Abstract

Phosphate-containing macromolecules are essential for bacterial pathogenesis and protein post-translational modification studies, but the widely used calf intestinal alkaline phosphatase (CIP) shows poor dephosphorylation activity against complex biomacromolecules like lipopolysaccharide (LPS) and protein. To overcome the limited activity of CIP for macromolecular substrates, we successfully expressed an alkaline phosphatase from Saccharomyces cerevisiae (sAP) in Komagataella phaffii . The recombinant sAP (rsAP) was well expressed and reached a protein yield of 1.12 g/L in the shake-flask fermentation supernatant. The rsAP exhibited excellent catalytic activity and stability over a wide pH range (7.0–11.0) and at high temperatures (up to 65 °C), with optimal activity at pH 10.0 and 65 °C. Notably, compared to CIP, rsAP demonstrated significantly higher dephosphorylation efficiency for macromolecular substrates, including LPS, phosphorylated proteins, and linearized DNA. Importantly, rsAP could effectively neutralize endotoxin activity. Pretreatment of LPS with rsAP markedly enhanced survival rates and reduced serum TNF-α levels in a murine endotoxemia model. The superior performance of rsAP is attributed to its unique structural features, including a deep, hydrophobic active pocket with complementary electrostatics and enhanced flexibility while CIP features a shallow, hydrophilic, and relatively rigid active pocket, which may hinder its ability to efficiently bind and process macromolecular substrates. This work not only highlights rsAP as a potent biocatalyst for LPS detoxification and phosphoprotein modification but also positions it as a promising tool for diverse biotechnological and therapeutic applications.