Study On Gene-Cloning, Expression And Property Of Hypericin Synthetase

Hypericin is the most active component in Hypericum.perforatum L. It has the function of anti-depression, anti-virus, anti-tumour and etc. Presently, hypericin is produced by extracted from H. perforatum L, which may cause the lack of wild plant resource of H. perforatum L. The extract contains small amount of hypericin, most of it are other compounds. These compounds may account for some of the reported side effects. Hypericin synthetase(Hyp) is an enzyme which can catalyze emodin to hypericin in a complex way. It's convenient to produce hypericin by the enzyme.The main result was as follows:(1) Total RNA of H. perforatum L was extracted by Trizol. Hyp gene was amplified by RT-PCR and cloned into the vector of pET30a, the recombinant plasmid pET30a-Hyp was transferred into E.coli BL21(DE3). The recombinant strain could express Hyp when it was induced by IPTG. Research showed that Hyp had the ability of converting emodin to hypericin.(2) The fermentative condition of the recombinant strain was studied. The suitable condition of shocking battle of the strain was as follows. The primary pH of medium was 7.0, the amount of inoculation was 1% and the strain was cultured at 37℃ for 3h, then Hyp was induced to express by 0.5mM IPTG at 30℃ > 120r/min for 8h.(3) Hyp was purified after cell collection, suspension, ammonium sulfate precipitation and Ni-NTA column chromatography. The enzyme exhibited higher activity when temperature was 30℃ and pH was 7.0. Mchihaelis constants of enzyme were K_m=5.049μmol/L, V_max= 0.0831 μg/h·mg。...