A synthetic reaction of dipeptide was carried out in organic solvents, using a thermostable neutral protease. The synthesis of benzyloxycarbonyl-L-aspartic-L-phenylalnine methyl ester, an aspartame precursor, is a typical protease-catalyzed reaction of peptide synthesis. Hydrolytic activity was reversed in nonaueous environment favoring synthesis of peptides, reversal of hydrolysis of peptide bond. Thermodynamic equilibria can be shifted toward condensation by introducing water-miscible and water-immiscible solvents. Synthetic yield and initial reaction rate can be influenced by toxicity and hydrophobicity of solvents. Various organic solvents were tested as reaction media. Thermolysin-catalyzed reaction was implemented in monophasic and biphasic system. There exists a optimal water content that gives the maximum yield. In water-immiscible solvents, distribution of reactants and products between organic and aqueous phase was shown to have effects on synthetic yield. Solvents may inhibit enzymatic activity and immobilization can be used to reduce solvent toxicity and stabilize enzyme and make it possible to use enzyme repeatedly in continuous operation.
