VOLTAGE-ASSISTED PEPTIDE-SYNTHESIS IN AQUEOUS-SOLUTION BY ALPHA-CHYMOTRYPSIN IMMOBILIZED IN POLYPYRROLE MATRIX

Potential-assisted enzymatic peptide synthesis in aqueous solution has been performed by cx-chymotrypsin immobilized in conducting polymer p olypyrrole matrix. Chymotrypsin is electrochemically adsorbed on a Pt electrode, and then a thin polypyrrole membrane is electrochemically p repared on the enzyme layer. The enzyme/polypyrrole on an electrode re tains chymotrypsin activity and the equilibrium of the enzymatic react ion shifts to the synthesis by ca. 25% due to the hydrophobic environm ent caused by polypyrrole matrix. Here, Ac-Phe-OEt and Ala-NH2 are use d as model substrates of acyl donor ester and a nucleophile acceptor, respectively, to synthesize Ac-Phe-AlaNH(2). Both the synthetic and th e hydrolytic activity of the immobilized chymotrypsin are found to be enhanced several times when a positive potential (0.4-0.8 V vs Ag/AgCl ) is applied to the electrode. In addition, the equilibrium of the enz ymatic reaction shifts further to synthesis. About 73% of synthetic yi eld of Ac-Phe-Ala-NH2 is obtained from equimolar concentration (200 mM ) of Ac-Phe-OEt and Ala-NH2 in aqueous solution. The effects of soluti on pH, temperature, organic solvent concentration, and substrates conc entration on the peptide synthesis are also described. An explanation of possible effects of the applied voltage which causes the remarkable increase of catalytic activity is also presented.