脂蛋白酯酶的分离纯化及其部分性质研究

Study on Purification and Characterization of Lipoprotein Lipase From Candida Rugosa

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。

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 CL4B 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 puri- fied lipoprotein lipase is a single protein band detected by reduced / non-reduced SDS-PAGE. The purity is above 95% ana- lyzed 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.