Purification OF THCA synthase from Cannabis callus

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The occurrence of DELTA^1-tetrahydrocannabinolic acid synthase was confirmed in the fresh leaf buds of marihuana (Cannabis sativa L.). The enzyme, assayd with cannabigerolic acid, was purified approximately 300-fold by chromatography on diethylaminoethyl-cellulose, phenyl Sepharose CL-4B and hydroxylapatite. The purified protein migrated during sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single band with a molecular mass of 75kD.The native molecular mass determined by gel-filtration chromatography was 74 kDa, indicating that DELTA^1-tetrahydrocannabinolic acid synthase is a monomeric enzyme. Using isoelectric focusing, we determined a pI of 6.4 for the enzyme. The enzyme activity was greatest between pH5.5 and 6.0. The enzyme catalyzed the conversion of cannabigerolic acid or cannabinerolic acid to DELTA^1-tetrahydrocannabinolic acid. The V_<max> values were 2.68 and 0.37 nkat/mg for cannabigerolic acid and cannabinerolic acid, respectively. These results indicated that DELTA^1-tetrahydrocannabinolic acid is predominantly formed from cannabigerolic acid.