Abstract: Trypsin is an important proteolytic enzyme，already used in large-scale processes by the detergent and dairy industries. Separation of trypsin directly from porcine pancreatic homogenate in aqueous two-phase systems (ATPS) of polyethylene glycol/ammonium sulfate was studied．The effects of polyethylene glycol 400 (PEG400) concentration，(NH4)2SO4 concentration，NaCl concentration，and pH value on the extraction of porcine pancreatic trypsin were investigated in terms of phase volume，partition coefficients of both enzyme activity and protein，and recovery．Orthogonal experiment was used to further analyze and optimize the separation of trypsin. The results indicated that the control of the concentration of (NH4)2SO4 and PEG400 were the most important for partitioning of trypsin．The NaCl addition had little effect on trypsin partition. Maximum partition coefficient of enzyme activity was 8.48 under the optimum separation conditions of 24% PEG400，20% (NH4)2SO4，and pH 4.2. The separation was successfully scaled up to 100 g system with the trypsin activity of 1，780 U/ mL at 20℃.

摘要： 采用聚乙二醇(PEG)/硫酸铵[(NH4)2SO4]双水相体系对猪胰蛋白酶分离进行了研究。通过综合考察酶分配系数、蛋白质分配系数、相比和回收率，探讨了PEG400质量分数、(NH4)2SO4质量分数、NaCl质量分数以及pH值对胰蛋白酶萃取的影响，并通过正交实验进一步优化实验条件，结果表明(NH4)2SO4质量分数和PEG浓度对胰蛋白酶的萃取影响大，在PEG400质量分数为24%、(NH4)2SO4质量分数为21%、pH值为4.2所组成的双水相体系下，可获得酶的高分配系数8.48，提取的胰蛋白酶活力达到1780 U/mL。