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

Abstract

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 α-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-α-chymotrypsin in d-ester hydrolysis was demonstrated. Furthermore, this particular CLIP-α-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 (kenz/knonenz) was in the same order of magnitude of about 104 −105 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).