The Characterization of the Purine Nucleoside Phosphorylase from Agaricus bisporus and Its Potential Application in Reducing Purine Content in Beer

Beer, the most popular alcoholic beverage, poses health risks for individuals with gout and hyperuricemia due to its high purine content. Herein, we identified a novel purine nucleoside phosphorylase (<i>Ab</i>PNP) from the edible mushroom <i>Agaricus bisporus</i> and heterologously expressed it in <i>Pichia pastoris</i>. The recombinant <i>Ab</i>PNP exhibited optimal activity at 60 °C and pH 7.0, retaining >80% activity at pH 6.0-9.0 and >85% activity after 3 h at ≤60 °C. Kinetic analysis revealed high catalytic efficiency (kcat/Km = 2.02 × 10⁶ s^-1⋅M^-1) toward inosine, with strong resistance to metal ions except for Co²⁺ and Cu²⁺. The application of <i>Ab</i>PNP (1.0-5.0 U/mL) during wort saccharification reduced purine nucleosides by 33.54% (from 151.53 to 100.65 mg/L) while increasing yeast utilization of free purine bases. The resulting beer showed improved fermentation performance (alcohol content increased by 3.6%) without compromising flavor profiles. This study provides the food-grade enzymatic strategy for low-purine beer production, leveraging the GRAS status of both <i>A. bisporus</i> and <i>P. pastoris</i>.