LIPASE ACTIVITY AND GENE CLONING OF ACINETOBACTER-CALCOACETICUS LP009

The production of lipase from Acinetobacter calcoaceticos LP009, a bac terium isolated from raw milk, was found to be best induced by Tween-8 0 at 1.0% concentration. It was efficiently secreted, and only a minut e amount of activity was detected at the cell surface and intracellula rly. A. calcoaceticus LP009 lipase exhibited maximum activity at pH 7. 0 and 50 degrees C, and was relatively stable upon storage at pH 5.0 t o 7.0 and at 4, 30, or 37 degrees C. The enzyme was found to be inacti vated by EDTA suggesting that it was a metalloenzyme. Its activity was reduced by less than 20% with the addition of various ions to reactio n mixtures, but long storage with them caused approximately 50% reduct ion in subsequent reactions under standard conditions. By contrast, th e addition of Fe3+ enhanced activity. The enzyme was highly stable upo n storage with 0.1% of Triton X-100, Tween-80 or Tween-20, but highly unstable with various organic solvents tested. PMSF, a serine enzyme i nhibitor, and P-mercaptoethanol, a reducing agent, did not affect enzy me activity. After extraction and transfer, the lipase gene was effici ently expressed in recombinant Aeromonas sobria. This recombinant stra in was shown to have increased hydrolyzing efficiency and have high po tential for lipid-rich wastewater treatment.