Purification and kinetics of extra and intracellular phospholipase-A of Salmonella enteritidis

Strain S-30 of Salmonella Enteritidis, isolated from goat liver, was used for the production of extracellular and intracellular phospholipase A. Crude fractions of the enzyme were precipitated by pure ammonium sulphate and then concentrated by dialyzing against polyethylene glycol before being applied to sephadex G-100 column on gel filtration chromatography. The specific activity of final pure extracellular and intracellular enzymes after gel filtration chromatography (25.1 units/mg; 11.2 units/mg, respectively) had increased by 46.48 and 40.17 fold, respectively in comparison to cell free supernatant. These purified extra and intracellular phospholipase A fractions were selected for further enzyme kinetic studies. The effect of various physical and chemical agents and variation in enzyme and substrate concentrations were studied using both enzyme fractions. Optimum activity of both enzyme fractions was at pH 7.0, temperature 40 degrees C and after 120 min incubation. The effect of different substrate concentrations on enzyme kinetics revealed that Kin values for extra and intracellular enzymes were 128 mu M and 370 mu M, respectively. The undiluted enzyme showed the maximum activity of both fractions and dilution gradually decreased the activity of enzyme fractions. All the organic solvents caused a decrease in the activity of both enzyme fractions. The addition of 1% surfactant to the enzyme assay mixture revealed that intracellular fraction was inactivated by SDS while triton X-100 enhanced its activity. Tween 20, tween 80 and lauricidin did not inhibit the enzyme activity of intracellular fraction to a significant extent. The extracellular phospholipase was not affected by triton X-100, tween-20 and tween 80 whereas other surfactants drastically reduced its activity. The addition of metal ions (100 mu g/ml) altered the enzyme activity of both enzyme fractions. Ca2+ stimulated the activity of both enzymes where as Zn2+ and Mg2+ did not show any marked change. Na+, K+ and Ba2+ showed a significant decrease in the activity while Mn2+ and Fe3+ were most detrimental. Both enzyme fractions remained active up to 4 h when stored at 4 degrees C, but there was a gradual decrease in the activity of both enzymes when stored at higher temperatures.