Cloning, biochemical identification and characterization of purine nucleoside N-ribohydrolase in Levilactobacillus brevis

Purine nucleoside N-ribohydrolase (PNase) plays a crucial role in purine metabolism and possibly in hyperuricemia management by degrading purine nucleosides. However, the genetic basis of the enzyme activity in lactic acid bacteria (LAB) remains largely unexplored. This study aimed to clone and express four putative PNase genes from Levilactobacillus brevis LABC170, and to identify the gene(s) responsible for degrading adenosine, guanosine, and inosine. Furthermore, we studied optimal reaction conditions and enzyme kinetics. The four putative PNase genes were cloned, and enzymatic activity was evaluated by high-performance liquid chromatography (HPLC). The optimal pH and temperature were determined, and kinetic parameters were analyzed. Among the candidates, the recombinant PNase 3 gene product exhibited purine nucleosidase activity with optimal activity at pH 7 and temperatures between 35 and 40 °C, while it failed to degrade pyrimidine nucleosides. Kinetic analysis showed the turnover number for adenosine, with a Kcat of 2.18 × 10³ ± 2.29 × 10² min⁻¹ and a catalytic efficiency of 2.79 × 10⁴ ± 2.94 × 10³ mM⁻¹·min⁻¹, followed by guanosine and inosine. PNase 3 from L. brevis LABC170 demonstrates promising potential for hyperuricemia management and application in functional foods aimed at modulating purine metabolism.