Continuous Synthesis of a Tripeptide by Successive Condensation and Transesterification Catalyzed by Two Immobilized Proteinases in Organic Solvent

The tripeptide Z-GlyPheLeuNH₂ was continuously synthesized in a high yield from three amino acid derivatives, Z-Gly, PheOMe, and LeuNH₂, by immobilized thermolysin (IMT) and immobilized α-chymotrypsin (IMC) in an organic solvent, ethyl acetate. The optimal conditions for the synthesis of Z-GlyPheOMe were established theoretically. The yield of Z-GIyPheOMe with IMT in ethyl acetate saturated with buffer was more than 88% after continuous synthesis for 116hr. The optimal conditions for the synthesis of Z-GlyPheLeuNH₂ from Z-GlyPheOMe and LeuNH₂ by IMC through transesterification was established in batch reaction experiments. When the concentration of water in the reaction solution was 17-20 μl/ml, the activity of IMC was highest. The equilibrium between the water concentration in the reaction solution and that in the resin used for enzyme immobilization depended on the resin and was not affected by the presence of the enzyme immobilized. Z-GlyPheLeuNH₂ was synthesized from Z-GlyPheOMe and LeuNH₂ with a yield of 100 %, by continuous reaction for 160 hr. The reactor for synthesis of this tripeptide was efficient and stable because of the use of transesterification and the choice of an appropriate organic solvent. The series plug-flow reactor was successfully operated for 220 hr with a yield of more than 80 %. The residual activity of IMT was 94%, and that of IMC was 100%.