In Vitro Recombinant Expression And Catalytic Study Of The Enzyme Related To The Synthesis Of ?-elemene By Mevalonate Pathway

?-elemene,the effective components of Elemene in anticancer drugs,is extracted from traditional Chinese Medicine Curcuma wenyujin.Because of its wide spectrum of anticancer,accurate curative effect and mild side effect,it can be used to treat many kinds of malignant tumors in clinic.Therefore,it is of great significance to improve the yield of ?-elemene in many fields,such as medicine,pharmacy and life science.In vitro multi enzyme catalysis is to add purified enzymes and coenzyme into the in vitro reaction system,so that the substrate can get the final product at a time by sequential multi-step reaction.With respect to the metabolic pathway for the synthesis of target product using traditional method,in vitro catalysis can reduce the metabolic demand of host cell itself to certain substances,and make all the enzyme in the reaction system to catalyze the formation of the final products.With high yield,high speed,easy to control the biological conditions,easy separation of products,and substrate selectivity,the method to study the development of synthetic biology is very fast,and it does' t need to clarify the cellular metabolism in organism complex,which improves the efficiency of the study.In this study,we use the related metabolizing enzymes of the sythesize of?-elemene through mevalerate pathway that had been elucidated.The 5 enzymes(MK,PMK,MVD1,IDI,ispA)of MVA downstream were overexpressed in vitro,and the soluble protein expressed in it are accounted for 90% of the total protein.By purifying the nickel column affinity chromatography,the 5 pure enzymes were obtained,and the concentration of which were 0.9 mg/mL,0.8 mg/mL,2.5 mg/mL,1.1 mg/mL and1.2 mg/mL,respectively.Germacrene A synthase(GAS)is the key enzyme to catalyze precursor farnesyl pyrophosphate(FPP)into germacrene A,then germacrene A can be transfered into?-elemene through simple Cope rearrangement in vitro.This study selected the GAS from Achillea millefolium,and successfully overexpressed it in vitro.The results show that the soluble protein expressed by Achillea millefolium GAS are accounted for 80% of the total protein,which are higher than those expressed by cyanobacterial GAS(inclusion of 76%).Thus we get a higher solubility GAS.We obtain the pure enzyme by nickel column purification with the concentration of 1.2 mg/mL.We construct an in vitro catalytic reaction with the purified MVA downstreampathways of the 5 enzymes and Achillea millefolium GAS,gaseous phase didn't appear obvious peak of target product.We preli minary conclude that the activity of the purified enzyme was decreased in vitro under normal temperature conditions,so it's catalytic ability is very weak,therefore how to improve the activity of enzyme reaction in vitro is our next research direction.For example,we can build substrate channels through DNA molecular scaffolds and protein scaffolds,or immobilize enzymes,which can improve the catalytic activity of enzymes.In summary of the experimental results,we obtained 5 enzyme of MVA downstream pathway and Achillea millefolium GAS to produce germacrene A with high solubility.Besides,we initialy realize an in vitro catalysis reaction of the related metabolic enzyme in MVA pathway to biosynthesis the precursors of ?-elemene,and laid a good foundation for further improvement of the in vitro catalysis reaction to generate ?-elemene.