Purification and characterization of a novel serine protease from the mushroom Pholiota nameko

Abstract A novel serine protease, with a molecular mass of 19 kDa and the N-terminal sequence of ARTPEAPAEV, was isolated from dried fruiting bodies of the mushroom Pholiota nameko . The purification protocol comprised ion exchange chromatography on DEAE-cellulose, Q-Sepharose and SP-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on DEAE-cellulose and Q-Sepharose but adsorbed on SP-Sepharose. It exhibited an optimum temperature at 50°C, an optimum pH at pH 8.8, a Km of 5.64 mg/mL and a Vmax of 0.98 μmol/min/mL against substrate casein. A number of metal ions inhibited the enzyme including Pb 2+ , Mn 2+ , Ca 2+ , Hg 2+ , Zn 2+ , Cu 2+ , Co 2+ , Fe 3+ and Al 3+ , with the inhibition of the last two cations being the most potent. K + and Mg 2+ slightly enhanced, while Li + moderately potentiated the activity of the protease. The protease was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting that it is a serine protease.