Purification and properties of a heat-stable enzyme of Pseudomonas fluorescens Rm12 from raw milk

Pseudomonas fluorescens Rm12 is a kind of Psychrotrophic bacteria growing in cold raw milk. It produced an extracellular heat resistant protease with an estimated molecular weight of 45 kDa by size exclusion chromatography and SDS-PAGE under both reducing and non-reducing conditions. The enzyme, designated Ht13, was purified to electrophoretic homogeneity from the culture supernatant by sequentially using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography and size exclusion chromatography. The specific activity of the enzyme increased 115.5-folds. The optimum pH value and temperature of Ht13 were 7.5 and 40 A degrees C, respectively. Based on its biochemical characteristics, Ht13 can be included in the group of metalloproteases, which was inhibited by 1, 10-phenanthroline and EDTA but not by pepstatin A, chymostatin, STI, E-64, BBI, PMSF and pAPMSF. Mn2+ has positive effect on activity and can increased the heat resistance capability, while Ca2+ had a negligible effect. For the hydrolysis of azocasein, the Km was 0.012 mg mL(-1). The enzyme showed typical heat-stable behavior. After treatment of 160 A degrees C 20 s, the residual activity was 9%. The half-life of the enzyme at 160 A degrees C in buffer with Mn2+ was approximately 12 s. Among several main milk proteins, Ht13 can cleave alpha(s)-casein, beta-casein and kappa-casein. The sequence of 1st-16th amino acids of N-terminal was MSKVKDKAIVSAAQAS, which was same as those proteases excreted from some other P. fluorescens. However, their molecular weights, the activation ion and amino acid composition were different, suggesting Ht13 from P. fluorescens Rm12 is a novel protease.