Secretome analysis of the chitinolytic machinery of Chitiniphilus shinanonensis and its implication in chitooligosaccharide production.

Chitin's robust structure poses significant challenges for degradation, necessitating the study of microbial processes in chitin-rich environments. We assessed the chitinolytic bacterium Chitiniphilus shinanonensis DSM 23277 (SAY3) for converting chitin biomass into valuable saccharides using various substrates (chitin flakes, α-chitin, and β-chitin) in shake flask cultures. The bacterium successfully grew on all substrates, achieving complete degradation, although chitin flakes required more time. Maximum growth was observed on β-chitin, followed by α-chitin and chitin flakes. Scanning electron microscopy confirmed bacterial colonization and potential hydrolytic activity on chitin flakes. Proteomic analysis via nanoLC-MS/MS identified 32 chitin-degrading enzymes distributed across secretome, periplasmic, and intracellular fractions, with a notable expression of glycoside hydrolases (families 18, 19, and 20), carbohydrate esterases (family 4), and auxiliary activity proteins (family 10). Among the family 18 chitinases, ChiM, ChiI, and ChiL were significantly upregulated on all chitinous substrates compared to glucose. The chitin-active-secretome exhibited optimal activity at pH 8.0 and 45 °C in 50 mM Tris-HCl. Moreover, the chitin-active-secretome effectively degraded chitin flakes, α-chitin, and β-chitin into chitobiose and GlcNAc, with β-chitin yielding the highest chitobiose levels. The diverse chitin-degrading enzymes of C. shinanonensis efficiently utilize recalcitrant chitin as a carbon and energy source, underscoring its industrial potential for chitin degradation.