Isolation and some properties of extracellular heat-stable lipases fromPseudomonas fluorescensstrain AFT 36

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Abstract
Aeration increased the growth and lipase production in milk by Pseudomonas fluorescens strain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented ∼ 71 % of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8·0 and 35 °C; it had a Km on tributyrin of 3·65 mM. and was inhibited by concentrations of substrate > ∼ 17 mM. The enzyme was very stable over the pH range 6–9; it was relatively heat-labile in phosphate buffer in the temperature range 60–80 °C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100–150 °C: the D values at 150 °C were ∼ 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the corresponding Z values in the temperature range 100–150 °C were ∼ 40 and ∼ 42 °C and the Ea for inactivation were 7·65 × 104 J mol-1 and 6·97 × 104 J mol-1 respectively.