Peptide synthesis catalyzed by α-chymotrypsin immobilized in the poly( N-isopropylacrylamide/acrylamide) gel

Abstract

The peptide syntheses were performed with α-chymotrypsin immobilized in the thermosensitive poly( N-isopropylacrylamide/acrylamide) (NIPAAm/AAm) gels. In the reaction between N-acetylphenylalanine ethyl ester (Ac-Phe-OEt) and alaninamide (Ala-NH 2), the product ratio of Ac-Phe–Ala-NH 2/Ac-Phe-OH increased with lowering temperature. The highest ratio was 3.3 in the reaction with immobilized enzyme at −10°C, while that was around 1.4 with free enzyme at 34°C. On the contrary, CBZ-Phe–Leu-NH 2 obtained from N-carbobenzoxyphenylalanine (CBZ-Phe) and leucinamide (Leu-NH 2) increased with increasing temperature (conversion: 13% at 10°C, 34% at 35°C) in Tris buffer without the organic solvent. The gel of NIPAAm copolymerized with AAm (NIPAAm/AAm=80/20) was effective as compared with the poly(NIPAAm) gel for the peptide production catalyzed by α-chymotrypsin using the ester substrate as the acyl donor. The reaction mechanism was discussed in connection with the hydrophobic surroundings inside the deswollen thermosensitive polymer gel.