was quite heat-stable, requiring 15 hr at 62.8 C, 8 hr at 71.4 C, and 9 min at 121 C for complete inactivation. Milk, whey, and casein each had a protective effect on the enzyme against heat inactivation. Purification was accomplished by growth of organisms in broth, centrifugation, sterilization by filtration, ammonium sulfate precipitation (55% saturation), dialysis (against six changes of water), and protein separation by passage through a Sephadex G-100 column. Ultracentrifugation revealed a single band with a sedimentation coefficient of 1.51 which suggested a molecular weight of approximately 23,000. As little as 0.2 unit of the purified enzyme caused detectable flavor defects during 30 days of storage at 4 C, and the Hull test for liberation of tyrosine compared favorably in sensitivity with the sensory method in milk.
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D000818:Animals; D002364:Caseins; D002474:Cell-Free System; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration; D005516:Food Microbiology; D005519:Food Preservation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008892:Milk; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D011551:Pseudomonas fluorescens / Q000201:enzymology; D013053:Spectrophotometry; D013649:Taste; D014461:Ultracentrifugation
D000596:Amino Acids; D002364:Caseins; D010447:Peptide Hydrolases; D000645:Ammonium Sulfate