Substrate affinity and spatial proximity synergistically guide multi-enzyme architecture rewiring for highly efficient chitin conversion

Multidimensional score guided design and tunable boost of LPMO-chitinase complex. • It integrated CBMs substrate affinity and catalytic domains spatial proximity. • The designed fusion CBM 11 enhanced synergistic activity on α-chitin and β-chitin. • Compared to Bc Chi2, NACOS yield increased 6.33 times and 20.16 times, respectively. N- acetylchitosan oligosaccharides (NACOS) show great promise in food, medicine, and agriculture. The synergistic effect of lytic polysaccharide monooxygenases (LPMOs) and chitinases can overcome kinetic barrier and boost the yield of NACOS from crystalline chitin. However, the enhancement level is highly dependent on the enzyme combination, lacking consistency and predictability. To address this challenge, this study developed a predictable multidimensional framework based on the substrate affinity of carbohydrate-binding modules (CBMs) and the spatial proximity of catalytic domains, guiding the rational design of LPMO-chitinase complex and the tunable enhancement of synergistic effect. The designed artificial fusion CBM 11 exhibited good catalytic activity. In the α-chitin and β-chitin systems, compared with chitinase ( Bc Chi2) alone, the addition of LPMO ( Bc LPMO1_T3) increased the yield of NACOS by only 0.17-fold and 1.97-fold, while fusion CBM 11 (a multi-enzyme complex of Bc LPMO1_T3 and Bc Chi2) achieved significant increases of 6.33-fold and 20.16-fold, respectively. The degradation rate of β-chitin by 3 μmol/L fusion CBM 11 reached over 85%. In addition, the fusion enzyme exhibited good thermostability and significant tolerance to high concentrations of H 2 O 2 , further demonstrating the application potential in complex reaction systems. This work presents a rational strategy for optimizing multi-enzyme synergistic systems to efficiently degrade chitin and provides a reference for the development of biocatalysts targeting other insoluble polysaccharides. Download: Download high-res image (143KB) Download: Download full-size image