Smart Nanoparticles for Selective Immobilization of Acid Phosphatases.

ABSTRACT

An easy to use method combining the selectivity of metal chelate affinity binding with strong covalent linking was developed for immobilization of non-specific acid phosphatases bearing a His-tag from crude cell lysate. Silica nanoparticles were grafted with aminopropyl functions which were partially transformed further with EDTA dianhydride to chelators. The heterofunctionalized nanoparticles charged with Ni2+ as the most appropriate metal ion were applied as support. First, the His-tagged phosphatases were selectively bound to the metal-chelate functions of the support. Then, the enzyme-charged silica nanoparticles were further stabilized by forming a covalent linkage between nucleophilic moieties at the enzyme surface and free amino groups of the support using neopentylglycol diglycidylether as the most effective bifunctional linking agent. The phosphatase biocatalysts obtained by this method exhibited better phosphate transfer activity with a range of alcohols and PPi as phosphate donor in aqueous medium applying batch and continuous-flow modes than the ones immobilized on conventional supports. Furthermore, this novel strategy opens up novel possibility for efficient immobilization of other His-tagged recombinant enzymes.