Jiang Zhi-Guo, Zhang Hai-De, Wang Wei-Tao
College of Food Science, Hainan University, Haikou, P. R. China.
J Sep Sci. 2015 May;38(8):1426-32. doi: 10.1002/jssc.201401371. Epub 2015 Mar 11.
A simple and inexpensive aqueous two-phase affinity partitioning system using metal ligands was introduced to improve the selectivity of commercial papain extraction. Polyethylene glycol 4000 was first activated using epichlorohydrin, then it was covalently linked to iminodiacetic acid. Finally, the specific metal ligand Cu(2+) was attached to the polyethylene glycol-iminodiacetic acid. The chelated Cu(2+) content was measured by atomic absorption spectrometry as 0.88 mol/mol (polyethylene glycol). The effects on the purification at different conditions, including polyethylene glycol molecular weight (2000, 4000, and 6000), concentration of phase-forming components (polyethylene glycol 12-20% w/w and sodium sulfate 12-20%, w/w), metal ligand type, and concentration, system pH and the commercial papain loading on papain extraction, were systematically studied. Under optimum conditions of the system, i.e. 18% w/w sodium sulfate, 18% w/w polyethylene glycol 4000, 1% w/w polyethylene glycol-iminodiacetic acid-Cu(2+) and pH 7, a maximum yield of 90.3% and a degree of purification of 3.6-fold were obtained. Compared to aqueous two phase extraction without ligands, affinity partitioning was found to be an effective technique for the purification of commercial papain with higher extraction efficiency and degree of purification.
引入了一种使用金属配体的简单且廉价的水相双相亲和分配系统，以提高商业木瓜蛋白酶提取的选择性。首先使用环氧氯丙烷活化聚乙二醇4000，然后将其与亚氨基二乙酸共价连接。最后，将特定的金属配体Cu(2+)连接到聚乙二醇-亚氨基二乙酸上。通过原子吸收光谱法测得螯合的Cu(2+)含量为0.88摩尔/摩尔（聚乙二醇）。系统地研究了不同条件对纯化的影响，包括聚乙二醇分子量（2000、4000和6000）、相形成组分的浓度（聚乙二醇12 - 20% w/w和硫酸钠12 - 20%，w/w）、金属配体类型和浓度、系统pH值以及商业木瓜蛋白酶在木瓜蛋白酶提取中的负载量。在系统的最佳条件下，即18% w/w硫酸钠、18% w/w聚乙二醇4000、1% w/w聚乙二醇-亚氨基二乙酸-Cu(2+)和pH 7，获得了90.3%的最大产率和3.6倍的纯化度。与没有配体的水相双相萃取相比，亲和分配被发现是一种用于纯化商业木瓜蛋白酶的有效技术，具有更高的提取效率和纯化度。
