Preparation of a flowerlike protein-inorganic nanohybrid biocatalyst via co-immobilization of cobalt phosphate with mutant cellobiose 2-epimerase

Abstract

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@Co3(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 PO43-. The resulting EDNP@Co3(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@Co3(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 °C). More importantly, a second hybridization process was first applied to further improve the total encapsulation yield up 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@Co3(PO4)2 has great potential in industrially lactulose bioproduction.