Purification of extracellular lipase from Pseudomonas aeruginosa.
Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s.
D001426:Bacterial Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000201:enzymology; D008049:Lipase / Q000302:isolation & purification*; D008070:Lipopolysaccharides / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D014304:Triolein / Q000378:metabolism
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D014304:Triolein; D008049:Lipase