Extraction And Preliminary Purification Of Diels-alderase From Mulberrv Leaf

Mulberry leaf,the leaf of medicinal plants Morus alba L.,has great therapeutical usage which includs the treatment of hypertension,anti inflammation,anti-virus,anti-tumor and improving diabets.Mulberry leaf contains large amounts of flavonoids,alkaloids,plant sterol,?-aminobutyric acid and polysaccharide of Mulberry leaf.Our preliminary study showed that by applying UV-induced enzyme-mediated natural products biosynthesis technology,more Moracin and Diels-Alder adducts emerged in mulberry leaves.The later period experiment of crude enzyme activity which catalyzes the reaction of morachalcone and moracin C,demonstrated that there was a key enzyme for the synthesis of active ingredients in mulberry leaf.The experimental subject of this research is the leaf of Morus alba L.Postharvest mulberry leaves were induced by UV light and then cultured in darkness at 25? for 36 h.Crude enzyme was extracted from treated mulberry leaves by buffer solution.Central composite test optimized the extraction process of crude enzyme.At last,Water dissolved proteins of mulberry leaves were isolated and purified to obtain electrophoretically pure enzyme with high activity.The main results of this paper are as follows:1.We determined the biochemical properties and optimum extraction technology parameters of Diels-Alderase.Firstly,the tolerance temperature and pH range of the Diels-Alderase were studied.The results showed that the crude enzyme is not heat resisting,the activity decreased sharply when the temperature is higher than 40 ?,and inactive at 70 ?.The pH tolerance range of the Diels-Alderase is from 5.0 to 10,and the optimum pH is 7.5.Secondly,the single factor test of optimum extraction technology parameters chose 4 factors,which included liquid material ratio,extraction time,extraction temperature and pH.According to the results of mono-factor experiments,the largely effect element of crude enzyme's extraction is conformed to be liquid material ratio,extraction time and temperature.Then we designed central composite test to determine the optimum extraction process,and the zero level of liquid material ratio,temperature and time respectively was 1/8,20?and 4 h?2.Based on the central composite test,we established the mathematical model which took the extraction rate of mulberry leaves' crude enzyme as target function.The result shows that the most significant to leaching rate is temperature,followed by time and liquid material ratio.According to the forecasting model,the optimal extraction technical condition is liquid material ratio 6:1,temperature 10?,time 4.84 h.The deviation of predicted and actual value is 0.76%.The mathematical model obtained by the central composite test is as follows:Y=-0.211+(1.74E-03)A+0.079B+0.422C-(9.35E-04)AB+(2.07E-03)AC-0.020B C-(1.62E-05)A2+(5.37E-04)B2-0.033C23.Research on the condition of Diels-Alderase's purification.We used different technology which including ammonium sulfate precipitation,PEG precipitation,ion-exchange chromatography,hydrophobic chromatography and gel chromatography,then the optimum purified conditions were determined respectively.Finally the procedure for purification of Diels-Alderase was determined:crude enzyme,ammonium sulfate precipitation,Phenyl-Sepharose HP,Sephadex 200 pg,Q-Sepharose HP.The major fraction with high activity we finally obtained named fraction 9.The result of SDS-PAGE shows that the fraction has high purity.This article determined the optimal extraction technical conditions and biochemical properties of mulberry leaves' crude enzyme,then the electrophoresis pure Diels-Alderase with high activity was obtained,it provides a basis for futher purification and heterologous expression of mulberry leaves' Diels-Alderase.