Enhancing activity and stability of a GH8 chitosanase through conserved N-terminal and peripheral residue mutations for bioactive chitooligosaccharide production

Abstract

Chitosan, the second most abundant biopolymer after cellulose, has vast application potential. However, its high viscosity and poor solubility have limited its full utilization. Chitosan degradation primarily depends on GH46 family chitosanases, while the more stable GH8 family chitosanases have received less attention. This study, for the first time, reveals the critical role of the conserved N-terminal region in the catalytic activity and substrate binding of the GH8 family chitosanase BcCn8A using truncation mutagenesis and molecular dynamics simulations. Through evolutionary analysis and computationally assisted semi-rational design, the optimized mutant BcCn8A-ΔN4-V319L was obtained, showing a 310 % increase in specific activity and a 5 °C increase in optimal temperature. The engineered enzyme efficiently hydrolyzes chitosan into chitooligosaccharides (COSs) with antioxidant and anti-browning properties, making it suitable for applications like juice preservation. This work provides new insights into the regulatory roles of the N-terminal region and distal amino acid residues in GH8 family chitosanases, advancing their potential in functional oligosaccharide production.