Selective Reverse Micellar Extraction of Three Proteins from Filtered Fermentation Broth Using Response Surface Methodology
Selective Reverse Micellar Extraction of Three Proteins from Filtered Fermentation Broth Using Response Surface Methodology
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使用响应面方法从过滤的发酵液中选择性反胶束提取三种蛋白质
DOI：
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发表时间：
2000
期刊：
影响因子：
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通讯作者：
D. Stuckey
中科院分区：
文献类型：
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作者：
S. Jarudilokkul;L. H. Poppenborg;D. Stuckey
三个系统参数[双（2-乙基己基）磺基琥珀酸酯（AOT）的浓度，pH值和温度]对细胞色素c，溶菌酶和核糖核酸酶A的选择性分离缓冲溶液和过滤的发酵液的影响进行了研究。结果发现，当从缓冲溶液中提取≥90%时，存在最小AOT浓度，并且该浓度取决于溶液的pH和蛋白质的疏水性。从过滤肉汤提取导致细胞色素c和溶菌酶的最低浓度的减少，而核糖核酸酶A，它是不变的。看来，某些肉汤成分作为助表面活性剂，并减少表面活性剂头基之间的电荷排斥，导致更小的胶束。这反过来导致pH范围缩小，但提取率没有改变。在与缓冲液、过滤的发酵液和分子量分级或提取的发酵液接触的反胶束相中测量含水量（W 0）。该数据得出的结论是，负责在最小AOT浓度和W 0的变化的肉汤成分是低分子量带正电荷的氨基酸和磷脂。最后，响应面法被用来优化关键系统参数，以最大限度地提高蛋白质提取，这种技术最大限度地减少了所需的实验。通过使用这些优化的条件，三种蛋白质的混合物可以从过滤的发酵液中以高收率（70-97%）和高纯度回收。此外，发现初始蛋白浓度不影响蛋白回收率。
The influence of three system parameters [bis(2-ethylhexyl)sulfosuccinate (AOT) concentration, pH, and temperature] on the selective separation of cytochrome c, lysozyme, and ribonuclease A from buffer solution and a filtered fermentation broth was examined. It was found that a minimal AOT concentration exists for ≥90% extraction from a buffer solution, and this concentration depends on the pH of solution and hydrophobicity of the protein. Extraction from filtered broth resulted in a reduction of the minimal concentration for both cytochrome c and lysozyme, while for ribonuclease A it was unchanged. It appears that certain broth constituents act as cosurfactants and reduce the charge repulsion between the surfactant headgroups leading to smaller micelles. This is turn led to a reduction in the pH range, although extraction yields were unaltered. The water content (W 0) was measured in a reverse micellar phase in contact with buffer, filtered fermentation broth, and molecular weight fractionated or extracted broth. This data led to the conclusion that the broth constituents responsible for changes in the minimal AOT concentration and W 0 were low molecular weight positively charged amino acids and phospholipids. Finally, response surface methodology was used to optimize key system parameters in order to maximize protein extraction, and this technique minimizes the experiments required. By using these optimized conditions, a mixture of the three proteins could be recovered from a filtered broth with high yields (70–97%) and high purity. Furthermore, the initial protein concentration was found not to influence protein recovery.