Expression Of Rutin Degrading Enzyme In Pichia Pastoris And Analysis Of Its Catalytic Mechanism

Rutin degrading enzymes?RDEs?are key enzymes in the flavonoid metabolism pathway of tartary buckwheat.RDEs exhibit strict substrate specificity and hydrolyze rutin's 3-O glycosidic bond to form rutinose and quercetin.RDEs play key roles not only in plants,but also show the rutin hydrolytic activity and transglycosylation activity in vitro,which make it the potential applications in food additives,drug synthesis and so on.In this study,RDE from tartary buckwheat?FtRDE?was recombinantly expressed in Escherichia coli and Pichia pastoris SMD1168H.Site-directed mutagenesis was carried out to investigate the RDE actives site and the effects of N-glycosylation on its stability and activity.In addition,in order to further study the biological function of RDE in plants,gene knockout vector pBSE401-RDE and the plant overexpression vector pCAMBIA3301-RDE were constructed.Transient expression was carried out in the mesophyll cells of tartary buckwheat and in tobacco.The main findings are as follows:In this study,the secretory expression of RDE was achieved in P.pastoris SMD1168H in the derection of the?-factor secretion signal.The expression level was21.5 mg/L.The optimum temperature was 50°C and the optimum pH was 5.5.The molecular weight of rRDE treated with PNGase F is smaller than that of nRDE,suggesting the existence of N-glycosylation after translation in secreted rRDE.By measuring the enzymatic activity of rRDE and nRDE at different substrate concentrations of rutin,the Km value of rRDE is 1.56 mmol·L^-1 and Vmax value is3.27 mmol·L^-1·s^-1,the Km value of nRDE is 2.04 mmol·L^-11 and Vmax value is 4.19mmol·L^-1·s^-1,showing that the post-translational modifications of rRDE and nRDE had effect on the enzyme activity of RDE.Two active sites E171 and E382 were predicted by multiple sequences alignment and SWISS-MODLE homology modeling combined with the existing structural and functional studies of?-glycosidase.Then mutations of E171L and E382L were inserted by quik change sitedirected mutagenesis.The rRDE_E171L and rRDE_E382L382L mutants were completely inactive,indicating that both E171 and E382 are essential for RDE activity.Site-directed mutagenesis was used to research the predicted Asn of N-glycosylation sites N249,390,N401,and N459 in RDE to Gln,and the secretory expression of each mutant was performed in P.pastoris.The enzymatic activities of the mutant enzymes N249Q,N390Q,and N401Q and N459Q were 1.86×10⁴,1.68×10⁴,1.79×10⁴ and 1.51×10⁴ U·mg^-1,respectively.The activity of the rRDE was1.96×10⁴ U·mg^-1 under the same enzyme conditions,indicating that N-glycosylation at different sites has an effect on RDE activity.The enzymatic activities of the mutant enzymes after incubating 3 min,6 min,9 min and 12 min at 65°C showed that the activity loss of rRDE and the mutant enzymes N249Q,N390Q and N401Q after 12min was 30%,28%,31%and 32%,respectively.The loss of activity of N459Q was62%,indicating that mutation of this single N-glycosylation site may lead to destruction of the conformational stability.A N-glycanase gene OsPNGase A was cloned from rice and expressed in P.pastoris,used as an assisted deglycosylation enzyme for rRDE.The optimal reaction temperature of OsPNGase A was 40°C pH was 6.0.OsPNGase A exhibited enzyme activity in the concentration range of 10-100mM of reducing agent DTT and?-mercaptoethanol.Although OsPNGase A could not deglycosylate the undenatured rRDE,it was able to remove N-glycans from the core?-1.3 fucose,which is abundant in plant glycoproteins,indicating that OsPNGase A has potential applications in the study of plant-derived glycoproteins.However,it cannot be used for the study of glycosylation modification on recombinase under undenatured conditions of rRDE.The research results broaden the source of N-glycanase.The gene knockout vector pBSE401-RDE and the plant overexpression vector pCAMBIA3301-RDE were successfully constructed and transiently expressed in protoplasts of buckwheat mesophyll cells and tobacco.Transgenic plants of tobacco were confirmed by PCR technique and basta resistance.