Protease Immobilization on γ‐Fe2O3/Fe3O4 Magnetic Nanoparticles for the Synthesis of Oligopeptides in Organic Solvents

The use of nanobiocatalysts, with the combination of nanotechnology and biotechnology, is considered as an exciting and rapidly emerging area. The use of iron oxide magnetic nanoparticles, as enzyme immobilization carriers, has drawn great attention because of their unique properties, such as controllable particle size, large surface area, modifiable surface, and easy recovery. In this study, various gamma-Fe(2)O(3)/Fe(3)O(4) magnetic nanoparticles with immobilized proteases were successfully prepared by three different immobilization strategies including A) direct binding, B) with thiophene as a linker, and C) with triazole as a linker. The oligopeptides syntheses catalyzed by these magnetic nanoparticles (MNPs) with immobilized proteases were systematically studied. Our results show that i) for magnetic nanoparticles immobilized alpha-chymotrypsin, both immobilization strategies A and B furnished good reusability for the Z-Tyr-Gly-Gly-OEt synthesis, the MNPs enzymes can be readily used at least five times without significant loss of its catalytic performance: ii) In the case of Z-Asp-Phe-OMe synthesis catalyzed by magnetic nanoparticles immobilized thermolysin, immobilization Strategy B provided the best recyclability: iii) For the immobilized papain, although Strategy A or B afforded an immobilized enzyme for the first cycle of Z-Ala-Leu-NHNHPh synthesis in good yield, their subsequent catalytic activity decreased rapidly. In general, the gamma-Fe(2)O(3) MNPs were better for use as an immobilization matrix, rather than the Fe(3)O(4) MNPs, owing to their smaller particle size and higher surface area.