Rational Design to Enhance Enzyme Activity for the Establishment of an Enzyme–Inorganic Hybrid Nanoflower Co-Immobilization System for Efficient Nucleotide Production

Increasing yields while reducing costs is one of the ultimate pursuits of industrial production. To achieve this goal in the enzymatic production of multiple nucleotides, in this study, a co-immobilized polyphosphate kinase-nucleoside kinase hybridized nanoflower system (PPK@NK) was constructed. To improve the productivity, the nucleoside kinase (NK) used was rationally designed, and a variant with significantly increased activity compared to the wild type was obtained. The polyphosphate kinase (PPK) and NK could be sequentially adsorbed on the surface of hybrid nanoflowers at room temperature (25 °C) through the interaction of Cu2+ and proteins without any other chemical pretreatment. The optimal preparation conditions and reaction parameters of PPK@NK hybrid nanoflowers were investigated. Under optimal reaction conditions, the newly prepared co-immobilization system could catalyze the conversion of 100 mM uridine, cytidine, and inosine to the corresponding nucleotides completely within 4 h and could be reused at least six times. The storage stability of the co-immobilized system was more than 2-fold higher than that of the free enzyme, and there was no significant difference in thermostability. PPK@NK hybridized nanoflowers have properties such as easy preparation and storage and low cost, indicating their suitability for the efficient production of nucleotides.