Genome-Driven Discovery of a Fe²⁺-Dependent Chitin Deacetylase from Bacillus pumilus B866 with Enhanced Thermostability

Chitin deacetylase (CDA) plays a pivotal role in converting chitin to chitosan, yet industrial applications remain constrained by low enzymatic activity, instability under process conditions, and insufficient understanding of metalloenzyme activation mechanisms. Addressing these challenges, we conducted a genome-driven investigation of 151 salt-tolerant Bacillus strains to identify robust CDAs tailored for industrial demands. Genomic analysis revealed 120 strains harboring CDA genes, with Bacillus pumilus B866 exhibiting the highest native activity (105.93 U/mL). Through systematic medium optimization—identifying lactose, yeast extract, and FeSO₄ as critical components—CDA production in B866 surged to 191.32 U/mL, a 2.39-fold increase over baseline. Heterologous expression of BpCDA in E. coli yielded a recombinant enzyme (123.27 U/mL) with superior thermostability (retaining > 42.9% activity after 24 h at 55 °C) and broad pH adaptability (>81.4% activity at pH 7.0–9.0). Notably, BpCDA demonstrated unique Fe²⁺-dependent activation (186.4% activity enhancement at 1 mM), contrasting with Mg²⁺-dependent systems in prior studies. Comparative genomic and pan-genome analyses underscored evolutionary adaptations linked to saline–alkaline niches, while biosynthetic gene cluster profiling revealed strain-specific metabolic potentials independent of genome size. This study resolves critical limitations in CDA performance by integrating genome mining, targeted screening, and metalloenzyme engineering, establishing a scalable platform for sustainable chitin valorization. The optimized BpCDA, with its industrial-compatible stability and novel activation mechanism, represents a significant advancement toward efficient, eco-friendly chitosan production.