碱性脂肪酶同工酶的分离纯化

Purification of Isoenzymes from PG37 Alkaline Lipase

对经疏水层析 (PhenylSepharoseCL 4L)和阴离子交换层析 (DEAE FastFlow)等部分纯化的样品 ,采用制备型聚丙烯酰胺凝胶电泳进一步将其分离纯化 ,获得了PG3 7碱性脂肪酶的 3种同工酶LipaseⅠ、LipaseⅡ和LipaseⅢ .用SDS 聚丙烯酰胺凝胶电泳和SephadexG 1 50凝胶色谱法分别测得同工酶的相对分子质量为 2 750 0和 2 90 0 0 .测定了LipaseⅠ的等电点为 5.4 ,动力学常数Km 和Vmax分别为 4 .54g/dL和 5.56μmol/ (min·μg) .

Partial purified lipase sample by Phenyl Sepharose CL 4B and DEAE Fast Flow from PG37 was separated further by preparative polyacrylamide gel electrophoresis. Three isoenzymes(Lipase Ⅰ、ⅡandⅢ ) bands were obfaind and had same molecular weight of 27 500 determined by SDS polyacrylamide gel electrophoresis or 29 000 by Sephadex G 150 gel filtration.These results indicated that lipase functions as a monomer ,and three isoenzymes had identical molecule and exists some difference in electric charge or glycosylation.The Lipase Ⅰshowed isoelectric point at pH 5.4.The K m and V max of the lipase Ⅰwere 4.54 g/dL and 5.56 μmol/(min·μg) respectively,as determined by Lineweaver Burk plot.