疏水层析法分离纯化脂肪酶的研究

PURIFICATION AND CHARACTERIZATION OF A LIPASE FROM CANDIDA LIPOLYTICA

采用疏水层析法对商业解脂假丝酵母的中性脂肪酶粗制剂进行了分离、纯化 ,实验以 Phenyl Sepharose为固定相 ,异丙醇 -哌嗪为流动相 ,经一次柱层析 ,从该粗酶中分离出的单一脂肪酶的比活可提高 1 0倍以上 .SDS-PAGE法测定其分子量约为 2 0 .8k D;管状等电聚焦法测其等电点约为 4 .50 .

Commerical lipase of Candida lipolytica was purified to homogeneity by a single chromatography on phenyl sepharose. The hydrophobic interaction chromatography conditions were designed and optimized. The activity of the enzyme was measured before and after purification; molecular weight and isoelectric point of purified enzyme were determined. Experimental results showed that:① With phenyl sepharose as a partitioner phase, the mixed solvent system of peperazine and 2 propanol as a mobile phase, the homogenous lipase was obtained directly from crude lipase of Candida lipolytica . ② By purificaiton, the specific activity of lipase could be raised 12 13 folds. ③This protein had a molecular weight of 20.8Kda as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. ④Isoelectric focusing in Coomassie Brilliant Blue R staining revealed a single protein band. The protein band is situated between the bands of 3.5 and 4.55, just below the band of 4.55; therefore the isoelectric point of the protein is about pH 4.50.