Site-directed chemically-modified magnetic enzymes: fabrication,improvements, biotechnological applications and future prospects

Numerous enzymes of biotechnological importance have been immobilized on magnetic nanoparticles (MNP) via random multipoint attachment, resulting in a heterogeneous protein population with potential reduction in activity due to restriction of substrate access to the active site. Several chemistries are now available, where the modifier can be linked to a single specific amino acid in a protein molecule away from the active-site, thus enabling free access of the substrate. However, rarely these site-selective approaches have been applied to immobilize enzymes on nanoparticles. In this review, for the first time, we illustrate how to adapt site-directed chemical modification (SDCM) methods for immobilizing enzymes on iron-based MNP. These strategies are mainly chemical but may additionally require genetic and enzymatic methods. We critically examine each method and evaluate their scope for simple, quick, efficient, mild and economical immobilization of enzymes on MNP. The improvements in the catalytic properties of few available examples of immobilized enzymes are also discussed. We conclude the review with the applications and future prospects of site-selectively modified magnetic enzymes and potential benefits of this technology in improving enzymes, including cold-adapted homologues, modular enzymes, and CO₂-sequestering, as well as non-iron based nanomaterials.