Enzymatic characteristics and origin tracing of nucleotidase and adenosine deaminase in Cordyceps sinensis based on HPLC

Adenosine is a key quality marker for the valuable fungus Cordyceps sinensis ( C. sinensis ). This study presents the first HPLC-based method for quantifying the activities and kinetic parameters of two adenosine-metabolizing enzymes—nucleotidase and adenosine deaminase—in C. sinensis . The enzyme extraction protocols, reaction systems, and assay procedures were optimized. Water was selected as the extraction solvent to preserve native enzymatic activity. Nucleotidase exhibited optimal activity at pH 6 and 35°C, while adenosine deaminase showed optimal activity at pH 8 and 45°C, revealing distinct catalytic profiles. Adenosine deaminase exhibited superior thermostability compared to nucleotidase. Kinetic analysis revealed that nucleotidase had a Michaelis constant of 0.24 µmol·mL -1 and a maximum reaction rate of 7.92 µmol·L -1 ·min -1 , while adenosine deaminase showed corresponding values of 0.14 µmol·mL⁻¹ and 0.61 µmol·L⁻¹·min⁻¹. Method validation confirmed the reliability of the analytical system. Source-tracing analysis revealed that nucleotidase originated primarily from the fungal component, whereas adenosine deaminase was exclusively derived from the host larva and detected only in insect tissues. Therefore, adenosine deaminase activity serves as a reliable marker for distinguishing insect-free fungus from fungus-insect complex. These findings provide a scientific basis for optimizing adenosine accumulation and improving the quality control and management of C. sinensis .