A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC50 of 25 µM. The protease did not have antifungal or ribonuclease activity.
In this study, a 39-kDa metalloprotease was purified from a rare edible mushroom with health-promoting activities, Oudemansiella radicata, using a purification protocol which entailed anion exchange chromatography on DEAE-cellulose and Q-Sepharose columns and gel filtration by fast protein liquid chromatography on a Superdex 75 column. Some peptide sequences were obtained by LC-MS/MS analysis and one of the sequences, DAWIQADVNR, manifested 90% identity to Coprinopsis cinerea metalloprotease. The optimal reaction pH and temperature for Oudemansiella radicata protease were pH 7.0 and 50°C, respectively. The protease was purified 79-fold and demonstrated a specific protease activity of 2.42 U/mg. The Km of the purified protease for the casein substrate was 0.65 mg/mL at pH 7.0 and 50°C. The activity of the protease was inhibited by Cd2+, Hg2+, Cu2+, Pb2+ and Fe3+ ions, but was enhanced by K+, Mn2+ and Fe2+ ions. The marked suppression of the protease activity by EDTA indicates that the protease is a metalloprotease.
An acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. By means of chromatographic procedures which involved chromatography on the cation-exchangers CM-cellulose and SP-Sepharose, chromatography on the anion-exchangers DEAE-cellulose and Q-Sepharose, and gel filtration on Superdex 75 using fast protein liquid chromatography, HSG was purified to electrophoretic homogeneity. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 revealed that the enzyme was a monomeric protein with a molecular weight of 38 kDa. Sequences of the inner peptides of the α-galactosidase obtained by MALDI-TOF-MS showed that HSG was a novel α-galactosidase since there was a little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50°C were optimal for the activity of the enzyme. The activity of HSG was inhibited by the chemical modification with N-bromosuccinimide (NBS) reagent. HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which were crucial to the α-galactosidase activity. The heavy metal ions Cd2+, Cu2+, Hg2+ and Zn2+ inhibited its activity. The Km and Vmax for the hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 μM min-1 mg-1. HSG also catalyzed the hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses a tremendous potential for food and feed industries in the elimination of indigestible oligosaccharides from leguminous products.
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