Expression and characterization of colanic acid-degrading enzyme from Escherichia phage phi92 and analysis of its hydrolysate composition and structure

Colanic acid (CA), an exopolysaccharide synthesized by bacteria, is known for its potential to extend the lifespan of nematodes. However, its high viscosity poses practical application challenges, which can be mitigated by employing colanic acid-degrading enzymes (CAEs). This study presents the expression and characterization of a novel CAE from Escherichia phage phi92, used for the production of CA oligosaccharides (CAOSs). Optimal CAE expression conditions were identified as induction at 20 °C for 24 h with 0.1 mM IPTG. The enzyme showed maximal activity at 55 °C and pH 6.0, with stability in the range of 4–50 °C and pH 4.0–7.0. Structural characterization of CAOSs was performed using GC–MS, LC–MS and NMR spectroscopy, revealing the composition of hexasaccharide and dodecasaccharide units, with CAE cleaving the β-1,4 glycosidic linkage between glucose and fucose. CAOSs were found to mitigate oxidative stress and inflammation induced by H 2 O 2 in RAW 264.7 macrophages, inhibiting NO and MDA production while enhancing CAT activity. Additionally, CAOSs modulated the mRNA expression of pro-inflammatory (IL-1β, COX-2) and anti-inflammatory (IL-10, HO-1) factors. This study deepens the understanding of CAE and facilitates the preparation of CA oligomers for applications in food, cosmetics, and pharmaceuticals.