Abstract: In order to study the immunomodulatory effects of peptides derived from edible mushroom, crude protein was extracted from dried powder of Pleurotus eryngii and sequentially hydrolyzed with pepsin and trypsin. The resulting hydrolysate was separated and purified by ultrafiltration with membranes with different molecular mass cutoffs (MMCOs of 3, 5 or 10 kDa), Sephadex G-15 column chromatography and high performance liquid chromatography (HPLC). The effects of the products obtained at the end of each step on the proliferation, NO release, phagocytic activity, and mRNA expression of inflammatory cytokines and inducible nitric oxide synthase (iNOS) in RAW264.7 macrophages were assessed. The results showed that among all ultrafiltration fractions, the one with a molecular mass of less than 3 kDa exhibited the highest immunoregulatory activity. After purification by Sephadex G-15 column chromatography and HPLC, fraction Peak 2-2 was obtained, with the highest immunomodulatory activity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for de novo sequencing of Peak 2-2, resulting in the identification of 51 peptide sequences with a confidence score greater than 99. Finally, molecular docking results confirmed the stable binding of P. eryngii peptides DFPALR and LLGVD to toll-like receptor 4 (TLR4), and molecular dynamics simulation showed that the binding was spontaneous and stable. This study indicated that P. eryngii peptides possessed significant immunomodulatory activity.

Key words: Pleurotus eryngii; active peptides; immunoregulation; molecular docking