Cloing And Experssion Of Myrosinase And It For Biosynthesis Of Sulforaphane

Myrosinase is a glycoside hydrolytic enzyme,which can catalyze glucosinolates to produce isothiocyanates such as sulforaphane,the substance with anti-cancer and anti-oxidation activities.Myrosinase mainly exists in cruciferous plants and plays a key role in the synthesis of sulforaphane in plants.Therefore,the efficient expression of myrosinase have great significance for the biosynthesis of sulforaphane.In this study,BMYR from cabbage aphid and Bo TGG1 from broccoli were expressed in P.pastoris and E.coli,the effects of p H and temperature on the myrosinase expression in different expression systems were studied,and use it to preparation of sulforaphane.Details are as follows:（1）Recombinant P.pastoris was constructed to express myrosinase.The myrosinase bmyr from cabbage aphid and the botgg1 from broccoli were cloned into plasmid p PIC9K,respectively,then the recombinant plasmids were expressed and induced in P.pastoris GS115.It was found that the protein content and enzyme activity of P.pastoris GS115/p PIC9K-bmyr were higher.Furthermore,the bmyr was cloned into plasmid p PICZαA,P.pastoris GS115 and P.pastoris X33 were used as hosts,respectively,recombinant P.pastoris GS115/p PICZαA-bmyr and P.pastoris X33/p PICZαA-bmyr were constructed.The results showed that P.pastoris X33 combining with vector p PICZαA were more conducive for expression of myrosinase than GS115 and p PIC9K,and the fermentation activity was 0.64 U·m L^-1 in 3 L fermenter.（2）Purification and catalytic synthesis of sulforaphane by BMYR expression in P.pastoris.The myrosinase was purified by anion exchange method,and a single target protein was obtained with specific enzyme activity of 14.32 U·mg^-1.The effects of p H and temperature on the activity of BMYR were investigated.It was found that the optimal p H of BMYR expression from P.pastoris was 4.5,and was stability in the range of p H 3.5～8.5,the stability was better in acidic condition than alkaline condition.The optimum temperature was 50℃,and its thermal stability was good at 20～45℃,but high temperature easily to reduced its activity.Conversion of 3 g·L^-1 glucoraphanin using myrosinase at p H 4.5 and 45℃,1.21 g·L^-1 of sulforaphane was obtained within 60 min.（3）A recombinant strain expressing myrosinase was constructed using E.coli as host.The bmyr and botgg1 were cloned into plasmid p ET-28a（+）and transfected into E.coli BL21（DE3）respectively.E.coli BL21（DE3）/p ET-28a（+）-bmyr was successfully expressed with myrosinase activity of 0.51 U·m L^-1.However,E.coli BL21（DE3）/p ET-28a（+）-botgg1 was failure to expressed myrosinase,the recombinant strain E.coli BL21（DE3）/p ACYCDuet-1-botgg1expressed the protein but existed as an inactive inclusion body.（4）Isolation and purification of BMYR produced by E.coli BL21（DE3）/p ET-28a（+）-bmyr,and whole cell synthesis of sulforaphane.A target myrosinase protein with specific enzyme activity of 15.45 U·mg^-1 was purified by affinity chromatography.Measured the influence of p H and temperature on the myrosinase activity,the optimal reaction p H and temperature of BMYR was 5.5 and 50℃,respectively,and was stability at 20～37℃.The thermal stability of BMYR expression in P.pastoris was better than that in E.coli.Using E.coli BL21（DE3）/p ET-28a（+）-bmyr for whole-cell synthesis of sulforaphane,the catalytic conditions of whole-cell were determined as substrate concentration 4 g·L^-1,the optimum concentration of biomass was5%,p H was 4.5,temperature was 45℃,and then 1.62 g·L^-1 sulforaphane was produced.The operation stability was improved successfully after cell immobilization.