Multi-module immobilization of Gluconobacter frateurii alcohol dehydrogenase and Lactobacillus brevis NADH oxidase with tethered nicotinamide adenine dinucleotide for D-allulose biosynthesis

Chemical tethering NAD + to GfADH enhances enzyme-cofactor spatial proximity. • The application of conjugate and NOX reduced the NAD + demand by 13-fold. • The NAD + -GfADH conjugate and NOX were co-fixed via sequential recruitment. • Co-immobilization enhanced enzyme stability with improved cofactor regeneration. The green biomanufacturing of high-value compounds is limited by enzyme instability and cofactor dependence. Herein, we developed a tailored multi-module immobilization strategy that integrated cofactor tethering with sequential multi-enzyme co-immobilization to facilitate the efficient conversion of allitol into D-allulose catalyzed by the Gluconobacter frateurii -derived dehydrogenase (GfADH). In this strategy, nicotinamide adenine dinucleotide (NAD + ) was covalently tethered to the surface of GfADH via a flexible linker, creating an NAD + -GfADH conjugate. Coupling the conjugate with NADH oxidase (NOX) established an efficient cofactor self-regenerating system, reducing the external NAD + demand by 13-fold. Furthermore, this conjugate was encapsulated within hybrid nanoflowers, thereby improving its reusability. To facilitate cofactor transfer, NOX was directionally recruited and colocalized with GfADH using the SpyTag/SpyCatcher system. The co-immobilized enzyme exhibited a high D-allulose yield of 88.7% from 110 mM allitol and retained 85.0% of its initial activity after 6–8 cycles. This work provides a valuable integrated strategy for cofactor regeneration and enzyme stabilization in cofactor-dependent biosynthesis. Download: Download high-res image (169KB) Download: Download full-size image