Crosslinking of imprinted proteases to maintain a tailor-made substrate selectivity in aqueous solutions

A covalent method to keep imprinted properties of proteins stable in aqueous as well as in organic environment is described. To stabilize the ligand induced acceptance for D-configured substrates by alpha-chymotrypsin or subtilisin Carlsberg, each protein was first vinylated by acylation with itaconic anhydride. Then, the tailoring of the derivatized proteins by precipitation in the presence of N-acetyl-D-tryptophan from an aqueous medium with 1-propanol, and the subsequent crosslinking of the enzyme preparations with ethylene glycol dimethacrylate in cyclohexane was carried out. The crosslinked imprinted proteins (CLIPs) obtained catalyzed the hydrolysis of N-acetyl-D-tryptophan ethyl ester in phosphate buffer and the corresponding back reaction in cyclohexane, respectively. The repeated use of CLIP-alpha-chymotrypsin in D-ester hydrolysis was demonstrated. Furthermore, this particular CLIP-alpha-chymotrypsin showed no loss in activity when it subsequently was used in the synthesis of N-acetyl-D-tryptophan ethyl ester in cyclohexane again. In the case of D-ester hydrolysis the reaction rate acceleration (k(enz)/k(nonenz)) was in the same order of magnitude of about 10(4)-10(5) mM(-1) for the two CLIP-proteases. The results suggest that enzymes tailored by imprinting technique do not lose their induced "new" property in the presence of water when they are prepared according to the described vinylation/crosslinking method (CLIP technique).