Preparation Of A Flowerlike Protein-Inorganic Nanohybrid Biocatalyst Via Co-Immobilization Of Cobalt Phosphate With Mutant Cellobiose 2-Epimerase

Hierarchically structured protein-inorganic hybrid nanoflowers hold promise for efficient enzyme immobilization. Herein, a facile and rapid method to synthesize efficient lactulose-producing enzyme-metal hybrid biocatalyst was developed by using mutant E161D/N365P (EDNP) as the organic component and cobalt phosphate as the inorganic component. The synthesis conditions and stepwise formation of the EDNP@Co-3(PO4)(2) biocatalyst were systematically investigated. With the characterization of surface morphologies, chemical bonding states, and element compositions analysis, a possible growth mechanism has been proposed based on the affinity interaction between Co2+ and his-tagged EDNP, and the electrostatic interaction between Co2+ and PI43-. The resulting EDNP@Co-3(PO4)(2) showed effective encapsulation yield (39.70-57.22%) and promising activity recovery (43.08-62.88%). Interestingly, the specific activity expression of the EDNP@Co-3(PO4)(2) was about 1.1-fold higher than that of the free EDNP. The immobilized biocatalyst also exhibited greatly improved catalytic activity and stability towards pH (pH 5-9) and a broad range of temperatures (40-75 degrees C). More importantly, a second hybridization process was first applied to further improve the total encapsulation yield up to similar to 70%. Furthermore, the unique flower-like nanostructure enabled enhanced kinetic characteristics. Over 70% of enzyme activity was retained after eight catalytic cycles. All those results suggested that the prepared EDNP@Co-3(PO4)(2) has great potential in industrially lactulose bioproduction.