Trypsin Purification by p-Aminobenzamidine Immobilized on Macroporous Chitosan Membranes

Membranes with suitable mechanical properties, chemical stability, and macroporosity which have a selectivity for trypsin were prepared by coupling a ligand (p-aminobenzamidine) to cross-linked chitosan membranes via a spacer (succinic acid). The chitosan membranes were cross-linked with ethylene glycol diglycidyl ether in order to avoid their dissolution in acidic media. The cross-linked chitosan membranes were then covalently coupled with succinic anhydride, at ambient temperature, to obtain hydrophilic spacers with carboxyl terminals. The amino groups of the chitosan membranes which have not reacted with the succinic anhydride were blocked with acetic anhydride to avoid the nonspecific interactions. Finally the ligand p-aminobenzamidine (PAB) was incorporated via the coupling between the carboxyl groups of the succinic acid and the amino groups of PAB, using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) as the activating agent. A PAB density of 2.7 × 10-3 mol/g of chitosan was achieved. Since the PAB−chitosan membranes have a much higher affinity toward trypsin than toward chymotrypsin, they have been employed for the purification of crude trypsin solutions. A purification factor > 7, a specific activity > 9500 BAEE units/mg, and activity yields between 60 and 80% were obtained. These results suggest that the PAB−chitosan affinity membrane can be efficient for trypsin purification.