Heat-stable protease from Pseudomonas fluorescens T16: Purification by affinity column chromatography and characterization

Abstract

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 K(m) of 0.05 mM. Heating crude enzyme and purified enzyme in buffer at 50, 90, and 120°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).