Doctor of Philosophy
Chih-Yu Cheng
Department of Marine Biotechnology, National Kaohsiung University of Science and Technology, Kaohsiung 81157, Taiwan
A Structure-Guided Engineering and High-Throughput Screening Platform for Chitosanase Specificity
Chih-Yu Cheng*, Chia-Huang Tsai, Pei-Jyun Liou, and Chi-Hang Wang,
Chitooligosaccharides (COS) are emerging bioactive molecules with promising applications in agriculture, biomedicine, and environmental protection. However, sustainable utilization of chitin-rich biomass—one of the world’s most abundant renewable biopolymers—remains limited by the inability of natural chitosanases to produce structurally uniform COS required for functional studies and high-value bioproducts. To address this bottleneck, we established ChiTuned™, a structure-guided enzyme engineering and high-throughput screening platform designed to generate product-specific chitosanases that support circular and low-carbon bioprocessing of chitinous waste. Using Bacillus circulans MH-K1 chitosanase as a model, we combined homology modeling, saturation mutagenesis, and loop-focused electrostatic tuning to reprogram substrate-length specificity with minimal experimental footprint. Subsite analysis revealed residues A174 and L175 as critical for chitotetraose recognition; saturation mutagenesis at this region yielded Csn-TI, which selectively reduced over-depolymerization and favored chitotriose (DP3) formation. Further refinement through a single aspartate insertion at the 75–76 loop produced Csn-TI-D, an engineered enzyme capable of generating 84–90% DP3—a dramatic improvement over the wild-type enzyme (~30%). Importantly, TI-D preserves endo-type catalytic behavior, enabling efficient bioconversion without excessive cleavage or energy-intensive downstream separation. Fluorescence binding analyses and closed-state docking models indicated that the engineered modifications weaken (−2) subsite affinity and shift substrate alignment, providing a mechanistic basis for length-selective hydrolysis. Together, these results demonstrate that ChiTuned™ enables rational development of precision biocatalysts that valorize crustacean and fungal biomass into high-purity COS. This sustainable biotransformation strategy aligns with global goals for resource efficiency, waste minimization, and bio-based circular economy, offering a scalable path toward environmentally friendly production of functional oligosaccharides.
Biography of the presenting author
Dr. Chih-Yu Cheng, Chair of the Department of Marine Biotechnology at NKUST, leads the Enzyme and Engineering Lab developing sustainable biotechnological innovations. Her work integrates protein engineering, fungal biotechnology, and chitin-based applications to create low-impact, high-value solutions. She reprograms chitosanase for precision chitooligosaccharide production, advances efficient mycelial cultivation, and applies enzymatic and biopolymer systems to bioremediation. Through the ChiTuned™ platform and circular use of marine biomaterials, she transforms chitin resources into functional ingredients and eco-friendly technologies that support a resilient blue economy and sustainable development.
Presenting author details
Full name: Chih-Yu Cheng
Contact number: +886-7-3617141~23817
Email: cycheng@nkust.edu.tw
Website:
Session name/ number:
Category: (Poster presentation)