Directional co-immobilization of artificial multimeric-enzyme complexes as a robust biocatalyst for biosynthesis curcumin glucosides and regeneration of UDP-glucose

Site-directed protein immobilization allows the homogeneous orientation of proteins while maintaining high activity, which is advantageous for various applications. In this study, the use of SpyCatcher/SpyTag technology and magnetic nickel ferrite (NiFe₂O₄ NPs) nanoparticles were used to prepare a site-directed immobilization of BsUGT2m from Bacillus subtilis and AtSUSm from Arabidopsis thaliana for enhancing curcumin glucoside production with UDP-glucose regeneration from sucrose and UDP. The immobilization of self-assembled multienzyme complex (MESAs) enzymes were characterized for immobilization parameters and stability, including thermal, pH, storage stability, and reusability. The immobilized MESAs exhibited a 2.5-fold reduction in UDP consumption, enhancing catalytic efficiency. Moreover, the immobilized MESAs demonstrated high storage and temperature stability over 21 days at 4 °C and 25 °C, outperforming their free counterparts. Reusability assays showed that the immobilized MESAs retained 78.7 % activity after 10 cycles. Utilizing fed-batch technology, the cumulative titer of curcumin 4’-O-β-D-glucoside reached 6.51 mM (3.57 g/L) and 9.45 mM (5.18 g/L) for free AtSUSm/BsUGT2m and immobilized MESAs, respectively, over 12 h. This study demonstrates the efficiency of magnetic nickel ferrite nanoparticles in co-immobilizing enzymes, enhancing biocatalysts' catalytic efficiency, reusability, and stability.