Preparation and mechanism of XOD inhibitory peptide from crayfish head based on ultrasound microwave-assisted enzymatic hydrolysis

Crayfish (Procambarus clarkii) head, a common processing by-product, has significant potential for high-value utilization to enhance overall product value. We employed ultrasound-microwave combined pretreatment assisted by alkaline protease hydrolysis of crayfish heads to prepare xanthine oxidase (XOD)-inhibitory peptides, and conducted a systematic investigation on the active component mining, peptide screening/validation, and XOD interaction mechanisms. Results demonstrate that compared with other pretreatments, 4-hour enzymatic hydrolysis after ultrasound-microwave pretreatment enhanced proteolytic efficiency and facilitated the release of low-molecular-mass oligopeptides. Ultrafiltration of the optimal hydrolysate yielded an active fraction (F3) exhibiting superior in vitro XOD inhibition. Four novel XOD-inhibitory peptides, which were WSPDAPF, SGGPWRPL, WTPDAPF, and DWSPPYPT, were identified with IC₅₀ values of 2.00–2.33 mmol·L⁻¹. Through structure-activity relationship analysis and molecular docking, tryptophan residues were identified as crucial for XOD inhibitory activity. The four peptides achieved stable binding by forming hydrogen bonds and hydrophobic interactions with key residues (Leu257, Ile353, Val345) in the XOD active pocket. Inhibition kinetics classified WSPDAPF, SGGPWRPL, and DWSPPYPT as reversible mixed-type inhibitors, while WTPDAPF functioned as a reversible non-competitive inhibitor. The results indicate that these bioactive peptides derived from crayfish heads have good potential for development as natural XOD inhibitors.