PURIFICATION AND CHARACTERIZATION OF LIPASE ENZYME EXTRACTED FROM Bacillus licheniformis 14T LOCAL ISOLATE
SAAD HUSSEIN KHUDHAIR *
Ministry of Higher Education, Al-Karkh University of Science, College of Science, Iraq.
MELAD KHALAF MOHAMMED
Ministry of Higher Education, Department of Biology, College of Science, Wasit University, Iraq.
*Author to whom correspondence should be addressed.
Abstract
In a previous study, the Bacillus licheniformis 14T thermophilic isolate was selected as the best lipase-producing isolate. The current work focuses on conducting the enzyme purification process and studying some of its properties.
Different steps of purification were achieved including the ammonium sulfate precipitation, dialysis, and gel filtration by Sephadex G-100 column to recovery the lipase enzyme from the culture broth of Bacillus licheniformis 14T which incubated for 3 days in liquid mineral salts medium at 50°C, thereafter the culture broth was precipitated by various saturations of ammonium sulfate.
Results have shown that the maximum specific activity was exhibited when using 70% ammonium sulfate saturation, while before and after this saturation the specific activity was decreased. It was also found that the gel filtration step of the Sephadex column gave a specific activity of 48.14 Unit/mg protein with 4.2 times of purification fold, and the yield has reached 26.4%.
Optimum conditions such as time, temperature, and pH for enzyme activity were determined using purified lipase. Obtained results from these experiments exhibited that the highest enzyme activity (18.89 U/ml) appeared after 30 min. at 50°C in the reaction mixture with a pH 7.
Keywords: Lipase, purification, optimization and Bacillus licheniformis