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

Abstract Hybrid magnetic Fe 3 O 4 @SiO 2 -poly(ethylene oxide)-maltose (Fe 3 O 4 @SiO 2 -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 Fe 3 O 4 @SiO 2 -PEO-mal nanoparticles were used for characterizing of MBP-Hep I immobilization. The results showed that all four kinds of Fe 3 O 4 @SiO 2 -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. Fe 3 O 4 @SiO 2 -PEO-mal nanoparticles with a PEO molecular weight of 100k were best able to immobilize MBP-Hep I (Fe 3 O 4 @SiO 2 -PEO 100k -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 Fe 3 O 4 @SiO 2 -PEO 100k -mal-MBP-Hep I were the same. Furthermore, Fe 3 O 4 @SiO 2 -PEO 100k -mal-MBP-Hep I exhibited reasonable reusability during enzymatic production of low molecular weight heparins (LMWHs).