Direct affinity immobilization of recombinant heparinase I fused to maltose binding protein on maltose-coated magnetic nanoparticles

• Fe3O4@SiO2-PEO-mal was used to immobilize MBP-Hep I from the crude enzyme directly.
• All Fe3O4@SiO2-PEO-mal nanoparticles exhibited excellent immobilization performance.
• Immobilized MBP-Hep I showed improved thermostability (20–50 times of free MBP-Hep I).
• Immobilized MBP-Hep I showed the same catalytic mechanism as free Hep I and MBP-Hep I.
• Fe3O4@SiO2-PEO100k-mal-MBP-Hep I showed suitable operating stability.

Hybrid magnetic Fe3O4@SiO2-poly(ethylene oxide)-maltose (Fe3O4@SiO2-PEO-mal) nanoparticles synthesized by our group can be used as affinity adsorption carriers for direct separation of maltose binding protein-fused Hep I (MBP-Hep I) from a crude enzyme solution in a magnetic field. In this work, different PEO molecular weights for Fe3O4@SiO2-PEO-mal nanoparticles were used for characterizing of MBP-Hep I immobilization. The results showed that all four kinds of Fe3O4@SiO2-PEO-mal magnetic nanoparticles (6k, 20k, 35k and 100k for PEO) exhibited excellent adsorption capacities and the adsorption ratio increased as the PEO molecular weight increased from 6k to 100k. All four kinds of immobilized MBP-Hep I exhibited significantly improved stability at 30 °C compared with free MBP-Hep I and their half-lives were 20–50 times that of the free MBP-Hep I. Fe3O4@SiO2-PEO-mal nanoparticles with a PEO molecular weight of 100k were best able to immobilize MBP-Hep I (Fe3O4@SiO2-PEO100k-mal-MBP-Hep I). The molecular weight distribution profiles and anticoagulant activities, obtained from heparin depolymerization by free Hep I, free MBP-Hep I and Fe3O4@SiO2-PEO100k-mal-MBP-Hep I were the same. Furthermore, Fe3O4@SiO2-PEO100k-mal-MBP-Hep I exhibited reasonable reusability during enzymatic production of low molecular weight heparins (LMWHs).