Rational structural design for the dual enhancement of catalytic activity and thermal stability of hyaluronidase from Citrobacter portucalensis.

Hyaluronidases (HAase) offer a practical route to produce valued low molecular weight hyaluronic acid (LMW-HA) and HA oligosaccharides (o-HA), which are valued across medical, cosmetic, and biomaterials applications due to their enhanced bioactivity and tissue penetration. However, reliable production and scale-up of LMW‑HA and o‑HA remain complicated due to inefficient production method, potential pathogenic hosts, enzymes with limited catalytic proficiency and prone to thermal denaturation. Here, we employed a structure-guided multi-strategy engineering approach to express and optimize a PL8 family hyaluronidase in E. coli, aiming to overcome these industrial bottlenecks. Using the HA tetrasaccharide binding site mapping, residue conservation analysis, and binding energy calculations, we obtained two mutants (T204R and Y97R) with significantly improved catalytic activities of 1.56 × 10⁶ U/mg and 1.43 × 10⁶ U/mg, respectively. Building on the best-performing variant (T204R), stability-focused design yielded T204R/N206H, which extended the inactivation half-life at 45 °C by up to 1.4-fold without loss of activity. This engineered enzyme efficiently generates LMW-HA and o-HA with tunable size distributions under mild conditions. These findings expand the hyaluronidase toolbox with a recombinant PL8 enzyme, enabling controlled enzymatic production of LMW-HA and o-HA for biomedical and personal care applications.