Coencapsulating Anionic Cofactor and Silica Nanoparticle-Bound Enzymes in Cationic Microgel for Multienzyme Cascade Reaction with Cofactor Recycling

Abstract

Large-scale applications of enzymes and cofactors remain a challenge due to their fragile property, nonrecyclability, and high cost. Herein, we report a coimmobilization strategy of multienzyme and cofactor based on a combination of microgel and silica nanoparticles (SNPs) to make the biocatalysis process economical and sustainable. As exemplified by a nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase and chimeric amine dehydrogenase cascade amination reaction, the dual-enzyme coimmobilization system, with 100% immobilization yield and 76% increased cascade activity, was constructed through the site-specific immobilization of silica binding peptide. The anionic dual-enzyme@SNPs (29.7 mg g SNPs/PVCL-Vim −1) and NADH (47.3 μmol g SNPs/PVCL-Vim −1 ) are then delivered into a cationic microgel through gel swelling and electrostatic interactions/ion exchanges. The system displays a 27 to 75% enhancement in the amination yield as compared to the free counterpart, possibly due to the site-specific immobilization of enzymes, the proximity effect resulting from the coimmobilization, and the mobility and accessibility of the immobilized cofactor. Moreover, the constructed coimmobilization system shows 8−43% increased stability over the free system and retains 97% of its original activity after eight recycles. Collectively, this work has demonstrated an efficient coimmobilization strategy of a cofactor-dependent multienzyme cascade for applications of sustainable biotransformation.