类芽孢杆菌胞外胆固醇氧化酶的分离纯化

Isolation and purification of extracellular cholesterol oxidase from Paenibacillus

【目的】探讨类芽孢杆菌产生胞外胆固醇氧化酶(COD)的分离、纯化方法,为其生产和应用提供技术支持。【方法】将类芽孢杆菌发酵液通过旋转蒸发浓缩、蔗糖透析浓缩、DEAE离子交换柱层析和凝胶层析,纯化目的蛋白,然后测定获得酶的基本酶学性质。【结果】类芽孢杆菌发酵液经DEAE离子交换柱层析和SephadexG-75凝胶柱层析后,纯化所得的COD比活力达6.83U/mg,酶活力回收为35.6%,纯化倍数为13.4倍。纯化酶经SDS-PAGE显示为均一条带,分子量约58kDa。将纯化的COD作用于不同浓度胆固醇,采用LineweaverBurk作图法,测得其Km=3.3×10-5mol/L。薄层层析结果显示,产物为胆甾-4-烯-3-酮,说明纯化所得酶是COD。【结论】纯化获得电泳纯类的芽孢杆菌胞外COD,其与底物亲和力较强,具有潜在的应用研究价值。

[Objective]The present study was conducted to investigate the isolation and purification method of extracellular cholesterol oxidase （COD） from Paenibacillus to provide technical support for Paenibacillus＇ production and application. [Method]The fermentation of Paenibacillus was treated through circumrotate evaporation evaporation, sucrose diallysis, DEAE-cellulose ion exchange column and Sephadex-G75 gel filtration column chromatography to purify target protein and determining its fundamental enzymological characteristics. [ Result ] After Paenibacillus fermentation broth was processed by DEAE-cellulose ion exchange column and Sephadex-G75 gel filtration column chromatography, the purified enzyme specific activity reached 6.83 U/mg, the recycling rate reached 35.6% and purification multiple was 13.4 times. The purified enzyme demonstrated to be a single band on SDS-PAGE, and its molecular weight was about 58 kDa. Applied to cholesterol in different concentrations, Km of the purified COD was 3.3×10^-5mol/L. using Lineweaver Burk mapping. The result of thin layer chromatography indicated that the product was cholesteric-4-olefin-3-ketone and the purified enzyme was COD. [Conclusion]Electrophoretical purity extracellular COD from Paenibacilhus was obtained through isolation and purification, and it is worthy of more application and studies for its positive affinity to cholesterol.