Identification, expression, and functional analysis of a chitin-degrading enzyme CtChi70 for N-acetyl Chitobiose production

Chitin, the second most abundant natural polysaccharide, is a renewable resource with great potential for producing functional oligosaccharides. In this study, we identified and characterized CtChi70, a glycoside hydrolase family 18 (GH18) exochitinase that selectively hydrolyzes chitin to produce N -acetyl chitobiose [(GlcNAc)₂] with exceptional purity (≥98 %). CtChi70 was heterologously expressed in Escherichia coli CtChi70 at a high yield of 228.2 ± 47.8 mg L −1 . The purified enzyme exhibited optimal activity at pH 5.0 and 45 °C, and demonstrated stability across a broad pH range (4.0–12.0). Time-course hydrolysis of colloidal chitin showed moderate catalytic efficiency but high product specificity, peaking at 24 h with minimal N -acetylglucosamine byproduct. The ability of CtChi70 to directly convert raw chitin powder to high-purity (GlcNAc)₂ suggests its potential for applications requiring pure chitooligosaccharides. These findings highlight CtChi70 as a promising biocatalyst for sustainable chitin valorization.