Highly efficient chitinase production from Chitinibacter mangrovi FCG-7T and immunomodulatory efficacy of generated GlcNAc in cyclophosphamide-induced immunosuppression.

Chitin, the second most abundant polysaccharide, is poorly soluble and underutilized, whereas its monomer N-acetylglucosamine (GlcNAc) has broad pharmaceutical applications. Efficient enzymatic conversion of chitin to GlcNAc remains challenging. Chitinibacter mangrovi FCG-7T, a novel chitinolytic strain isolated from mangrove sediments, exhibited exceptional chitin degradation, achieving a hydrolysis zone ratio (H/C) of 4.75 and maintained genetic stability over ten successive passages. Sequential optimization using One-Factor-at-a-Time (OFAT), Plackett-Burman designs (PBD), and Box-Behnken designs (BBD) enhanced chitinase production by 49.43-fold, yielding a final activity of 1.211 U/mL under optimized conditions (pH 7.4, 20 °C, 12 g/L powdered chitin). Purification yielded a 100 kDa bifunctional chitinase CmChi, exhibiting dual chitobiosidase and N-acetylglucosaminidase (NAGase) activities. The purified enzyme showed a specific activity of 7.96 U/mg toward colloidal chitin. CmChi demonstrated a high affinity for colloidal chitin with a Km value of 0.24 mg/mL and retained over 80% stability across a broad pH range 4.0-11.0. Furthermore, CmChi completely converted colloidal chitin to N-acetylglucosamine (GlcNAc) within 48 h and demonstrated tolerance to various reagents, including 1% methanol, acetonitrile, Tween 20, and Tween 80 (v/v). Notably, in cyclophosphamide (CTX)-induced immunosuppressed mice, GlcNAc (200 mg/kg) significantly restored body weight, thymic/splenic indices, and tissue architecture while elevating serum TNF-α, IL-2, IgG, and IgM (p < 0.05). This study establishes CmChi as an efficient biocatalyst for industrial GlcNAc production and validates the immunomodulatory potential of GlcNAc, highlighting its dual applicability in biotechnology and immunotherapy.