Isolation and characterization of an extracellular lipase from Pseudomonas tolaasii

Pseudomonas tolaasii, a plant pathogen, produced an extracellular, heat-stable lipase in reconstituted skim milk (10%, wv) which was purified by ion-exchange chromatography on DEAE-52 cellulose and gel filtration on Sephadex G-150. The purified enzyme showed one major protein peak on a TSK G3000 SW column, from which a native molecular weight of ~670 kDa was estimated. Maximum enzyme activity was at pH 7.0 and 35 °C when assayed on β-naphthyl caprylate. The enzyme lost no activity at pH 7.0 for 48 h at 21 °C and was more stable over the pH range 5–6.5 than in the range 8–11. The lipase was quite heat-stable (D-values ranged from 1171 s at 100 °C to 79 s at 140 °C; the Z-value in the temperature range 100–140 °C was ~31 °C and the corresponding activation energy (Ea) for inactivation was 94 kJ mol−1. The enzyme was inhibited more strongly by EDTA than by o-phenanthroline but not by phenylmethylsulphonylfluoride (PMSF) and was strongly reactivated by Ca2+. The lipase hydrolysed β-naphthyl esters in the order C8 > C6 > C4 > C2 > C10.