Biological Removal Of Heavy Metal Ions Based On CsgA Protein Functionalization Modification

Heavy metal pollution in water brought about by industrial development poses a huge challenge to environmental remediation technology.However,the current technology for removing heavy metals in water is prone to secondary pollution and expensive,so it is urgent to develop new technologies that are green,efficient,and low-cost.The in vitro self-assembly and multifunctional properties of amyloid fibrils reveal their potential to bind heavy metal ions.Based on this,this study constructed a programmable and recyclable microbial amyloid（CsgA）system for the bioremediation of heavy metal pollution in water.First,the CsgA extracellular self-assembly engineering strain was successfully constructed to achieve Pb²⁺adsorption;And phytochelatin synthase（PCS）was fused with CsgA protein to remove low-concentration heavy metals;In addition,a bioactive hybrid membrane was prepared;Finally,an artificial protein fiber-Fe₃O₄ complex was constructed,which could efficiently remove a variety of low-concentration heavy metal ions and achieve material recyclability.The main contents and conclusions are as follows:（1）To achieve overexpression of CsgA protein,this study constructed strain E.coli BL21（DE3）/p ET22b-CsgA,and the congo red（CR）staining method was used to determine that the optimal concentration of isopropyl-β-D-thiogalactoside（IPTG）was 0.5 m M.By exploring the adsorption performance of the strain on Pb²⁺,it was shown that the optimum p H value for removing Pb²⁺was between 5 and 7.When the initial lead concentration was 50 mg·L^-1,the removal rate reached up to 98.26%after adsorption for 6 h,which was much higher than that of the wild-type strain.Combining the isothermal adsorption model,it was concluded that the adsorption of Pb²⁺by the strain was more in line with the freundlich model.（2）In order to realize the high-efficiency adsorption of low-concentration heavy metal ions by recombinant strains,this study fused PCS with CsgA protein through the Spy Tag Spy Catcher strategy.By exploring its adsorption performance for heavy metals,it was determined that the strain removal of lead ions reached the maximum at 6 h,and the optimum p H value was between5-7.In the removal of different heavy metal ions(Pb²⁺,Cu²⁺,Cd²⁺),the fusion protein strain had the highest removal rate of Cd²⁺with an initial concentration of 1 mg·L^-1,reaching 91.85%.（3）In order to improve the practicability of the adsorption material,the bioactive hybrid adsorption membrane was prepared by mixing the successfully constructed fusion protein strain with activated carbon.When the initial concentration was 1 mg·L^-1,the removal rates of Cd²⁺,Pb²⁺and Cu²⁺were 98.70%,95.33%and 85.67%,respectively.And after the same composite membrane was repeated 6 times,the removal rate of Pb²⁺,Cu²⁺and Cd²⁺remained above 80%.In the simulated wastewater,the Cd²⁺removal capacity of the composite membrane containing the fusion protein PCS was still greater than 90%.It was indicating that the composite membrane has strong stability for the adsorption of heavy metal ions and environmental adaptability.（4）In order to achieve more convenient recycling of adsorption materials,artificial protein fiber-Fe₃O₄ complexes were prepared.By exploring its adsorption performance for heavy metal ions,the results showed that the maximum removal rate of heavy metal ions by the CsgA-Fe₃O₄complex could reach 98.73%(Cu²⁺)>98.43%(Cd²⁺)>93.00%(Pb²⁺)after 150 min adsorption equilibrium.The optimum p H value was 6.5.The adsorption of Cd²⁺by adsorption materials was mainly specific adsorption.The adsorption mechanism of Pb²⁺and Cu²⁺was dominated by electric adsorption,supplemented by specific adsorption.At the end of six cycles,the removal capacity of Pb²⁺,Cu²⁺and Cd²⁺was still more than 80%.In multi-component simulated wastewater,the removal effect of heavy metal ions with initial concentration of 1 mg·L^-1 was94.73%(Cu²⁺)>93.10%(Cd²⁺)>91.57%(Pb²⁺).（5）Analysis of adsorption mechanism.The analysis of fourier transform infrared spectroscopy（FT-IR）and X-ray photoelectron spectroscopy（XPS）showed that the characteristic vibration of the hydroxyl group of CsgA-Fe₃O₄ composite adsorbed heavy metal ions shifted towards the low wavenumber direction by about 30 cm^-1 and the O 1s moved to the direction of low binding energy.Oxygen participated in the adsorption of heavy metal ions by CsgA-Fe₃O₄ complex,in which-OH played a major role.