Heat-stable protease from Pseudomonas fluorescens T16: purification by affinity column chromatography and characterization.
A heat-stable extracellular protease from Pseudomonas fluorescens T16, a psychrotroph, was purified by affinity column chromatography on a carbobenzoxy-D-phenylalanine-triethylene tetramine-Sepharose-4B column. The purified enzyme is a monomer with a molecular weight of 38,905 +/- 2,000. In an analytical ultracentrifuge, the Schlieren profile revealed a single symmetrical peak. The sedimentation coefficient was estimated to be 3.93S. Alpha-casein was the preferred substrate, with a Km of 0.05 mM. Heating crude enzyme and purified enzyme in buffer at 50, 90, and 120 degrees C resulted in a rapid initial loss of more than 50% of the initial activity followed by a gradual inactivation which exhibited first-order kinetics. The activation energy for the hydrolysis of casein was calculated to be 3.2 kcal/mol (13.4 kJ/mol).
D000818:Animals; D002364:Caseins / Q000378:metabolism; D002846:Chromatography, Affinity; D006358:Hot Temperature; D008892:Milk / Q000382:microbiology; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000302:isolation & purification; D013379:Substrate Specificity
D002364:Caseins; D010447:Peptide Hydrolases