Immobilization of a polyphosphate kinase 2 by coordinative self-assembly of his-tagged units with metal-organic frameworks and its application in ATP regeneration from AMP.

Abstract

Self-assembly of the functional units onto the surface of nanoparticles is a powerful approach to generate functional nanosystems. In this work, we first expressed a recombinant class III polyphosphate kinase 2 (ArPPK2) with his-tag. It is able to synthesize ATP from AMP by a single enzyme, simplifying two-step reaction of ATP regeneration from AMP. Then we chose the Fe-based metal-organic frameworks (MOF)s as carriers to produce the enzyme-MOF biocomposite, based on the interaction between the his-tags and coordinatively unsaturated metal sites present on the external surface of MOFs by a self-assembly process. It was found that ArPPK2@MIL-101-NH2@Fe3O4-COOH exhibited better reusability than other candidates during cycle analysis, preserving 70.1% of initial activity after reusing thirteen times, and also retained high storage stability. The optimum pH of the enzyme-MOF biocomposite was increased from 8.0 to 9.0 and the optimum temperature was increased from 30℃ to 45℃. Compared to free ArPPK2, the enzyme-MOF biocomposite showed increased thermal and pH stability. In addition, we successfully constructed an ATP regeneration system from AMP using the enzyme-MOF biocomposite, coupled with amide bond formation catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). The immobilized ArPPK2 will provide a promising route for ATP regeneration from AMP in industrial processes. And the generation of the enzyme-MOF biocomposite by the self-assembly approach can be extended to efficiently immobilize other recombinant his-tagged enzymes.