Domain-Function Analysis of the Endolytic Ulvan Lyase PcPL40A: Elucidating the Impact on Enzymatic Properties

Ulvan lyases play a crucial role in the high-value utilization of ulvan. In this study, we expressed and characterized a PL40 family ulvan lyase, PcPL40A, from the marine bacterium Pseudotamlana carrageenivorans. Its optimal reaction temperature and pH were 40 °C and 8.0, respectively. Additionally, PcPL40A exhibited a high substrate affinity (Km = 1.61 ± 0.14 mg/mL). It endolytically degrades ulvan into unsaturated oligosaccharides (disaccharides to octasaccharides). The enzyme employs a Tyr/His catalytic mechanism, where R459 neutralizes the carboxyl group, H429 acts as the catalytic base, and Y260 serves as the catalytic acid. Domains D1 and D2 are catalytic; truncating D3/D5 inactivates the enzyme, while truncating D4/D6 reduces the thermostability and catalytic efficiency. MD simulations revealed that truncating D4 and D6 can increase enzyme flexibility while also causing unstable hydrogen bonding between key sites and the ligand. Our findings demonstrate that PcPL40A is an efficient tool for preparing unsaturated ulvan oligosaccharides.