Potential-assisted enzymatic custom peptide synthesis in aqueous solution has been performed by α-chymotrypsin immobilized in conducting polymer polypyrrole matrix
Potential-assisted enzymatic custom peptide synthesisin aqueous solution has been performed by α-chymotrypsin immobilized in conducting polymer polypyrrole matrix.Chymotrypsin is electrochemically adsorbed on a Pt electrode, and then a thin polypyrrole membrane is electrochemically prepared on the enzyme layer.The enzyme/polypyrrole on an electrode retains chymotrypsin activity and the equilibrium of the enzymatic reaction shifts to the synthesis by ca. 25% due to the hydrophobic environment caused by polypyrrole matrix.Here, Ac-Phe-OEt and Ala-NH2 are used as model substrates of acyl donor ester and a nucleophile acceptor, respectively, to synthesize Ac-Phe-Ala-NH2.Both the synthetic and the 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 enzymatic reaction shifts further to synthesis.About 73% of synthetic yield of Ac-Phe-Ala-NH2 is obtained from equ... <h3>Abstract</h3> <p>In order to combine the high potential of frozen state custom peptide synthesis and the advantages of the application of immobilized proteases, the capability of carrier‐bound α‐chymotrypsin (CT, EC 22.214.171.124) to form peptide bonds in frozen aqueous reaction mixtures was investigated. The properties of the support materials strongly influenced the peptide yields. CT bound to rather hydrophobic supports catalysed peptide synthesis as effectively as the soluble enzyme. The immobilized enzyme preparations could be successfully re‐used at least 15 times. Peptide synthesis catalysed by immobilized chymotrypsin was also studied in low water organic reaction mixtures at sub‐zero temperatures. The advantages and limitations of the different approaches are discussed.