Engineering a high-performance isopropyl alcohol biosensor using a bio-nanocomposite multienzyme platform for...

The spatial organization of multi-enzyme cascades offers a powerful strategy to overcome diffusion limitations and cofactor regeneration bottlenecks in industrial biocatalysis. In this study, a cellulosome-inspired complex was engineered to facilitate the efficient, self-sufficient oxidation of isopropanol (IPA). By exploiting high-affinity cohesin-dockerin interactions, a dockerin-fused secondary alcohol dehydrogenase (S-ADH_doc) was co-immobilized with a dockerin-fused alkyl hydroperoxide reductase (AhpF_doc), serving as a robust NADH regeneration partner. Kinetic characterization of the individual recombinant modules revealed that S-ADH_doc exhibited a Vmax of 5.59 ± 0.80 μmol·minute^-1 and a turnover number (kcat) of 46.6 s^-1. Crucially, the regeneration module, AhpF_doc, displayed superior kinetics with a Vmax of 27.0 ± 0.60 μmol·minute^-1 and a kcat of 225 s^-1, ensuring that cofactor recycling was not rate-limiting. Upon assembly, the complex demonstrated synergistic catalytic efficiency driven by proximity-induced substrate channeling and rapid electron transfer and thereby improving the overall enzyme kinetics (Vmax of 1834.8 μmol·minute^-1). The engineered system exhibited high selectivity for IPA against structurally related alcohols and demonstrated remarkable operational stability, retaining over 85% of its initial activity after 15 days with high reproducibility (RSD = 3.87%). These findings underscore the potential of artificial scaffoldin-based architectures as versatile, scalable platforms for constructing stable enzymatic modules for use in industrial bioprocess monitoring and sustainable chemical synthesis.