木瓜蛋白酶在PEG-IDA-Fe^(3+)/(NH_4)_2SO_4双水相体系中亲和分配行为研究

Study of affinity partition behavior of papain in PEG-IDA-Fe^(3+)/(NH_4)_2SO_4 aqueous two-phase system

目的:研究木瓜蛋白酶在PEG-IDA-Fe3+/(NH4)2SO4亲和双水相体系中分配行为。方法:采用浊点法,构建双水相相图,考察各成相剂对木瓜蛋白酶活性的影响;研究不同PEG4000和(NH4)2SO4的质量分数、不同PEG-IDA-Fe3+的取代量时,木瓜蛋白酶在两相体系中分配行为的变化;用响应面法优化亲和双水相萃取木瓜蛋白酶的最佳分离条件;同时对木瓜蛋白酶在萃取体系中的结构进行表征。结果:当两相体系中p H6.9,PEG4000质量分数16%,(NH4)2SO4质量分数20%,PEG-IDA-Fe3+为3%时,木瓜蛋白酶的上相酶活性回收率达90.5%,蛋白分配系数达1.85,而其在PEG/(NH4)2SO4中的酶活回收率及蛋白分配系数都低于PEG-IDA-Fe3+/(NH4)2SO4亲和双水相体系。结论:PEG-IDA-Fe3+/(NH4)2SO亲和双水相系统能有效提高木瓜蛋白酶的分配效果,采用Fe3+螯合PEG成相剂的双水相萃取木瓜蛋白酶条件温和,不影响酶的特征结构。

Objective.To study the partion behavior of papain in affinity aqueous two-phase system（ATPS） which compossed of PEG,PEG-IDA-Fe3＋ and （NH4）2SO4. Methods.. Investigated the phase diagrams of these two type of ATPS. Considered the influence of reagents on papain activity,and the effect of PEG4000 and （NH4）2SO4 concentration,replacement rate of PEG-IDA-Fe3＋ on partition behavior were investigated. Optimization conditions of papain affinity partitioning were annalyzed using response surface methodology,and enzyme conformation before and after extraction was examinated. Results.The recovery（Y） and partition coefficient（Kp） of papain were achieved 90.5% and 1.85 respectively under the condition of pH6.9,PEG4000 16% （W/W）,ammonium sulfate 20% （W/W）, PEG-IDA-Fe3＋ 3% （w/w）. Conclusion： PEG-IDA-Fe3＋/（NH4）2SO4 system could significantly improve the recovery（Y） and partition coefficient（Kp） of papain. The condition of affinity aqueous two-phase system which compossed of PEG-IDA-Fe3＋ were mild,would not damage the structure features of papain.