Enzyme variants in biosynthesis and biological assessment of different molecular weight hyaluronan

In the present study, low- and high-molecular-weight hyaluronic acids (LMW-HA and HMW-HA) were synthesized in vitro by truncated Streptococcus equisimilis hyaluronan synthases (SeHAS). The enzyme kinetic parameters were determined for each enzyme variant. The MW, structure, dispersity, and biological activity of polymers were determined by electrophoresis, FTIR spectroscopy, carbazole, cell proliferation, and cell migration assay, respectively. The specific activities were calculated as 7.5, 6.8, 4.9, and 2.8 µg_HA µg_enzyme^-1 min^-1 for SeHAS, HAS₁₂₃, HAS₂₃, and HAS_Intra, respectively. The results revealed SeHAS produced a polydisperse HMW-HA (268 kDa), while HAS₁₂₃ and HAS₂₃ produced a polydisperse LMW-HA (< 30 kDa). Interestingly, HAS_Intra produced a low-disperse LMW-HA. Kinetics studies revealed the truncated variants displayed increased K_m values for two substrates when compared to the wild-type enzyme. Biological assessments indicated all LMW-HAs showed a dose-dependent proliferation activity on endothelial cells (ECs), whereas HMW-HAs exhibited an inhibitory effect. Also, LMW-HAs had the highest cell migration effect at 10 µg/mL, while at 200 µg/mL, both LMW- and HMW-HAs postponed the healing recovery rate. The study elucidated that the transmembrane domains (TMDs) of SeHAS affect the enzyme kinetics, HA-titer, HA-size, and HA-dispersity. These findings open new insight into the rational engineering of SeHAS to produce size-defined HA.