Functional capsule membranes. Part 29. Thermolysin-immobilized capsule membranes as bioreactors in the synthesis of a dipeptide (precursor of aspartame) in an organic solvent

Abstract

Thermolysin (TLN) was covalently immobilized onto a large, ultrathin nylon capsule membrane grafted with poly-[p-(aminomethyl)styrene] using glutaraldehyde. When the TLN-immobilized capsule containing a buffer solution (pH 7) in the inner aqueous phase was immersed in a chloroform solution of N-benzyloxycarbonyl-L-aspartic acid (Z-L-Asp) and L-phenylalanine methyl ester (L-PheOMe) with shaking at 40 °C, the dipeptide (Z-L-Asp-L-PheOMe) was produced efficiently in the outer chloroform solution. From the Lineweaver–Burk plot, condensation in the aqueous–organic solvent involves initial binding of Z-L-Asp to the enzyme to form the Z-L-Asp–enzyme complex and then attack by L-PheOMe on the complex as the rate-determining step to form the peptide linkage. The TLN–capsule system can be used repeatedly without denaturation of protein structures by organic solvents because the enzyme on the capsule membrane is protected by buffer solutions coming from the inside. The enzyme-immobilized capsule membrane is a new bioreactor in aqueous–organic heterophases.