胆固醇氧化酶的三相分离及其血清总胆固醇的测定

On Determination of Serum Total Cholesterol Cholesterol Oxidase by Three-Phase Partitioning and its Clinic Application

对一株胞外胆固醇氧化酶高产菌 COX48进行了三相分离,结果表明：最优分离条件为：硫酸铵异丙醇沉淀,硫酸铵饱和度为70%,异丙醇与发酵上清液的体积比为0.8∶1时,pH 值为9,在20℃下静置30 min,12000 r/min 离心10 min,纯化倍数4.5倍,回收率高达95%.酶动力学方法检测胆固醇氧化酶测定血清总胆固醇含量,其线性回归方程为 y =0.0064x＋0.0027,R 2=0.9654,线性范围0～7.16 mmol/L.结果表明了该酶具有作为临床诊断试剂的潜力,并为临床上利用酶动力学方法测定血清总胆固醇含量奠定了实验基础.

A strain of extracellular cholesterol oxidase high bacteria COX4-8 by the three phase separation technology has been studied in this paper.Results show that the pH of the supernatant is adjusted to 9.0. Ammonium sulfate （saturation 70%）is added and vortexed gently,followed by addition of isopropanol （80%）.The mixture is then incubated at 20 ℃ for 30min for the three-phase formation.These phases are then separated by centrifugation at 12 000 r/min for 10min.The results show that 95% recovery of activi-ty with 4.5 fold purification for cholesterol oxidase has been achieved.