Novel cofactor regeneration-based magnetic metal–organic framework for cascade enzymatic conversion of biomass-derived bioethanol to acetoin

[Display omitted] •Biomass-derived bioethanol is used to produce acetoin.•An efficient magnetic multienzyme cascade system is developed.•Immobilized NAD+ enhances the cofactor regeneration.•Maximum conversion rate of 90.4% is obtained under cofactor regeneration.•Magnetic co-immobilization system retains better operational stability. Upgrading biomass-derived bioethanol to higher-order alcohols using conventional biotechnological approaches is challenging. Herein, a novel, magnetic metal–organic-framework-based cofactor regeneration system was developed using ethanol dehydrogenase (EtDH:D46G), NADH oxidase (NOX), formolase (FLS:L482S), and nicotinamide adenine dinucleotide (NAD+) for converting rice straw-derived bioethanol to acetoin. A magnetic zeolitic imidazolate framework-8@Fe3O4/NAD+ (ZIF-8@Fe3O4/NAD+) regeneration system for cell-free cascade reactions was introduced and used to encapsulate EtDH:D46G, NOX, and FLS:L482S (ENF). ZIF-8@Fe3O4/NAD+ENF created an efficient microenvironment for three-step enzyme cascades. Under the optimized conditions, the yield of acetoin from 100 mM bioethanol using ZIF-8@Fe3O4/NAD+ENF was 90.4 %. The regeneration system showed 97.1 % thermostability at 50 °C. The free enzymes retained only 16.3 % residual conversion, compared with 91.2 % for ZIF-8@Fe3O4/NAD+ENF after ten cycles. The magnetic metal–organic-framework-based cofactor regeneration system is suitable for enzymatic cascade biotransformations and can be extended to other cascade systems for potential biotechnological applications.