The Purification and Characterization of a Novel Neutral Protease from Volvariella volvacea Fruiting Bodies and the Enzymatic Digestion of Soybean Isolates.

Abstract

A novel protease was isolated from the fruiting bodies of the straw mushroom Volvariella volvacea. The protease was purified 13.48-fold using a series of techniques, including ammonium sulfate precipitation, ultrafiltration, diethylaminoethyl fast-flow (DEAE FF) ion-exchange chromatography, and Superdex 75 gel filtration chromatography, resulting in a specific enzyme activity of 286.82 U/mg toward casein as a substrate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the purified protease had a molecular weight of 24 kDa. The enzyme exhibited optimal activity at pH 7 and 50 °C, showing sensitivity to alkaline conditions and instability at elevated temperatures. The presence of Ca²⁺ significantly enhanced enzyme activity, whereas Ni²⁺ and Cu²⁺ exerted strong inhibitory effects, with other metal ions showing weak inhibition. β-mercaptoethanol, Tween-80, and Triton X-100 had more pronounced inhibitory effects, whereas PMSF, EDTA, and CTAB had weaker inhibitory effects. The Michaelis constant (Km) and maximum velocity (Vm) of the protease were determined to be 1.34 g/L and 3.45 μg/(mL·min), respectively. The protease exhibited a greater degree of enzymatic degradation of soybean-isolate protein (7.58%) compared to trypsin (5.24%), with the enzyme product containing a high percentage of medicinal amino acids (73.54%), particularly phenylalanine (Phe) and arginine (Arg), suggesting their presence at the enzyme's active site. These findings suggest that the protease from V. volvacea holds promising potential for applications in the food industry, particularly in protein hydrolysate production and flavor enhancement.