脂蛋白酯酶的分离纯化及其部分性质研究

将Candida rugosa用橄榄油诱导培养,所得上清液依次经过截留分子量10 000中空纤维柱浓缩,85%硫酸铵沉淀,然后经过DEAE-Sepharose F.F.离子柱和Phenyl Sepharose CL-4B柱进行交换层析和疏水层析,得到了活力达6.04 U/mg的脂蛋白酯酶,纯化倍数和回收率分别为13.8倍6.6%。所得纯化酶经还原和非还原SDS-PAGE分析为单一蛋白染色带,HPLC显示纯度为95%以上。该酶的最适温度为40℃～50℃之间,最适pH为7.5,具有较强的热稳定性和酸碱稳定性,在最适温度和pH条件下该酶Km值为3.4×10-4mol/L。

Study on Purification and Characterization of Lipoprotein Lipase From Candida Rugosa

Candida rugosa is cultivated with inducement of substrate including olive oil.The obtained supernatant is treated by the ways of hollow fiber concentration(10,000cut M.W.) and 85%(NH4)SO4,then the crude lipoprotein lipase is purified by DEAE-Sepharose F.F.chromatography and Phenyl Sepharose CL-4B chromatography.The pure enzyme is obtained which reaches 6.04U/mg,and the yield of enzyme activity is 6.6% with a 13.8-fold purification factor.The purified lipoprotein lipase is a single protein band detected by reduced / non-reduced SDS-PAGE.The purity is above 95% analyzed by HPLC.The enzyme is found to have good pH stability and thermal stability,the optimum pH is 7.5 and the optimum temperature is 40℃~50℃.The Km of purified lipoprotein lipase is 3.4×10-4 mol/L at the optimum temperature and pH.