| Panax ginseng,as the representative plant of Panax,has been applied in China for thousands of years,it is a traditional previous Chinese herbal medicine which belongs to perennial herb of Araliaceae.Ginsenoside is the main active ingredient in Panax including Panax ginseng,existing in roots,stems,and leaves of Panax ginseng.Ginsenoside Rh2 is one kind of ginsenosides that has very high medicinal value,especially in prevention and treatment of cancer,yet its producting technology has not been found.The study indicates that ginsenoside Rh2 is synthesized through a series of metabolism,which goes through squalene,2,3-oxidospualene,dammarenediol,protopanaxadiol,and protopanaxadiol is finally covered into ginsenoside Rh2 by enzyme catalyzing of glycosyltransferase UGT-D-3OH-1.Industrial production of protopanaxadiol has been realized through fermentating by now.The purpose of this study is to construct engineering bacterias of prokaryotic expression of glycosyltransferase UGT-D-3OH-1 using modern molecular biology technology.Because of high-efficiency expression of prokaryotic expression system,glycosyltransferase UGT-D-3OH-1 could be produced by fermentation,in order to achieve converting protopanaxadiol into ginsenoside Rh2 by enzyme engineering.In this study,the gene of glycosyltransferase UGT-D-3OH-1 was cloned through genetic engineering technology from Panax ginseng hairy roots that had been subcultured in the lab,and the gene was ligated respectively into the prokaryotic expression vectors pET-22b?+?and pET-28a?+?to construct recombinant vectors.Then the two constructed recombinant vectors were transformed into E.coli BL21?DE3?respectively to build engineering bacterias BL21-UGT-22 b and BL21-UGT-28 a of prokaryotic expression.After culturing the engineering bacterias with IPTG inducing expression,verifying target protein through SDS-PAGE,renaturating inclusion body and assaying enzyme activity,the target protein with enzymatic activity was finally obtained,ginsenoside Rh2 was produced by enzymic catalytic reaction in vitro using protopanoxadiol as substrate.Fermentation condition of the engineering bacteria was optimized further to provide solid foundation for realizing industrialized production of ginsenoside Rh2.The main contents of this study are shown as follows:?1?The total RNA was extracted from hairy roots of Panax ginseng that has cultured for 28 d,the c DNA was synthesized by RT-PCR from the total RNA.Then the specific primers for UGT-D-3OH-1 were designed based on the known sequence in NCBI and the requirements of introducing restriction enzyme cutting site,with the obtained c DNA as template,the gene of glycosyltransferase UGT-D-3OH-1 was successfully cloned finally.?2?The gene of UGT-D-3OH-1 was ligated into the cloning vector of pMD18-T to construct the recombinant vector,which is named pT-UGT.After that,transforming the recombinant vector into E.coli JM109.Then performing bacterial fluid PCR and restriction enzyme digestion firstly,gene sequencing secondly,confirmed that the recombinant vector pT-UGT were successfully constructed.?3?BamHI and Sal I were used to digested the recombinant vector pT-UGT,the prokaryotic expression vectors pET-22b?+?and pET-28a?+?were linearized by this two restriction enzymes respectively at the same time.Then the gene UGT-D-3OH-1 from vector pT-UGT was ligated into prokaryotic expression vectors pET-22b?+?and pET-28a?+?using T4 DNA Ligase to construct recombinant vectors pET-22b-UGT and pET-28a-UGT.Gene sequencing was used to confirm the correction of the target gene sequence which has been ligated into the two prokaryotic expression vectors.The recombinant vectors pET-22b-UGT and pET-28a-UGT were then transformed into E.coli BL21?DE3?respectively.The two engineering bacterias BL21-UGT-22 b and BL21-UGT-28 a of prokaryotic expression were built as expected at last.?4?The two built engineering bacterias were cultured respectively.After IPTG induced,the expression of the target protein from engineering bacterias was verified by SDS-PAGE.The result indicated that the target protein could be expressed as insoluble inclusion body in both of the two engineering bacterias.The molecular weight of target band in BL21-UGT-22 b was about 50 KDa,which was 5 KDa less than the predicted molecular weight.The molecular weight of target band in BL21-UGT-28 a was the same as the predicted one,about 55 KDa.?5?Renaturation was performed to activate the target protein which was expressed as inclusion body in BL21-UGT-28 a.After renaturating,the corresponding substrate was added to conduct enzymatic reaction.HPLC was used to verify the reaction product of ginsenoside Rh2 and the result was 6.657×10-3 mg/L.It indicated that the inclusion body after renaturation had normal enzymatic activity,which was about 1.78×10-4 U/m L.?6?Fermentation condition optimization for BL21-UGT-28 a was preformed from four aspects including the initial induced OD600 nm,the concentration of IPTG,the inducing time,the inducing temperature.The optimal fermentation conditions of BL21-UGT-28 a were the initial induced OD600nm=0.8,the concentration of IPTG= 0.1 m M,the inducing time 6 h,the inducing temperature 37 ?.Under this fermentation condition,the expression quantity of the target protein took up53.5% in the expression quantity of the total protein. |
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