Enhancement Of The Catalytic Activity Of Dye Decolorizing Peroxidase TfuDyP In Recombinant E.coli

At present,a new member of this family,the dye decoloring peroxidases?DyPs?,has attracted much attention because it can decolorize multiple dye wastewater.However,the limitation of prosthetic heme results in the low activity of the proteins in heterologous expression.Therefore,while increasing the expression of DyPs,the content of prosthetic heme should be increased as much as possible.Finally,the combination of enzyme protein and more prosthetic groups becomes the key factor.The object of this research is the dye decoloring peroxidase?TfuDyP?derived from Thermobifida fusca,which is co-expressed with key enzyme genes that play important role in the heme C5 synthesis pathway.With the heme content increasing,the binding of the enzyme and prosthetic heme is enhanced and ultimately improves the enzyme activity.Then,three kinds of refractory dyes were chose to explore the decolorization ability of TfuDyP.The dyes are Reactive Blue 19,Congo Red and Bromocresol Green,belonging to anthraquinone,disazo and triphenylmethane categories,respectively.We selected Bromocresol green as a substrate and optimize the reaction conditions.Since TfuDyP enzyme is an intracellular enzyme that is hard to be deployed in industrial applications,we used OsmY as a fusion vector to promote the extracellular secretion of the recombinant protein TfuDyP.The main contents are as follows:?1?The effect of overexpression of key enzyme genes of heme synthesis pathway on the content of heme in E.coli.Genes of hemA,hemL,and hemH were overexpressed in E.coli,respectively.The results showed the heme content was significantly increased in the over-expressing hemA strain,reaching 24.2?mol?L^-1,while the heme content in the strains overexpressing hemL and hemH did not change significantly.hemA was co-expressed with hemL and hemH,and it was found that the heme content after co-expression did not increase compared with the single overexpression of hemA.?2?The effect of co-expressing heme and TfuDyP genes on TfuDyP enzyme activity.Porphyrins has obvious characteristic absorption peaks at 400?405 nm.Quantitative analysis of the content of porphyrins in each bacterial solution after induction indicated that the maximum absorption value of pAD reached 0.342,which was higher than the pD absorption value of 0.126.In addition,the heme concentration of the pD strain was determined to be 3.4?mol?L^-1,while the heme content of the strain pAD was significantly increased to 9.8?mol?L^-1.Spectral analysis of the pure TfuDyP enzyme found Soret value of TfuDyP in the co-expressed strain pAD was 0.794 compared with that of 0.637 in pD strain.Detection of specific enzyme activity of TfuDyP showed that the enzyme activity of pD strain was 4.5U?mg^-1,while that of pAD reached 9.2 U?mg^-1,increased by 110%.Exogenous addition of FeCl₂ and Glu to the pAD strain increased the enzyme activity of TfuDyP to 11.8 U?mg^-11 and13.2 U?mg^-1,respectively.?3?Taking Bromocresol Green as the research object,the optimal conditions for decolorization of TfuDyP were investigated.It was found that the optimum reaction temperature,pH,reaction time,enzyme concentration and mass concentration of Bromocresol Green were:25?,4.5,60 min,40 U?L^-11 and 80 mg?L^-1.?4?Secretion of recombinant TfuDyP in E.coli.OsmY protein was selected as a fusion carrier to mediate the extracellular secretion expression of recombinant protein TfuDyP.The results showed that OsmY protein and TfuDyP were successfully fused and expressed in E.coli.After 12 hours of induction in 100 mL LB liquid medium,the fusion protein activity in the extracellular supernatant was 5.7±0.25 U?L^-1.