Abstract
Chitosan oligosaccharides (COSs) with defined degrees of polymerization (DP) exhibit distinct bioactivities with promising applications in food, pharmaceutical, and agricultural industries. However, the specific and sustainable production of COSs remains challenging due to the broad product distribution of wild-type chitosanases and the difficulties in enzyme recovery and reuse. In this study, we employed rational design to engineer a GH46 chitosanase (CsnB) from Bacillus sp. BY01 for chitobiose production. Through homology modeling and molecular docking analysis, 15 mutants were designed by targeting key residues structurally critical for substrate stabilization, product release, and active-site geometry in the substrate-binding subsites. The D78Y mutant exhibited exclusive specificity for chitobiose, demonstrating a specific activity of 102.4 U/mg and yielding chitobiose with a purity exceeding 98%, thereby surpassing the previously reported enzymes for chitobiose production. To address the challenges of enzyme stability, purification costs, and product separation, we developed a ReELP system by integrating elastin-like polypeptides (ELPs) with a ReverseCatcher/ReverseTag peptide pair. This system enabled one-step purification and co-immobilization of CsnB-D78Y directly from cell lysate onto biomimetic silica nanoparticles, achieving 96.8% immobilization efficiency and 90.7% activity recovery. The immobilized enzyme exhibited enhanced thermal and pH stability, retaining approximately 50% activity after 12 h at 40 °C compared to only 5.7% for the free enzyme. In reusability assays, the immobilized CsnB-D78Y maintained efficient chitobiose production over 5 consecutive cycles. This work provides a green and cost-effective strategy for the specific and sustainable production of chitobiose, offering new insights into enzyme engineering and immobilization for industrial COS production.