Purification,Identification And Heterogenous Expression Of Diels-alderase In The UV+D Treated Mulberry Leaves

Moraceae have a total of 37 genera and 1100 species worldwide,including mulberry,breadfruit trees,Artiaris toxicaria et al.Mulberry leaves are the food source of silkworm and it's important for the silk formation.Because of the abundant active substrate,the leaves,the fruits and the bark are used for traditional medicine.The previous studies in our lad found that the contents of moracin C,moracin N,morachalcone,guangsangon E and chalcomoracin in UV+D treated leves were significantly increased.Chalcomoracin is a kind of Diels-Alder adduct with antioxidant activity,antiviral activity,antibacterial activity,antineoplasmic activity and hypoglycemic activity.Unfortunately,according to our knowledge,there's no mature chemical synthesis nor biosynthesis route for chalcomoracin and chalcomoracin can only be extracted from mulberry bark and mulnerry leaves.Although the content of chalcomoracin in mulberry leaves increased after UV+D treatment,the yield is still low.Diels-Alderase is a kind of enzyme in mulberry,which can catalyze moracin C and morachalcone to form chalcomoracin.In this study,the specific activity of Diels-Alderase and the result of SDS-PAGE were set as the index to purify the Diels-Alderase in UV+D treated leaves.The results are as follows.The UV+D mulberry leaves were treated with 100 mM PBS at a solid-liquid ratio of 1:10 and then extracted at 4 ? for 2 h.The supernatant was collected after centrifuged at 12000 g 4 ? for 20 min.The supernatant was subsided by Ammonium sulfate fractionation at an ammonium sulfate saturation during 40 and 60 percent.The protein pellet was redissolved with PBS(pH = 7.5)containing 0.8 M ammonium sulfate.After hydrophobic interaction chromatography,anion exchange chromatography,cation exchange chromatography and gel filtration chromatography I got purified Diels-Alderase.Throughout the purification process,the specific activity of Diels-Alderase increased,the purification fold increased and the recovery decreased.After purification by gel filtration chromatography,protein purification folds up to 25.29 times,the protein recovery rate was only 0.04%.The highly-purified protein provides crutial information for the identification of the target protein.The purified protein was analyzed by LC-MS/MS and MALDI-TOF/TOF.The target protein chloroplast sedoheptulose-l,7-bisphosphatase(SBPase)was finally determined by comparing with the mulberry leaf transcriptome data.The complete open reading frame was 1179 bp with 392 amino acids.The predicted molecular weight was 42521.55 Da and the theoretical isoelectric point was 5.96.In addition to the predicted target protein,there are 26 common proteins found in the LC-MS/MS result.In the study of SBPase cloning and expression,pET-19b was used as the vector,and the expression strain was BL21.SDS-PAGE showed that a large amount of soluble recombinant protein was expressed after 4 h l mM IPTG induction at 37 ?.The results showed that the recombinant protein could catalyze the formation of chalcomoracin utilizing moracin C and morachalcone to form chalcomoracin non-efficiently in the presence of FAD.In this thesis,Diels-Alderase was isolated from UV+D treated mulberry leaves,and the amino acid sequence of candidate proteins was obtained by MALDI-TOF/TOF and LC-MS/MS.The prokaryotic expression system was used to heterogenously express the suspected target protein and verify its activity.The establishment of the screening criteria of the Diels-Alderase in mulberry leaf lays the foundation for the study of chalcomoracin biosynthesis pathway and structural biology of the Diels-Alderase in chalcomoracin biosynthesis pathway.