|
Hypericin Hypericum perforatum important biologically active substances , has anti-depressant , anti-viral , anti- tumor , anti-bacterial and improve immunity function . Hypericin mainly from Hypericum extract obtained extract hypericin content , and the inclusion of many substances such as flavonoids , phenols, tannins , these substances are often side effects of Hypericum extract sources. The hypericin synthase is a key enzyme in the synthesis of hypericin can catalyze the emodin generated hypericin . Enzymatic production of hypericin mild reaction conditions , the product of a single purification , etc. , there are good prospects for development . The main results of the paper are as follows : 1, obtained by RT-PCR synthase gene encoding hypericin ( Hyp) and cloned into the prokaryotic expression vector pET30a recombinant plasmid pET30a -Hyp transforming Ecoli BL21 works strain engineering Upon induction with IPTG , high-level expression of recombinant proteins that hypericin synthase (Hyp), to determine Hyp has been tested the function of the catalytic emodin synthetic hypericin . 2 engineering optimization of fermentation conditions been more appropriate culture conditions are as follows : an initial pH of 7.0 TB medium , inoculum size 1 % at 37 ° C 140r/min OD 600 about 0.6 (approximately 3H ) , and then IPTG was added to a final concentration of 0.5mM induced , induction temperature of 30 ° C , the rotational speed of 120r/min , induced cells were collected 8h later . 3, the fermentation broth by centrifugation to collect the cells , and the suspended bacterial cells with the buffer , after sonication , ammonium sulfate fractionation , and Ni-NTA affinity chromatography , to obtain electrophoretically pure Hyp . The results show that of the enzymatic properties of the enzyme optimum reaction temperature of 30 ° C , the appropriate reaction pH 6.0 , emodin suitable substrate dissolved in ethanol , Michaelis constant K m < / sub> = 5.049μmol / L, V max = 0.0831μg / h · mg.
|