A protease-resistant α-galactosidase characterized by relatively acid pH tolerance from the Shitake Mushroom Lentinula edodes.

A monomeric α-galactosidase with a molecular weight of 64 kDa was purified from fresh fruiting bodies of Lentinula edodes. The purification protocol involved ion-exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose and a final gel-filtration on Superdex 75. The purified α-galactosidase (LEGI) was identified by LC-MS/MS. It demonstrated the optimum pH of 5.0 and temperature optimum of 60 °C towards pNPGal. It was inhibited by Cd²⁺, Fe³⁺, Pb²⁺, Zn²⁺, Al³⁺, Hg²⁺, Cr²⁺, Ba²⁺. The LEGI activity was strongly abolished by the chemical modification N-bromosuccinimide (NBS) at 1 mM, while significantly enhanced by the thiol-reducing agents dithiothreitol (DTT). Moreover, LEGI showed strong resistance to protease pepsin, papain, acid protease and neutral protease. LEGI demonstrated hydrolysis towards melibiose (13.27%), raffinose (4.75%), stachyose (2.58%), locust bean gum (0.82%) and guar gum (1.29%). The Km values of LEGI for pNPGal, stachyose, raffinose, and melibiose were found to be 1.08, 17.24, 13.80 and 8.05 mM, respectively. Results suggest that LEGI demonstrates potential for elimination of indigestible oligosaccharides.