Novel cofactor regeneration-based magnetic metal-organic framework for cascade enzymatic conversion of...

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.