The Kinetic Separation of Protein Mixtures Using Reverse Micelles
The Kinetic Separation of Protein Mixtures Using Reverse Micelles
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使用反胶束对蛋白质混合物进行动力学分离
DOI：
10.1081/ss-100100196
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发表时间：
2000
影响因子：
2.8
通讯作者：
D. Stuckey
中科院分区：
文献类型：
--
作者：
L. H. Poppenborg;Aristidis A. Brillis;D. Stuckey
近年来，对可以连续分离、浓缩和纯化蛋白质并且容易放大的经济有效的方法的商业兴趣显著增加。已经发现使用反胶束有机溶剂的液-液萃取具有这种潜力。本工作的总体目标是研究使用反胶束的蛋白质混合物的动力学分离。在搅拌槽（刘易斯槽）中研究了溶菌酶、细胞色素c和核糖核酸酶A的同时正萃取动力学。此外，Graesser接触器用于从缓冲溶液以及从已添加细胞色素c的稀释鸡蛋白色中同时提取溶菌酶和细胞色素c。的刘易斯细胞实验表明，总的传质系数，k 0，同时提取的蛋白质在pH 5.5的相互影响是显着的，在某些情况下相当大。细胞色素c或核糖核酸酶A的存在有助于防止溶菌酶沉淀在界面处，而细胞色素c的加入改变了溶菌酶的pH依赖性模式，并且溶菌酶的存在降低了细胞色素c的k 0值。使用Graesser接触器可以实现溶菌酶和细胞色素c的动力学分离，并且在低转子速度（2- 3rpm）、低温（4°C）和接近两种蛋白质的pI的pH（pH 10）下最大化; 30分钟后，约80%的溶菌酶和仅10%的细胞色素c被提取到反胶束相中。在Graesser接触器中测量的萃取速率不同于在刘易斯池中测量的萃取速率，并且该观察结果表明反胶束转移机制的不同步骤控制转移。使用鸡蛋白色（溶菌酶的天然来源），溶菌酶和添加的细胞色素c的动力学与缓冲溶液不同;细胞色素c提取更快，使动力学分离更困难。此外，用鸡蛋白色形成稳定的乳液，这在工业应用中是不希望的，因为它导致额外的分离步骤。
Commercial interest in cost-effective methods that can separate, concentrate, and purify proteins continuously, and be easily scaled-up, has increased markedly in recent years. Liquid–liquid extraction using reverse micellar organic solvents has been found to have this potential. The overall objective of this work was to investigate the kinetic separation of protein mixtures using reverse micelles. The kinetics of simultaneous forward extraction of lysozyme, cytochrome c, and ribonuclease A were investigated in a stirred cell (Lewis cell). In addition, a Graesser contactor was used for the simultaneous extraction of lysozyme and cytochrome c from a buffer solution as well as from diluted hen egg white to which cytochrome c had been added. The Lewis cell experiments showed that the mutual effect on the overall mass transfer coefficient, k 0, of the three simultaneously extracted proteins at pH 5.5 was significant, and in some cases quite large. The presence of cytochrome c or ribonuclease A helped to prevent lysozyme precipitation at the interface, while the addition of cytochrome c altered the pH dependency pattern of lysozyme, and the presence of lysozyme reduced the k 0-value of cytochrome c. The kinetic separation of lysozyme and cytochrome c was possible with a Graesser contactor, and maximized at low rotor speed (2–3 rpm), low temperature (4°C), and a pH close to the pI of both proteins (pH 10); after 30 minutes about 80% of the lysozyme and only 10% of the cytochrome c were extracted into the reverse micellar phase. The extraction rate measured in the Graesser contactor differed from that measured in the Lewis cell, and this observation indicates that different steps of the reverse micellar transfer mechanism are controlling the transfer. Using hen egg white (the natural source of lysozyme), the kinetics of lysozyme and added cytochrome c were different from those with buffer solutions; cytochrome c extraction was faster, making kinetic separation more difficult. In addition, a stable emulsion formed with hen egg white, which is not desirable in an industrial application since it results in an additional separation step.