Production and stabilization of a solvent-tolerant alkaline lipase from Pseudomonas pseudoalcaligenes F-111

Abstract

An extracellular alkaline lipase from Pseudomonas pseudoalcaligenes F-111 was identified by screening using a rhodamine B solid medium containing Na 2CO 3 by detection of orange fluorescent halos around the colony. For enzyme production, the inclusion of olive oil, soymeal, Triton X-100 and sodium ion in the medium was found to be essential. The optimal culture conditions for maximum production of alkaline lipase by P. pseudoalcaligenes F-111 were investigated and shown to be as follows: a culture medium composed of (g· l −1) olive oil, 4; soymeal, 10; Bacto-peptone, 15; yeast extract, 5; Triton X-100, 2; K 2HPO 4, 3; MgSO 4·7H 2O, 0.04; Na 2CO 3, 1.0; with an incubation period of 24 h at 30°C under shaking conditions. The production kinetics of the enzyme were also monitored in this study. Approximately 68% of the initial crude enzyme activity was lost during storage of at 4°C for 24 h. Calcium ion added to crude enzyme broth stabilized the initial enzyme activity. Furthermore, addition of calcium ion to broth resulted in the co-precipitation of the non-protein components and facilitated the enzyme purification. The purified alkaline lipase was activated even after incubation at 30°C in 40% water-immiscible solvents for 6 h.