Study on purification and characterization of Lipoprotein lipase from Candida rugosa

Candida rugosa was cultivated with inducement of substrate including olive oil. Crude extraction was obtained using the techniques of concentration (10,000 cut M.W.) by hollow fiber and ammonium sulfate precipitation. Lipoprotein lipase (LPL) was purified to electrophoretic homogeneity from liquid cultured cells of Candida rugosa through the following separation procedures which including DEAE-Sepharose F.F. chromatography and Phenyl Sepharose CL-4B chromatography. The specific activity of pure preparation reached 6.04U/mg, and the yield of enzyme activity is 6.6% with a 13.8-fold purification factor. The purified Lipoprotein lipase migrated as a single protein band on reduced/non-reduced SDS-PAGE. The purity analyzed by HPLC is above 95%. The native molecular weight of purified lipoprotein lipase was about 32.0kDa measured by Sephacryl S-200 chromatography and molecular weight of Single peptide chain under non-reduced SDS-PAGE conditions demonstrated 34.0kDa. The enzyme was found to have good pH stability and thermal stability, with 7.5 as the optimum pH of enzyme activity and 45°C as the optimum temperature. The Km of purified Lipoprotein Lipase is 3.4×10^-4 mol/L at pH 7.5 and 45.0V.