Summary auto-generated
Researchers purified extracellular lipase from Pseudomonas aeruginosa EF2 grown in Tween 80-limited continuous culture using ultrafiltration and fast protein liquid chromatography (FPLC). The purified enzyme is a single-subunit protein with molecular weight of 29,000 Da and isoelectric point of 4.9. The lipase demonstrates substantial catalytic activity toward olive oil (triolein) with a turnover rate (kcat) of approximately 3,000 s⁻¹, and also exhibits weaker esterase activity on soluble substrates. Notably, this enzyme is more thermostable than other characterized Pseudomonas lipases, retaining activity longer at 60°C with a half-life of 17.5 minutes. The lipase shows regiospecificity for the 1,3-ester bonds of triolein and is resistant to metal chelation by EDTA, suggesting it does not require metal cofactors. N-terminal amino acid sequencing reveals significant similarity to lipases from other Pseudomonas species, indicating these enzymes are evolutionarily related. The weak inhibition by serine-active reagent 3,4-dichloroisocoumarin suggests either absence of an essential active-site serine or limited accessibility of this residue.
Key findings
- Ps. aeruginosa EF2 lipase is a 29-kDa single-subunit enzyme with high specific activity (6,606 LU/mg) and superior thermostability compared to other bacterial lipases
- The enzyme demonstrates regiospecificity for 1,3-oleyl residues of triolein and exhibits 8-fold higher activity on olive oil than on soluble esterase substrates
- Unlike most other Pseudomonas lipases, this enzyme is not inhibited by the metal chelator EDTA, indicating independence from divalent metal cofactors
- N-terminal amino acid sequencing shows striking similarity to multiple Pseudomonas lipase species, suggesting close evolutionary relationships among these enzymes
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Abstract
Summary: 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 (Mr29000, 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-1for 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 °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.