Abstract: The key steps for preparing antitumor peptide from enzymatic hydrolysates of Perinereies aibuhitensis were investigated in the present work. The most suitable enzyme preparation was selected and the optimal hydrolysis conditions allowing high inhibition of cancer cell proliferation were determined using one-factor-at-a-time method and orthogonal array design. A fraction containing peptides with molecular weights of 1?3 ku was obtained by ultrafilation of hydrolysates, and it was further purified by sequential ion exchange chromatography, gel filtration chromatography and preparative chromatography. Then, lung cancer A549 cells were used to test the anticaner effect of the purified peptide PAP by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The morphological changes were observed through an inverted microscope, dual acridine orange/ethidium bromide (AO/EB) fluorescent staining and Hoechst fluorescent staining. The results showed that alcalase was found to be the optimal enzyme for the production of anticancer peptide and that the optimal hydrolysis conditions were determined as follows: temperature, 50 ℃; pH value, 11; solid-to-liquid ratio, 1:1; hydrolysis time, 6 h; and enzyme dosage, 300 U/g. The peptide PAP was identified as Ile-Glu-Pro-Gly-Thr-Val-Gly-Met-Met-Phe. Moreover, our results demonstrated that PAP suppressed the proliferation of A549 cells in a time- and dose-dependent manner with morphological features of apoptosis being observed. Therefore, our findings suggest that PAP could inhibit the proliferation of lung cancer A549 cells and induced apoptosis.

Key words: Perinereies aibuhitensis, polypeptide, anti-cancer, A549 cell lines, cell apoptosis