A novel serine protease, designated as cordysobin, was purified from dried fruiting bodies of the mushroom Cordyceps sobolifera. The isolation procedure utilized ion exchange chromatography on DEAE-cellulose and SP-Sepharose followed by gel filtration on Superdex 75. The protease did not adsorb on DEAE-cellulose but bound to SP-Sepharose. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protease resolved as a single band with an apparent molecular mass of 31 kDa. Its optimal pH was 10.0, and the optimal temperature was 65 degrees C. The protease displayed a K-m value of 0.41 mu M and 13.44 mu M min(-1) using Suc-Leu-Leu-Val-Tyr-MCA as substrate at pH 10.0 and 37 degrees C. Protease activity was enhanced by the Fe2+ ion at low concentration range of 1.25-10 mM and was strongly inhibited by Hg2+ up to 1.25 mM. The protease was strongly inhibited by chymostatin and phenylmethylsulfonyl fluoride (PMSF), suggesting that it is a serine protease. It manifested significant inhibitory activity toward HIV-1 reverse transcriptase (RT) with an IC50 value of 8.2 x 10(-3) mu M, which is the highest anti-HIV-1 RT activity of reported mushroom proteins. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.