Research Initiation Award: Protein Separation Utilizing Utilizing Temperature-Sensitive Mecroemulsions

这项提案研究旨在开发一种提取技术，利用温度敏感的微乳液相从水溶液中回收蛋白质。该技术可作为大规模生物分离的主要回收步骤。通过利用液相传质，可以最大限度地减少一次分离的停留时间，并消除蛋白酶的作用。与两相水相系统相比，微乳液具有几个处理此类和萃取技术的优势，包括易于相分离(不需要资本密集型离心机)和低粘度材料(允许更快的传质)。通过使用非离子表面活性剂，可以利用温和的升温来以浓缩的形式回收蛋白质，而不是像目前离子表面活性剂反胶束系统所采用的那样利用化学加成。非离子温度敏感型微乳液需要结合亲和配体才能获得所需的高度特异性。一种模型酶的可逆酶抑制剂&gt；-半乳糖苷酶将被化学连接到非离子表面活性剂的环氧乙烷加合物上，以形成温度敏感的微乳液。~gt；-半乳糖苷酶在所配制的微乳液中的分配将作为温度的函数进行研究。该酶的溶解预计在较低温度(4℃)下发生，而破乳和酶恢复将在较高温度(35℃)下发生，远低于&gt；-半乳糖苷酶的变性温度(50%变性发生在57℃)。然后，该微乳液系统将被用于在实验室规模的连续分离装置上研究温度敏感型微乳液的处理优势。

The proposal research is directed at developing an extraction technique to recover proteins from an aqueous solution utilizing a temperature-sensitive microemulsion phase. The technique may be utilized as a primary recovery step for large-scale bioseparations. By utilizing liquid phase mass transfer, residence times for primary isolation can be minimized and protease action eliminated. Microemulsions possess several processing advantages over two-phase aqueous systems for such and extraction technique, including ease of phase disengagement (eliminating the need for capital- intensive centrifuges) and low viscosity materials (allowing for faster mass transfer). By utilizing a nonionic surfactant, an mild temperature increase can be utilized to recover the protein in a concentrated form instead of utilizing chemical addition as currently practiced for ionic surfactant reverse micellar systems. Nonionic temperature- sensitive microemulsions require the incorporation of an affinity ligand to obtain the high degree of specificity desired. A reversible inhibitor of a model enzyme, >- galactosidase, will be chemically attached to the ethylene oxide adduct of the nonionic surfactant employed to form the temperature-sensitive microemulsion. The partitioning of >- galactosidase into the formulated microemulsion will be investigated as a function of temperature. Solubilization of the enzyme is expected to occur at a low temperature (4>C), while demulsification and enzyme recovery will occur at a higher temperature (35>C) which is well below the denaturation temperature for >-galactosidase (50% denaturation occurs at 57>C). This microemulsion system will then be utilized to study the processing advantages of a temperature-sensitive microemulsion on a bench-scale continuous separation unit.