Immobilized Trypsin-catalyzed Semisynthesis Of Human Insulin Through One-step Reaction

In this paper, a study on preparation of monocomponent human insulin by immobilized trypsin-assisted transpeptidation reaction is reported. O-tert-butyl threonine tert-butyl ester was prepared by the addition of liquid isobutylene to threonine in the presence of sulfuric acid catalyst dissolved in 1,4-dioxane. The product conformed to the requirement of transpeptidation and the preparing technique was feasible and convenient. Its purity can reach 98.5%. The activity of immobilized trypsin prepared through rapid technique reached 5990.5U/g and the whole process was more rapid and convenient than the technique before improvement, therefore it could be used for transpeptidation in industry successfully. Porcine insulin is converted into human insulin with Thr(Bu~t)OBu~t through transpeptidation catalyzed by immobilized trypsin. The yield of human insulin ester in the transpeptidation reaction is 75.1%. The product is found to be homogenous by the analysis of PAGE, SDS-PAGE and HPLC. These results reach to the level of monocomponent human insulin.