Production, purification and partial characterization of the extracellular proteinase from Pseudomonas fluorescens 22F

Abstract

Abstract Pseudomonas fluorescens 22F was inoculated into tryptone-lactose medium, and incubated at 20 °C for 8 days. Proteinases were produced during the exponential and stationary phase. Cells were removed by centrifugation, and from the supernatant an extracellular proteinase from the bacteria was purified to electrophoretic homogeneity by ammonium sulfate precipitation, hydrophobic interaction chromatography, ultrafiltration and gel filtration. The increase in specific activity was 57-fold, the yield was 40%. The purified enzyme had an apparent molar mass of 52 kDa as determined by SDS-PAGE and 47 kDa as determined by gel filtration. The proteinase was characterized as an alkaline metalloproteinase with an optimum pH for activity on sodium caseinate of 9.5–10. The p I of the proteinase was 7.4. The optimum temperature of the enzyme was 50 °C; activity decreased rapidly at temperatures > 50 °C. With sodium caseinate concentrations up to 1%, Michaelis-Menten-type reaction kinetics were observed, whereas at higher concentrations substrate inhibition occurred. V max and K m were estimated at 1560 TNBS units mL −1 and 0.24% (0.11 mM) sodium caseinate, respectively.