β-Cyclopiazonate oxidocyclase from Penicillium cyclopium. I. Assay methods, isolation and purification

1. 1. Spectrophotometric and colorimetric assay methods for the dehydrogenation of β-cyclopiazonic acid and the conversion of β- into α-cyclopiazonic acid, both catalysed by β-cyclopiazonate oxidocyclase, were developed. Paper chromatography, ultraviolet spectrophotometry and mass spectrometry were also used in identifying the product, α-cyclopiazonic acid, formed in the above conversion. 2. 2. Five isoenzymes of β-cyclopiazonate oxidocyclase were isolated and purified from the mycelium of Penicillium cyclopium Westling by homogenization and extraction, differential centrifugation, (NH₄)₂SO₄ fractionation, DEAE-cellulose chromatography, Sephadex G-100 gel chromatography and CM-Sephadex C₅₀ chromatography. The final yield was 64% and the average final enrichment for the five isoenzymes 133. 3. 3. The results of microzone electrophoresis, polyacrylamide gel electrophoresis and analytical ultracentrifugation indicated pure and homogeneous preparations of all five isoenzymes. 4. 4. The optimum pH for the dehydrogenation reaction and the conversion of β- into α-cyclopiazonic acid was found to be 6.8. 5. 5. The five isoenzymes had essentially the same sedimentation coefficients (s^o_20,w approx. 4.0·10^-13 sec); diffusion coefficients (D_20,w approx. 7.3·10^-7 cm²·sec^-1) partial specific volumes (ν, 0.73 cm³·g^-1) and molecular weights (mol. wt. approx. 50 000). 6. 6. The isoenzymes showed a similar specific activity for both the dehydrogenation and the conversion of β- into α-cyclopiazonic acid and occurred in a constant relative ratio.