Improved purification process of beta- and alpha-trypsin isoforms by ion-exchange chromatography

摘要

The purpose of this work was to improve the separation and yield of pure beta- and alpha-trypsin isoforms by ion-exchange chromatography and to characterize some physical-chemical properties of these isoforms. Purification of trypsin isoforms was performed by ion-exchange chromatography in 0.1 mol/L tris-HC buffer, pH 7.10 at 4 degrees C. The sample loading, salt concentration, flow rate and pH of mobile phase were varied to determine their effects on the resolution of the separation. The resolution was optimized mainly between beta- and alpha-trypsin. Pure isoforms were obtained by chromatographying 100 mg of commercial trypsin during seven days, yielding 51 mg of high purity beta-trypsin and 13 mg of alpha-trypsin partially pure, with small amounts of contaminating of psi-trypsin. Thus, time and resolution of purification were optimized yielding large amounts of pure active enzymes that are useful for several research areas and biotechnology.