Optimal covalent immobilization of α-chymotrypsin on Fe3O4-chitosan nanoparticles

This study investigated the immobilization of α-chymotrypsin onto magnetic Fe3O4-chitosan (α-chymotrypsin-Fe3O4-CS) nanoparticles by covalent binding. The response surface methodology (RSM) with a 3-factor-3-level Box–Behnken experimental design was employed to evaluate the effects of the manipulated variables, including the immobilization time, temperature, and pH, on the enzyme activity. The results indicate that the immobilized temperature and pH significantly affected enzyme activity. In a ridge max analysis, the optimal condition for α-chymotrypsin immobilization included a reaction temperature of 21.7 °C, a pH of 7.6, and an incubation time of 1.1 h. The predicted and the experimental immobilized enzyme activities were 354 and 347 ± 46.5 U/g-support, respectively, under the optimal condition. Besides, the synthesis reactions of the dipeptide derivative using the free and immobilized α-chymotrypsin were compared. The yields of the dipeptide derivative via the free or immobilized α-chymotrypsin catalyzed were almost the same. The α-chymotrypsin-Fe3O4-CS nanoparticles exhibited a good acid-resisting ability and the less reaction time was required for dipeptide synthesis. After twelve repeated uses in dipeptide synthesis, the immobilized α-chymotrypsin still retained over 60% of its original activity. The magnetic α-chymotrypsin-Fe3O4-CS nanoparticles can be easily recovered by magnetic field will have potential application in industry.

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► α-Chymotrypsin immobilized onto magnetic Fe3O4-chitosan nanoparticles by covalent binding.
► RSM design to optimize immobilized conditions.
► Characterization of magnetic α-chymotrypsin-Fe3O4-CS nanoparticles was analyzed.
► A dipeptide derivative synthesis using a α-chymotrypsin-Fe3O4-chitosan nanoparticles catalyst.
► The reusability of the immobilized α-chymotrypsin for synthesis of N-Ac-Phe-Gly-NH2 was studied.