Box-Behnken响应面法优化[C_nPy]Cl(n=2,4,6)-K_2HPO_4双水相体系萃取木瓜蛋白酶  

Optimization of [C_nPy]Cl(n=2,4,6)-K_2HPO_4 ionic liquid aqueous two-phase system extraction of papain using response surface methodology with Box-Behnken design

作　　者：朱新儒 张海德[1] 余垒 ZHU Xin-ru;ZHANG Hai-de;YU Lei(College of Food Science and Engineering,Hainan University,Haikou 57022)

机构地区：[1]海南大学食品学院,海口570228

出　　处：《食品科技》2018年第9期290-297,共8页Food Science and Technology

基　　金：国家自然科学基金项目(31660444);2017年海南省普通高校研究生创新科研课题(Hys2017-34)

摘　　要：以[C_nPy]Cl(n=2,4,6)-K_2HPO_4离子液体双水相体系萃取木瓜蛋白酶。通过单因素实验研究了K2HPO4浓度、离子液体浓度、酶添加量、pH、温度对木瓜蛋白酶分配行为的影响。采用BoxBehnken响应面法优化萃取工艺,以酶活性回收率和分配系数的总评归一值(OD)为响应指标。最后运用SDS-PAGE电泳实验分析木瓜蛋白酶的纯化效果。结果表明:最佳萃取条件为0.35 g/mL K_2HPO_4,0.25 g/mL [C4Py]Cl,pH7.87,酶添加量2.17 mg/mL,温度30℃;在此条件下OD值高达0.8985。经双水相萃取后的木瓜蛋白酶的比活力为4120.17U/mg,与商品酶相比,纯化倍数达到1.88倍,且经SDS-PAGE电泳实验可观察到纯化后的酶杂条带明显减少。研究结果为该体系的萃取工艺参数优化以及高活力酶的制备提供理论指导。The papain was extracted by [CnPy]Cl（n=2,4,6）-K2HPO4 ionic liquid aqueous two-phase system. The effects of the concentration of K2 HPO4, concentration of ionic liquid, dosage of papain, pH and temperature on the partition behaviour of papain were investigated by the single factor experiment. BoxBehnken design of RSM was developed to optimize the extraction conditions. The overall desirability（OD） of the activity recovery of the enzyme and partition coefficient was considered as the response variable. Finally, the purification results of papain was analyzed by SDS-PAGE electrophoresis experiment. Results showed that： the optimized extraction conditions were 0.35 g/mL K2HPO4, 0.25 g/mL [C4Py]Cl, pH 7.87, dosage of papain 2.17 mg/mL, temperature 30 ℃. Under this optimized conditions, the OD value was 0.8985. The specific activity of papain extracted by the optimized ILATP system was 4120.17 U/mg. The purification fold reached 1.88 compared with that of commercial enzyme. It was obviously that through the SDS-PAGE electrophoresis experiment, the miscellaneous lane of the purified enzyme decreased. This work provided theoretical guidance for the optimized extraction process parameters and preparation of high activity enzymes.

关 键 词：离子液体双水相 萃取 木瓜蛋白酶 总评归一值 纯化倍数 

分 类 号：TS255.1[轻工技术与工程—农产品加工及贮藏工程]