Purification and properties of extracellular lipase from Pseudomonas aeruginosa EF2.
Extracellular lipase was purified from a Tween 80-limited continuous culture of Pseudomonas aeruginosa EF2 by ultrafiltration of the culture supernatant followed by anion-exchange and gel-filtration FPLC. The lipase was composed of a single subunit (Mr 29,000, pI 4.9), which was capable of a variable degree of aggregation, and which exhibited both lipase activity, measured with the insoluble substrate olive oil (predominantly triolein), and esterase activity, measured with the soluble substrates p-nitrophenyl acetate and Tween 80. Lipase activity was approximately eight times higher than either type of esterase activity (kcat approximately 3000 s-1 for the hydrolysis of olive oil). The enzyme showed a marked regiospecificity for the 1,3-oleyl residues of radiolabelled triolein, was relatively stable at moderate temperatures (exhibiting a biphasic loss of activity with an initial t1/2 of 17.5 min at 60 degrees C) and was very stable to freezing and thawing. Lipase activity was only weakly inhibited by the serine-active reagent 3,4-dichloroisocoumarin, and was not inhibited by the chelating agent EDTA (1 mM). The N-terminal amino acid sequence of the Ps. aeruginosa EF2 lipase showed a marked similarity to those of several other bacterial lipases.
Gilbert
E J
EJ
Department of Biochemistry, University of Leicester, UK.
Cornish
A
A
Jones
C W
CW
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D008049:Lipase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D008049:Lipase