| On the background of the separation and purification of high-value protein products, precipitation assistant, ethanol, was introduced into the isoelectric precipitation process using compressed CO2 as a volatile acid, which was put forward recently. A precipitation process of proteins from ethanol-water solution using compressed CO2 as a volatile acid was presented, which was characterized as environment friendly and readily controllable. In a word, the process is in accord with the development of bioseparation, and has the potential to be applied in the downstream processing of proteins.Firstly, feasibility and rules of the protein precipitation process were preliminarilyinvestigated where alkaline proteinase 2709 were used as a model protein. The results indicated that the process could be used to remove impurity from the crude alkaline proteinase. The enzyme would decompose while pH value of the solution was lower. However, the enzyme remained stable at certain operating conditions. Pressure of CO2, initial pH of solution, enzyme concentration, and ethanol content have influences on the precipitation and the change of pH, while temperature has little effect. Processing time, Pressure of CO2, initial pH of solution, enzyme concentration, and ethanol content influences the stability of protein in the process.Secondly, the effects of temperature, initial pH, and ethanol content on the ternary system of compressed CO2-ethanol-water were determinated. It showed that the curves of pH as a function of pressure was little influenced by temperature and ethanol content. The pH-pressure curve of the CO2-ethanol-water ternary system was analyzed with the simplified pH calculation model, and the result indicated that ethanol decreased the activities of CO2 and water, which revealed the reasons why ethanol changed the pH-pressure curve little although it increased CO2 solubility in solution. The expansion results of ethanol-water solutions under compressed CO2 demonstrated that liquid phase expanded tinily when ethanol content was less than 30% (wt). It was concluded that the antisolvent effect of CO2 contributes little to proteins precipitation in such a system.Thirdly, the precipitation phenomena and pH-curves of the compressed COi-ethanol-water system with BSA or lysozyme were studied, and it was found that: BSA would precipitate when pH of solution decreased close to isoelectric point, and the precipitate would redissolve when pH decreased further. As to lysozyme, the pi, 11.0, couldn't be reached within the experimental range, so lysozyme couldn't be precipitated, but the precipitation of impurities in crude lysozyme with pi of about 5.0 could be obtained. All the results implicated that the precipitation mainly attributed to isoelectric precipitation, the action of antisolvent precipitation could not be a dominant factor in the experiments investigated.Fourthly, ethanol acts as a precipitating aid in the process. The data of proteins conformation implied that ethanol changed the conformation to some extent also. As a result, characteristic of proteins such as buffer capacity altered. Since ethanol could drive down the pH curve of the compressed CCh-ethanol-water-protein multi-component system, lower pH could be reached with the same protein concentration in this system than in compressed CCV water-protein system correspondingly, and higher protein concentration could be dealt with. However, in the experiments investigated the changes of conformation are reversible, such contents of ethanol have little negative effect on the enzymes activities. In the compressed COi-ethanol-water-protein multi-component system, the regulation of initial pH changed the adjustable pH extent, and improved pH controllability.Fifthly, the buffer capacity of proteins made pH of solution with proteins higher than that of solution without protein under the same pressure. It was found that the pH ratio of solution with protein to solution without protein remained constant with increasing of CC2 pressure. Ethanol reduced buffer capacit...
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