Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2

Abstract

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomunas ueruginusa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5,35.5 "C, at a dilution rate of 0.04 h-l . Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-' h-* (1 LU equalled 1 p o l fatty acid released min-l). Esterase activity, measured with pnitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.

Purification and properties of extracellular lipase from Pseudomonas aeruginosa EF2

“…aeruginosa EF2 was isolated, i.e. at a relatively high temperature (50 "C) in an olive oil minimal medium supplemented with the chelating agent nitrilotriacetic acid (Gilbert et al, 1991).…”

“…Pseudomonas aeruginosa EF2 was grown in continuous culture under Tween 80 (polyoxyethylene sorbitan monooleate) limitation (dilution rate, D 0-05 h-l, pH 6.5, 37 "C) as described previously (Gilbert et al, 1991).…”

“…Discontinuous SDS-PAGE and determination of protein using the Bradford method was carried out as described previously (Silman et al, 1989;Gilbert et al, 1991). The gels were stained for protein with Kenacid blue R, then destained and, where appropriate, scanned at 633 nm using an LKB laser densitometer linked to a recording integrator.…”

“…Lipase activity was assayed titrimetrically at pH 9.0 with a standard olive oil emulsion as substrate (Sigma), and esterase activity was assayed spectrophotometrically at pH 7.0 with pnitrophenyl acetate as substrate, both at 37 "C, as described previously (Gilbert et al, 1991). Esterase activity was also measured titrimetrically at pH 9.0 and 37 "C, as described for lipase, except that the olive oil emulsion was replaced by 20% (v/v) Tween 80 as substrate.…”

“…The physiological regulation of lipase expression by this organism, and the optimization of lipase production during growth in continuous culture, have recently been reported (accompanying paper : Gilbert et al, 1991). This paper describes the purification of the Ps.…”