Immobilized Thermolysin

Immobilization of thermolysin was also expected to reduce enzyme inactivation because it is protected from autolysis. However, an application of immobilized thermolysin in the current production process seems difficult, because the reaction for Z-APM production takes place in the presence of a dense solid precipitate, that is, an insoluble addition compound of Z-APM and PheOMe. If both the immobilized enzyme and the reaction product occur in the reaction mixture as solids, they cannot be separated by a simple procedure. Additionally, if the reaction would be performed in a continuous process equipped with an immobilized thermolysin-packed column, the precipitate would block the flow of the reaction solution in the column. Z-APM forms its insoluble addition compound with PheOMe quite easily in water.

To overcome this problem, Oyama et al. (9,22) attempted to perform the reaction in ethyl acetate. Because ethyl acetate dissolves Z-APM without formation of the insoluble addition compound, it was expected that Z-APM could be recovered in solution and that the immobilized thermolysin could be recovered as a solid, thus enabling complete recycling of enzyme (9). Moreover, because the equilibrium state of the synthesis and hydrolysis of Z-APM shifts toward the synthesis side in organic solvents, it was expected that Z-APM could be obtained in high yield in ethyl acetate even though the product is not removed from reaction system in situ. Unfortunately, significant inacti-vation of immobilized thermolysin was observed in both batchwise operation and continuous operation. Additionally, Z-APM synthesis was disturbed by channeling of organic solvent and water in the immobilized thermolysin column when it was operated continuously (9); thus, high production yield could not be obtained by this system. Only if these problems can be solved, immobilized thermolysin can be applied in the APM production. Studies for APM production by immobilized thermolysin are being continued by various researchers (23,24).

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