Selective ene-reductase immobilization to magnetic nanoparticles through a novel affinity tag.

Abstract

Magnetic nanoparticles (MNPs) are becoming more important as carriers, because of their large specific surface area and easy separability. They are increasingly used in enzyme technology, diagnostics, and drug delivery. For the directed and almost irreversible immobilization of proteins on MNPs, we have developed a new selective (His-Arg)4 peptide-tag, that binds fusion proteins directly from an E. coli cell lysate to non-functionalized, low-cost MNPs. Using the immobilization of an ene-reductase as an example, we could demonstrate that the fusion with this tag increases thermostability without reducing overall activity (ER w/o tag: t1/2 =3.7 h, (HR)4 -ER: t1/2 =9.9 h). Immobilization by adsorption in Tris buffer resulted in very high enzyme loads with approx. 380 mg g-1 and 67% residual activity. The immobilization on the MNPs allowed a fast concentration, buffer exchange, and reuse. While about 50% of the activity was lost after the first reuse, we were able to show that the activity did not decrease further and was stable for another ninecycles. According to our studies, our tag highly works for any kind of immobilization on MNPs and holds the potential for enzyme immobilizations as well as for drug delivery and sensors.