Purification and recovery of trypsin using Fe3O4@PDA@BTI magnetic affinity adsorbent

● Recombinant BTI was immobilized on PDA-coated Fe₃O₄ nanoparticles. ● Fe₃O₄@PDA@BTI enabled one-step trypsin purification from porcine pancreatic homogenate. ● The maximum trypsin adsorption capacity was 13.78 ± 0.34 mg/g. A magnetic affinity adsorbent, Fe₃O₄@PDA@BTI, was prepared by immobilizing recombinant buckwheat trypsin inhibitor (BTI) on polydopamine (PDA)-coated Fe₃O₄ nanoparticles. TEM, FTIR, XRD, VSM, BTI-loading analysis, and retained inhibitory activity collectively supported the successful construction of the material. The adsorbent recovered trypsin from porcine pancreatic extract in a single batch purification step, with a maximum adsorption capacity of 13.78 ± 0.34 mg/g. Fe₃O₄@PDA@BTI retained more than 90% of its inhibitory activity at pH 4–10, more than 85% of its inhibitory activity after 3 h at 30–80 ℃, and approximately 94% after 30 d at 4 ℃. It also exhibited good stability against ethanol, dimethyl sulfoxide (DMSO), urea, and sodium dodecyl sulfate (SDS). However, the relative adsorption capacity decreased to 45.35 ± 0.19% after 10 reuse cycles, indicating that ligand coupling and regeneration require further optimization. These results demonstrate the feasibility of combining recombinant inhibitor recognition with magnetic recovery for trypsin purification.