皱褶假丝酵母脂蛋白脂酶的分离纯化及酶学性质的研究

目的从皱褶假丝酵母的发酵液中纯化脂蛋白脂酶,并对其酶学性质进行研究。方法利用硫酸铵盐析、DEAE-Sepharose Fast Flow和Phenyl Sepharose CL-4B层析等方法进行纯化,利用SDS-PAGE、PAGE和HPLC法进行鉴定。结果获得的脂蛋白脂酶为单一组分,相对分子质量为34 kDa;此酶在50℃、pH6～9时保持稳定;在最适条件下测得其米氏常数(Km)为3.4×10-4mol·L-1;用等电点沉淀法测得脂蛋白脂酶的pI=5.5;Hg+、Cu2+、Ag+和SDS是脂蛋白脂酶的抑制剂,Mg2+和Triton X-100对此酶有激活作用。结论文中方法获得了皱褶假丝酵母脂蛋白脂酶的酶学性质,对此酶在食品、医药和生物技术等领域的应用提供了理论支持。

Purification and characterization of the lipoprotein lipase from Candida rugose

OBJECTIVE To purify lipoprotein lipase from the fermentation supernatant of Candida rugose,and characterize this lipase by the Candida rugose.METHODS Lipoprotein lipase was purified by ammonium sulfate precipitation,DEAE-Sepharose Fast Flow and phenyl Sepharose CL-4B chromatographies.The purity was identified by SDS-PAGE,PAGE and HPLC.RESULTS A single protein fraction with lipoprotein lipase activity was obtained from fermentation supernatant.The lipoprotein lipase with relative molecular weight of 34 kDa was tested by SDS-PAGE and gel filtration.Dynamic analysis showed that the lipoprotein lipase from Candida rugose was stable at 50 ℃ and at pH6.0-9.0.The Michaelis-menten constant(K_m) of lipoprotein lipase determined by Linewear-Burk plots was 3.4×10~-4 mol·L~-1.The pI of lipoprotein lipase determined by pI precipitation method was 5.5.Hg~+,Cu~2+,Ag~+ and SDS were inhibitors of lipoprotein lipase,but Mg~2+,Triton X-100 were its activators.CONCLUSION These properties of Candida rugosa lipoprotein lipase in present work provided theoretical support for medical application.