Efficient acidophilic chitinase from a carnivorous plant for the depolymerization of pretreated chitin into a reproducible spectrum of chitooligosaccharides

Chitin-rich biomass represents an abundant renewable resource for sustainable valorization into functional chitooligosaccharides. Here, we report the recombinant production and functional evaluation of a thioredoxin-fused class I GH19 chitinase from the forked sundew Drosera binata (rDbChitI-3+Trx) and assess its suitability for controlled chitin depolymerization. The enzyme exhibited strong endochitinase activity toward structurally accessible substrates (swollen-, glycol-, and colloidal chitin), while showing minimal activity on crystalline chitin and highly deacetylated chitosan. The rDbChitI-3+Trx is strongly acidophilic with an optimum at pH 3.0 and temperature maximum at 15–30°C. Its apparent kinetic analysis yielded a catalytic efficiency of 2.8 × 10³ M⁻¹ s⁻¹ ( k cat / K m,app ) and demonstrated improved substrate affinity relative to its thioredoxin-free version. The enzyme showed limited sensitivity to most metal ions at 2 mM, whereas Fe²⁺ and Pb²⁺ strongly inhibited activity. Reducing agents, toluene, and dimethylformamide enhanced the catalytic performance of rDbChitI-3+Trx, although most tested organic solvents reduced activity. Mass spectrometry and polyacrylamide gel electrophoresis revealed that recombinant chitinase converts partially acetylated swollen chitin into a reproducible spectrum of chitooligosaccharides (degree of polymerization 4–16). The combination of high-yield production, acidophilic cold-active profile, and activity enhancement by selected additives highlights rDbChitI-3+Trx as a promising biocatalyst for controlled chitooligosaccharide production.