Biochemical characteristics and high-efficiency expression of a novel PL24 family ulvan lyase

Ulvan lyase can degrade ulvan into ulva oligosaccharides with higher water solubility and better biological activity. This study characterized the enzymatic properties of a novel ulvan lyase ALT559 and enhanced its expression level. ALT559 was composed of 530 amino acid residues and exhibited the highest identity to ulvan lyases of the PL24 family. It has a high affinity for ulvan, with a K_m value of 0.95 mg·mL^-1. The activity of ALT559 was increased by 40.9% in the presence of 50 mM Ba²⁺, whereas it was almost completely inhibited by 50 mM Cu²⁺, Fe³⁺, or Zn²⁺. The optimal reaction temperature and pH for ALT559 are 45 °C and 8.5, respectively. ESI-MS analysis showed that the primary product of ulvan degradation by ALT559 was disaccharides. Based on the preliminary shake-flask optimization, a combined exponential and pH-stat feeding strategy was implemented in a 7-L fermenter, which achieved a high cell density of OD₆₀₀ = 69.2. The enzyme activity reached 24.29 U/mL, representing a 17.4-fold increase over the pre-optimized shake-flask level. In conclusion, this study reports a novel PL24 family ulvan lyase ALT559 and successfully realizes high-density fermentation in a 7-L fermenter, which is of great significance for its industrial application.