Biosynthesis Of Bimetallic Nanoparticles Using Deinococcus Radiodurans Protein Extracts And Their Degradation Mechanism On Polybenzene Dyes

Metal nanoparticles are widely applied in biology and medicine fields due to their nanoscale size and novel physicochemical properties.Among various synthesis methods,the biosynthesis of nanoparticles,especially bimetallic nanoparticles,using microbial bacterial and their extracts,has great advantages such as excellent biocompatibility,good dispersity,high specific surface area and functional diversity,and has attracted much attention in the research fields of durg carrier,industrial catalysis and environmental remediation.The extremophile-Deinococcus radiodurans?DR?has exceptional radiation,oxidation resistance and damage repair ability.With rich reducing substances in its cells,it has great application potential in the biosynthesis of functionally modified metal nanoparticles.This study used D.radiodurans protein extracts to directly mediate synthesis of gold and silver bimetallic nanoparticles.We detailed the material structure,surface characteristics,synthesis mechanism and application potential based on biochemical and molecular biology,biophysics,analytical chemistry and nanoscience.Its value and application potential in the degradation of polybenzene toxic dyes were further explored,and the specific functions and mechanisms of functional-related proteins capping on nanoparticles were investigated and discussed.The following conclusions are obtained:?1?Au-Ag bimetallic nanoparticles capped and functionalized using extremophilic Deinococcus radiodurans proteins?Drp-Au-AgNPs?were biosynthesized.In the synthesis process,Drp-AgNPs was generated by the reduction reaction of the protein with the silver nitrate?AgNO₃?,and then HAuCl₄·3H₂O was added to form Drp-Au-AgNPs.The characteristics of bimetallic nanoparticles were characterized by means of X-ray diffraction?XRD?,dynamic light scattering?DLS?,Fourier transform-infrared spectroscopy?FT-IR?and X-ray photoelectron spectroscopy?XPS?.The synthesized nanoparticles were spherical,with an average hydration particle size of 149.8 nm and a polydispersity index?PDI?of 0.397±0.021.The synthesis amount and particle size were affected by temperature,pH conditions.The surface of the bimetallic nanoparticles was covered with capping proteins,with characteristic crystal faces of Ag and Au,which are the characteristics of the bimetallic materials.The hydroxyl and amide bonds,phosphorus-containing oxygen functional groups and protonated amine groups in the protein of Deinococcus radiodurans were all involved in the metal reduction process.First,Ag?I?was reduced to Ag?0?,then Au?III?was reduced to Au?I?and further reduced to Au?0?.Drp-Au-AgNPs had good stability in the solution,and the metal release rates of Drp-Au-AgNPs at room temperature for 48 hours were Ag%=16.17%and Au%=3.69%,respectively,which are relatively low compared with materials of the same kind.More than 80%MCF-10A survived under 25?g/mL Drp-Au-AgNPs treatment,indicating that Drp-Au-AgNPs is low-toxic and biocompatible.?2?Drp-Au-AgNPs has the ability to degrade and detoxify toxic polybenzene dyes.It can decolorize polybenzene dyes such as malachite green?MG?,reactive black?RB-5?and safranin O?SO?efficiently.Drp-Au-AgNPs has higher decolorization efficiency compared with cell culture,protein extract and single metal nanoparticles from D.radiodurans.GC-MS analysis showed that Drp-Au-AgNPs caused the oxidative cleavage of the central carbon bond between benzene rings of MG,and generated low toxic product 4-?dimethylamino?benzophenone?4-DMABP?.The degradation and detoxification were attributed to the capping proteins on Drp-Au-AgNPs.FT-IR and XPS showed that the hydroxyl group,amide bond,phosphorus-containing functional group and carboxyl group of the Drp-Au-AgNPs surface proteins might be involved in the interaction with MG degradation.The used Drp-Au-AgNPs can be regenerated by centrifugation and washing.After regeneration,the elements composition,crystal structure,particle size and other characteristics of the NPs remained unchanged.The degradation capacity of Drp-Au-AgNPs can be maintained at more than 60%of the original material in 3 cycles.?3?Drp-Au-AgNPs capping proteins were identified by mass spectrometry.A variety of related redox proteins and enzymes were identified,which might provide strong reduction capacity for the synthesis of metal nanoparticles and degradation of polybenzene dyes.DRA0145 is a C-type Dye-decolorizing peroxidase?DyP?homolog,and has several highly conserved sites in its sequence,such as Asp189 and Arg348.In addition,through homologous sequence alignment,it was found that DR1966 in D.radiodurans,a homolog of polyphenol oxidase-laccase,might also have the function of polybenzene dyes degradation.The evolutionary characteristics of these two proteins were predicted by analyzing their molecular evolutionary tree.The survival rate of gene mutants?drA0145 and?dr1966 was lower than that of the wild type under hydrogen peroxide stress,indicating that these two proteins were related to the oxidative resistance of D.radiodurans.Heterogenously expressed and purified DRA0145 and DR1966 were obtained.It was proved that in vitro both DRA0145 and DR1966 could directly degrade MG to the low toxic product 4-DMADP,in which the degradation percentage of DRA0145 was 91.86%and that of DR1966 was 80.10%.In summary,this thesis investigated biosynthesis of Deinococcus radiodurans protein extracts mediated gold-silver bimetallic nanoparticles.The characteristics and synthesis mechanism of the Drp-Au-AgNPs were clarified.The role of Drp-Au-AgNPs in degrading and detoxifying polyphenyl dyes and related genes and proteins of D.radiodurans involved in the degradation were studied.The results provided new ideas and theoretical references for the study of synthesis and values of functionalized metal nanomaterials.Compared with bacterial cells used for polyphenyl dye degradation,Drp-Au-AgNPs have the functionalized modification with capping proteins,and have good stability,dispersion and biological safety in solution.This study will provide support for the application of nanomaterials,including the functionalized D.radiodurans mediated bimetallic nanoparticles,in the field of environmental remediation and biomedicine.