为了改进辣根过氧化物酶的提纯方法，在双水相萃取的基础上使用聚乙二醇（ PEG6000）在高饱和度（ NH4）2 SO4中沉淀辣根提取液中的过氧化物酶，利用磷酸盐溶液复溶解共沉淀物形成的双水相萃取体系能高效回收高纯度酶蛋白。研究了pH、PEG浓度和（ NH4）2 SO4饱和度对酶活性的影响，并考察不同液固比、pH和NaCl浓度对目标酶在双水相体系中的分配行为，并通过响应面法优化出最优萃取条件。结果表明：在液固比0．3 mL／g、pH 7．02和NaCl 42 g／L的优化萃取条件下，辣根过氧化物酶回收率达88．1％，酶纯度较优化前提高了21．7倍。该方法的建立对于微量蛋白质的高效率提纯具有重量的参考价值。%To improve the extraction and purity of horseradish peroxidase ( HRP ) by aqueous two⁃phase extraction, polyethylene glycol( PEG6000) was used to precipitate the protein enriched with HRP in the high saturation ammonium sulfate. Then, an aqueous two phase system of PEG/phosphate was formed based on the Co⁃precipitation resolution to extracted higher composition HRP . The influence of different pH,PEG concentration,( NH4 ) 2 SO4 saturation on the enzyme activity and the effect of different liquid⁃solid ratio, NaCl and pH concentration on the distribution of the target enzyme in aqueous two⁃phase system were explored by single factor experiments. The extraction conditions were optimized by response surface method. The optimal extraction conditions were:liquid⁃solid ratio 0. 3 mL/g, pH of phosphate buffer saline 7. 02,NaCl 42 g/L.Under the optimal extraction conditions,the recovery rate of the enzyme activity was 88. 1%,and the purity was increased by 21. 7 times. The establishment of this method is of great significance for improving the economic benefit of the biological downstream process.
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