Abstract: In this study, a novel N-acetylgalactosaminyltransferase (GalNAcT) was developed for in vitro modification of mucins by O-glycosylation. A recombinant expression vector containing the ppGalNAcT gene derived from the common swift (Apus apus), AappGalNAcT11 was constructed, with heterologous expression in Pichia pastoris GS115 cells. The catalytic activity was determined using an enzymatic reaction system and its biochemical properties, including optimum reaction temperature and temperature stability, optimum reaction pH and pH stability, metal ion dependence, and storage stability, were analyzed. The molecular weight of the target protein is approximately 42.1 kDa. AappGalNAcT11 specifically transferred GalNAc from UDP-GalNAc to the peptide, EA2, and also added 1-3 GalNAc residues to the receptor protein, SUMO-MUC5AC, forming three distinct Tn antigen structures. The enzyme had optimal activity at 37 ℃ and retained >95% of its activity after incubation at 20 ℃ to 40 ℃ for 75 minutes. The optimal reaction pH was 9.0, and the enzyme retained >80% of its residual activity after incubation at pH 7 to 9 for five days. The catalytic activity was significantly dependent on the presence of manganese ions. In addition, the enzyme had excellent stability at 4 ℃, maintaining 80% of its relative activity after 21 days of storage. The K_m values of AappGalNAcT11 for EA2 was 1.152 mmol·L⁻¹,and the K_cat and K_cat/K_m value for EA2 were 21.145 min⁻¹ and 18.6 μmol·L⁻¹·min⁻¹, indicating the AappGalNAcT11 has high catalytic efficiency for EA2.This study provides a new enzyme, AappGalNAcT11, for in vitro modification of mucins by O-glycosylation and provides a theoretical foundation for its application in biosynthesis and the food industry.