Establishment Of An Enzymatic Synthesis System Of Flavonoids Apigenin And Optimization Of Reaction Conditions

Apigenin,is a naturally plant flavonoid,also known as apiolin.It has been reported that apigenin possesses a variety of pharmacological activities,such as anti-cancer,anti-oxidation,anti-inflammatory,anti-bacterial,anti-virus and other activities.Because of a wide range of applications in medicine,there are large demands for apigenin.However,traditional extraction method consumes of a large amount of organic reagents and plant raw materials,and the yield is low.The method of chemical synthesis of apigenin is accompanied by extreme reaction conditions and many by-products.In vitro,enzymatic synthesis method of apigenin is simple,environmental-friendly,and mild,which can make up for the shortcomings of the first two methods.Therefore,the method of enzymatic synthesis has a good application prospect.This study focused on the establish of a reaction system for the enzymatic synthesis of apigenin in vitro to replace the traditional methods.Firstly,based on the pathways of synthesis of flavonoids in plants,a pathway for enzymatic synthesis of apigenin in vitro was designed.In this process,four main enzymes were involved,followed by 4-coumarate:CoA ligase 1(4CL1),chalcone synthase 2(CHS2),chalcone isomerase(CHI),flavone synthase I(FNSI).CHI(gm)and 4CL1(gm)genes were cloned from roots and leaves of Glycine max(L.)Merr,and CHS2(sb)and FNSI(ag)genes were cloned from leaves of Sorghum bicolor(L.)Moench and Apium graveolens(L.).Four main enzymes activities were identified in Escherichia coli,and the results showed that the presence of corresponding flavonoids in the fermentation broth,which indicated that these four kinds of key enzyme genes can be functionally expressed in Escherichia coli.The genes encoding four enzymes were successfully constructed into the prokaryotic expression vector pET-32a,and the correct recombinant expressin vectors were transformed into E.coli BL21(DE3)to induced the expression of fusion protein by isopropyl thiogalactoside(IPTG).The soluble recombinant proteins were purified by Ni-agarose resin.SDS-PAGE results showed that the purity of each fusion protein reached about 90%.Then,purified fusion proteins and substrates were mixed to synthesize apigenin through enzymatic reaction,the reaction liquid was detected by polyamide TLC(thin layer chromatography)and LC-QTOF-ESI/MS(liquid chromatography-quantitative time of flight-electrospray ionization mass spectrometry)analysis.The results displayed that the four purified recombinant proteins were biologically active in vitro and could catalyze the production of the corresponding outcomes,finally leading to synthesis of apigenin.Finally,reaction conditions were optimized in order to increase production of apigenin.Through optimization of components,time,pH value,temperature and substrate concentration,reaction conditions were determined as follows:the reaction mixture,consisting of 200 mM Tris-HCl(pH 7.2),10%glycerol,5 mM MgCl2,5 mM ascorbic acid,5 mM ATP,300?M CoA,280?M malonyl-CoA,300?M 4-coumaric acid,0.1 mg/mL bovine serum albumin,0.25 mM?-oxoglutarate,0.1 mM ferrous ion,and four fusion proteins(20?g/mL each),was incubated at 28? for 2h.In summary,we established an enzymatic synthesis method of apigenin in vitro,which provides technical means for production of apigenin and also provides a reference for the synthesis and application of other flavonoid compounds.