Construction Of Rhamnosyltransferase Expression Gentic Engineering Bacteria And Optimization Of Its Expression Conditions Study On

Neohesperidin content in plants is low, its isomer, hesperidin contents in plants is much higher, differences in structure between the two is only flavone ring substituted medium glucose and rhamnose glycosidic bond. The aim of this study is using the biological conversion method of hesperidin into neohesperidin. In this paper,by constructing a former nuclear engineering strain to express rhamnose transferase,the enzyme can be catalytic rhamnose and glucose by 1,2 glycosidic bond connection, in order to synthesize neohesperidin, and study on its enzymatic properties, also the fermentation conditions of the engineering bacteria were optimized. The main research contents and results are as follows:Firstly, Construction and expression of prokaryotic bacteria:The GT2 gene was amplified from Lactobacillus plantarum WCFS1, then the expression plasmid pET-DsbA-GT2 was constructed by linked the GT2 gene and pET-DsbA plasmid. A GT2 expressed engineering strain BL21(pET-DsbA-GT2) was constructed by transforming pET-DsbA-GT2 into BL21. The recombinant strains of BL21(pET-DsbA-GT2) was induced cultivation by IPTG, it was found that DsbA and GT2 were fusion expressed, and the molecular weight of the fusion protein was about 57kDa(GT2:35.7 kDa,DsbA:21 kDa). The expression pattern was partly inclusion body, partly soluble expression.Secondly, The separation and purification of rhamnose transferase and enzymology properties: Inclusion have been obtained respectively through purification protease(GT2-1),soluble(GT2-2), two kinds of rhamnose transferase.The enzyme activity assay showed that the specific activity of GT2-1 was 0.41 U/mg,which was much lower than that of the GT2-2 1.92 U/mg. The research of enzymology characteristics showed: GT2-1, GT2-2 two kinds of enzyme the optimal reaction temperature 38-40 ?, the optimum pH value of 6.0-6.5; GT2-1, GT2-2 in 20 to 40 ? treatment after 1 h of the enzyme activity was reached more than 90%, whenthe temperature of more than 40 ?, GT2-1 enzyme activity decreases rapidly with increasing of the temperature, when the temperature reached 70 degrees has completely lost the enzyme activity and the temperature of the GT2-2 reached 80 ?to complete loss of enzyme activity. PH stability, GT2-1, GT2-2 in the pH value in the range of 5-10 have better stability.Finally, Optimization of expression conditions of recombinant strain BL21(pET-DsbA-GT2): The expression conditions of recombinant strain BL21(pET-DsbA-GT2) were optimized by the expression coefficient F and the expression amount Q. Finally the optimal expression conditions were gotten: induced occasion for the training of 10-12 h after induction; induction time of 5-6 hours; induced by the temperature of 30 ?; induced IPTG at a final concentration of 100 UG / ml, shaker speed to 160 rpm.