Effect Of Heterologous Expression Of Mcp Gene On The Tolerance And Adsorption Of Cadmium In Escherichia Coli And Its Mechanism

Heavy metal pollution of farmland has become a major environmental problem to be solved urgently in our country.Due to the difference of soil texture in different regions,the conventional treatment technology is difficult to meet the needs of safe utilization of farmland with moderate and mild pollution in a large area of our country.The use of microbial remediation technology to control heavy metals in farmland soil has a good application prospect,but the single species of microorganisms,poor specificity and the remediation efficiency needs to be improved,which limits the large-scale application and popularization of this technology.Therefore,in this paper,we constructed a heterologous expression plasmid of mcp gene and transformed it into E.coli BL21(DE3)pLysS to study the effect of heterologous expression of mcp gene on the cell interface properties of this strain and the mechanism of its cadmium tolerance and cadmium adsorption capacity.The conclusions of this paper are as follows:(1)A heterologous expression system of mcp gene stably inherited in E.coli BL21(DE3)pLysS was constructed and named as GEM01-MCP strain.The protein(MCP)encoded by the mcp gene(Gen Bank accession number:MW854801)is 32.03k Da in size,which is predicted to bind divalent metal ions.The optimum expression condition was as follows:1.0 mmol/L IPTG,16?for 15 h.(2)The heterologous expression of mcp gene in E.coli BL21(DE3)pLysS improved the tolerance to Cd²⁺(20 mg/L)and adsorption capacity(8.087 mg/g,was2.32 times higher than that of E.coli BL21(DE3)pLysS).Adsorption kinetics and isotherm analysis showed that the Cd²⁺adsorption strength,affinity and reaction rate of mcp heterologous expression bacteria were stronger than that of E.coli BL21(DE3)pLysS,and it was difficult to re-release Cd²⁺after acid leaching or cell autolysis.Also,the adsorption capacity of Pb²⁺,Zn²⁺,Cu²⁺and Ni²⁺was 17.52,7.81,1.63 and 0.76 mg/g,respectively.In addition,with the increase of Cd²⁺concentration(0?5 mg/L),at 96 h,theg_DPH(reflecting cell membrane fluidity)increased from0.098 to 0.195,the cell hydrophobicity increased from 1.41%to 6.52%,and the Zeta potential increased from-49.97 m V to-43.16 m V,thus affecting the expression of mcp gene.After treated with 2 and 5 mg/L Cd²⁺for 96 h,the expression level of mcp gene was 5 times and 3.5 times higher than that of the control group,respectively.(3)MCP protein plays an important role in the adsorption of Cd²⁺by GEM01-MCP bacteria.FTIR and 2D-FTIR-COS proved that the binding order of active sites and functional groups with Cd²⁺in GEM01-MCP was as follows:amide I band>cell wall>MCP>C-C,C-O,and C-N>PO₄^3-and amino.Molecular docking further showed that polar and hydrophilic amino acid residues such as aspartic acid(37/94/98),glutamic acid(45/49/128/166),serine(3/163)and histidine(33/124)on MCP protein were binded with Cd²⁺by electrostatic attraction as binding sites.