Reconstruction of a cross-family chitinase cocktail for efficient chitobiose production guided by the secretome of Streptomyces sp. F-3.

Abstract

The enzymatic conversion of chitin to chitobiose is of significant interest due to its broad biological applications. However, the limited functional diversity and insufficient synergism of chitinase systems constrain catalytic efficiency and industrial applicability. In this study, we rationally reconstructed a cross-family chitinase cocktail by elucidating the synergistic mechanisms between GH18 and GH19 enzymes from Streptomyces sp. F-3. The three GH18 chitinases and one GH19 chitinase displayed complementary substrate specificity toward insoluble polysaccharides and oligosaccharides, suggesting potential for synergistic chitin degradation. Guided by the pattern of chitinase secretion, we designed the in vitro four-enzyme cocktail (FR48) based on the native stoichiometry at 48 h. FR48 exhibited remarkable performance, yielding 3814 mg/L chitobiose with 93.1% purity and achieving an 8.86-fold increase in reducing-sugar production over commercial chitinase under the tested conditions. This study provides a generalizable biomimetic strategy for designing cross-family multi-enzyme systems for recalcitrant biomass valorization in green biomanufacturing.