A novel β-primeverosidase-like enzyme, originating from the hypocotyl of soybeans, was isolated and characterized. This enzyme, with an estimated molecular weight of 44 kDa, was identified as a monomer and exhibited peak activity at 55 °C and pH 5.5. It demonstrated a specific and efficient hydrolysis of 1-octen-3-yl β-primeveroside (1-octen-3-yl prim) and 3-octanyl β-primeveroside (3-octanyl prim) but did not act on glucopyranosides. Mn2+ significantly enhanced its activity, while Zn2+, Cu2+, and Hg2+ exerted inhibitory effects. Kinetic analysis revealed a higher hydrolytic capacity toward 1-octen-3-yl prim. Partial amino acid sequences were determined and the N-terminal amino acid sequence was determined to be AIVAYAL ALSKRAIAAQ. The binding energy and binding free energy between the β-primeverosidase enzyme and its substrates were observed to be higher than that of β-glucosidase, thus validating its superior hydrolysis efficiency. Hydrogen bonds and hydrophobic interactions were the main types of interactions between β-primeverosidase enzyme and 1-octen-3-yl prim and 3-octanyl prim, involving amino acid residues such as GLU-470, TRP-463, GLU-416, TRP-471, GLN-53, and GLN-477 (hydrogen bonds) and PHE-389, TYR-345, LEU-216, and TYR-275 (hydrophobic interactions). This study contributes to the application of a β-primeverosidase-like enzyme in improving the release efficiency of glycosidically conjugated flavor substances.
Keywords: molecular docking; primeverosically bound precursors; purification; soybean hypocotyl; β-primeverosidase-like enzyme.