Purification and characterization of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti

Abstract

The sesquiterpene cyclase, aristolochene synthase, has been purified from Penicillium roqueforti by gel filtration and anion-exchange chromatography. Isolation was facilitated by a change in the elution behavior of the enzyme during gel filtration at different steps in the purification. The purified enzyme had a specific activity of 70 nmol/min/mg protein. The molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was M r 37,000. The native molecular weight as determined by gel filtration chromatography was M r 48,000. The requirement for Mg 2+ could be partially substituted with 0.01 m m Mn 2+, but higher concentrations were inhibitory. Pyrophosphate, a competitive inhibitor of most terpene cyclases, had no effect on enzyme activity up to a concentration of 5.0 m m. The maximum activity was observed between pH 6.25 and pH 7.50, and the K m for farnesyl pyrophosphate was 0.55 ± 0.06 μ m.