Study On Precipitation Of Proteins From Ethanol-Aqueous Solution Using Compressed Carbon Dioxide

On the background of the separation and purification processes of high-value protein products, supercritical antisolvent (SAS) precipitation technology was integrated with conventional precipitation of organic solvent, thus a new separation process of precipitation of proteins in mixed solvent using compressed carbon dioxide was formed. The process is in accord with development of bioseparation by its characteristic of environment friendly and integration, and enjoys the value of potential application.Firstly, a set of experimental apparatus for SAS precipitation processing has been established. The influence of the cosolvent, ethanol, on the precipitation of target protein, from crude milk in the mixed solvent using near- and supercritical CO2 antisolvent was investigated. The results indicated that the precipitation of proteins in milk occurred above critical temperature (32@) and critical pressure (2.76MPa). Ethanol could strengthen the precipitation effect and the critical precipitation temperature and pressure fall with the increase of ethanol concentration, It may be due to the solubility of CO2 in aqueous solution increased by theaddition of ethanol. Therefore, it seemed that a double precipitation effects are provided with both CO2 and ethanol. The critical precipitation temperature and pressure decreased to 30@ and 0.98 MPa when the concentration of ethanol reached 30~40wt.%. The results also demonstrated that our experimental apparatus are operable and SAS process is feasibility to precipitate proteins in aqueous mixed solvent.Secondly, the precipitation phenomena and the influence enzyme activity of some proteins ( cellulase, a -amylase, acid proteinase and basic proteinase ), in ethanol-water mixed solvent at near- and supercritical CO2 pressure were investigated. Different enzymes showed different rules on the change of enzyme activities and phenomena of precipitation. Three preconditions of the precipitation of protein in ethanol-water mixed solvent at near- and supercritical C℃2 pressure were proposed. The operation conditions including temperature, C℃2 pressure, ethanol concentration of SAS process for cellulase, acid proteinase and basic proteinase have been obtained.Thirdly, the solubilities of total proteins of milk, cellulase, acid proteinase and basic proteinase have been measured under the experimental conditions by precipitation of protein in mixed solvent using near- and supercritical CO2.Fourthly, a new method of MESV was proposed and its ability for a prediction of phase equilibria for CO2- C2H5OH-H2O ternary system have been demonstrated. It can be applied to engineering calculation and obtain the data of phase equilibria under experimental conditions with good precision.Fifthly, a new model for correlating the solubility of total protein in liquid phase at near- and supercritical CO2 pressure has been established. Using the phase equilibria dada base on the MESV equation, the new model successfully correlates solubility of total protein from milk and cellulase in ethanol-aqueous solution, and showed good agreement with average absolute relative deviation (AARD) of 11.5% (303.15K), 18.1% (308.15 K) and 7.0% (303.15K), 7.7% (308.15 K) .Finally, the new process with precipitating of proteins from mixed solvent using compressed carbon dioxide was applied to purification of cellulase, acid proteinase and basic proteinase. A "Fractionation Precipitation Diagram" was introduced in the analysis of the purification factor (PF) of cellulase process. The results indicated that the maximum purification factor and the maximum yield reach 6.33 and 62% respectively. The process could also get rid of protein impurity from acid proteinase or basic proteinase mixture.On general, near- and supercritical CO2 precipitation technology is quite new and valuable for researching deeply in the future, which has potential and practical significance on separation and purification of protein.