Purification and characterization of alkaline lipase from a pseudomonas sp.isolate

Abstract

One bacterial strain was isolated from soil as alkaline lipase producing microoganism. Isolated strain was identified as Pseudomonas pseudoalcaligenes. When this strain was cultivated in medium containing soybean meal extract and olive oil, this secreted a large amount of alkaline lipase. This strain showed maximum lipase production at 32℃ and at pH 10.0 in 3days. Sodium acetate and sodium citrate was effective carbon source for lipase production, whereas glycerol decreased the lipase production remarkably. Soybean meal extract as nitrogen source was more effective than any others used. The lipase was inducibly produced in the presence of oil. The extracellular lipase was purified treating ammonium sulfateprecipitation and applying the chromatography on Sephadex G-150 and DEAE-Sephacel column. The lipase was purified from culture broth 3.6 fold giving a yield of 15.6% and purified enzyme was homogeneous on SDS-polyacrylamide gel electrophoresis. The molecular weight to the lipase was estimated to be 80,000-85,000 under reducing conditions. The purified enzyme was most active at pH 10.5 and 50℃. The lipase was stable between pH 8 and 11, while more than 70% of enzyme activity remained after incubation 65℃ for 30min. The activity was inhibited by iron ions. It was affected by bile salt.