Cordysobin, a novel alkaline serine protease with HIV-1 reverse transcriptase inhibitory activity from the medicinal mushroom Cordyceps sobolifera(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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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 31kDa. Its optimal pH was 10.0, and the optimal temperature was 65℃. The protease displayed a K_m value of 0.41μM and 13.44μM・min^<-1> using Suc-Leu-Leu-Val-Tyr-MCA as substrate at pH 10.0 and 37℃. Protease activity was enhanced by the Fe^<2+> ion at low concentration range of 1.25-10mM and was strongly inhibited by Hg^<2+> up to 1.25mM. 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 IC_<50> value of 8.2×10^<-3>μM, which is the highest anti-HIV-1 RT activity of reported mushroom proteins.