Fast Purification Of Biopolymers By Chromatographic Pie

Chromatographic pie is applied successfully in the separation and purification of the down-stream products in biotechnology. In the thesis, the chromatographic pie was used in the fast separation and purification of the native proteins in the organizations of animals and the industrial grade enzyme. High purity target proteins were obtained in a relatively short time. The chromatographic pie was also characterized by its low pressure during the separation process and rapid, high recovery of proteins. The thesis includes five parts as follows:1. Review of literature2. Purification of human serum albumin (HSA)The optimization of the kinds, concentration, buffer, pH and flow rate of mobile phase by using chromatographic pie was investigated in this thesis. In the optimized conditions, the separation of standard protein was compared in different flow rates. From the results, it indicates that the chromatographic pie packed with small particles still has great separate ability using high flow rate, the rapid purification of HSA was also been done in 10 minutes. The purity was more than 88% and the recovery was over 65%.3. Purification of lysozyme(Lys) in egg whiteFirstly, Lys was separated and purified by using high performance hydrophobic interaction chromatography (HPHIC). The retention time of Lys is near to many proteins in egg white, so Lys can not be separated completely by HIC. In this thesis, Ion Exchange chromatography was used to purity Lys as a new method. The separation was proved to be effective and the recovery of bioactivity reached 94%. The method was a rapid and effective separation way.4. Purification of a-AmylaseIndustrial grade a-Amylase was purified by HIC and the optimized condition was discussed in this chapter. In a give condition, the bioactivity recovery of a-Amylase can reach to 91% and specific activity can reach 120U/mg. This is a simple, rapid andeffective separation method for cc-Amylase. And this procedure can be used to purity not only Industrial grade a-Amylase, but those from other sources. 4. Purification of cytochrome(Cyt-C)Cytochrome-C in pig heart was purified by HPHIC and HPIEC, respectively in this thesis. The great hydrophilic ability of Cyt-C made it can be separated effectively from other proteins in pig heart and the purity can reach to 90%. In addition, pI of Cyt-C is 10.8, it impossible to retain well in cation exchange column. In the optimized conditions, the purity of Cyt-C can reach to 85%.