Novel (R)-Hydroxynitrile lyase enzyme of Pyrus communis: Purification and characterization of its physicochemical and...

Hydroxynitrile lyases (HNLs) play a vital role in the asymmetric synthesis of drug precursors and plant defence. In this study, an R-specific HNL (PycHNL) was isolated and purified from Pyrus communis (pear) seeds using ammonium sulphate precipitation, gel filtration, and ion exchange chromatography, achieving a 14.31 % yield and 6.9-fold purification. Native PAGE estimated a molecular mass of ∼92 kDa, and SDS-PAGE revealed heterodimeric subunits of 52 and 39 kDa. High-Performance Liquid Chromatography confirmed the presence of flavin adenine dinucleotide (FAD). Peptide sequencing showed no significant similarity with other Rosaceae HNLs but indicated partial identity with serine carboxypeptidases and α/β hydrolase fold proteins from Arabidopsis species. Optimal enzyme activity was observed at pH 5.5 and 30 °C, with stability for up to 6 h. Kinetic analysis revealed a K_m of 11.75 mM, V_max of 227.27 μmol/min/mg, k_cat of 101.46/min, and a half-life of ∼1.9 days. Chiral HPLC analysis demonstrated that PycHNL preferentially synthesized (R)-mandelonitrile with 96.33 % enantiomeric excess and 86.83 % molar conversion, indicating its potential for biocatalytic applications in producing enantiopure nitriles.