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Yulan Weng, 1 Jiulong Li, 1 Xingcheng Ding, 2 Binqiang Wang, 1 Shang Dai, 1 Yulong Zhou, 3 Renjiang Pang, 1 Ye Zhao, 1 Hong Xu, 1 Bing Tian, 1, 3 Yuejin Hua 1 1MOE Key Laboratory of Biosystems Homeostasis & Protection, Zhejiang University, Hangzhou, People’s Republic of China; 2Zhejiang Runtu Chemical Research Institute, Shaoxing, People’s Republic of China; 3Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, Xinjiang, People’s Republic of ChinaCorrespondence: Bing TianZhejiang University Zijingang Campus West Part, A403 Biophysics Building, 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of ChinaTel/Fax +86-571-86971215Email tianbing@zju.edu.cnBackground: Biodegradation of toxic organic dye using nanomaterial-based microbial biocatalyst is an ecofriendly and promising technique.Materials and Methods: Here, we have investigated the novel properties of functionalized Au-Ag bimetallic nanoparticles using extremophilic Deinococcus radiodurans proteins (Drp-Au-AgNPs) and their degradation efficiency on the toxic triphenylmethane dye malachite green (MG).Results and Discussion: The prepared Drp-Au-AgNPs with an average particle size of 149.8 nm were capped by proteins through groups including hydroxyl and amide. Drp-Au-AgNPs demonstrated greater degradation ability (83.68%) of MG than D. radiodurans cells and monometallic AuNPs. The major degradation product was identified as 4-(dimethylamino) benzophenone, which is less toxic than MG. The degradation of MG was mainly attributed to the capping proteins on Drp-Au-AgNPs. The bimetallic NPs could be reused and maintained MG degradation ability (> 64%) after 2 cycles.Conclusion: These results suggest that the easilyprepared Drp-Au-AgNPs have potential applications as novel nanomedicine for MG detoxification, and nanomaterial for biotreatment of a toxic polyphenyl dye-containing wastewater.Keywords: bimetallic nanoparticles, Deinococcus radiodurans, biodegradation, toxic triphenylmethane dye, malachite green, detoxification

Background Biosynthesis of noble metallic nanoparticles (NPs) has attracted significant interest due to their environmental friendly and biocompatible properties. Methods In this study, we investigated syntheses of Au, Ag and Au–Ag bimetallic NPs using protein extracts of Deinococcus radiodurans, which demonstrated powerful metal-reducing ability. The obtained NPs were characterized and analyzed by various spectroscopy techniques. Results The D. radiodurans protein extract-mediated silver nanoparticles (Drp-AgNPs) were preferably monodispersed and stably distributed compared to D. radiodurans protein extract-mediated gold nanoparticles (Drp-AuNPs). Drp-AgNPs and Drp-AuNPs exhibited spherical morphology with average sizes of 37.13±5.97 nm and 51.72±7.38 nm and zeta potential values of −18.31±1.39 mV and −15.17±1.24 mV at pH 7, respectively. The release efficiencies of Drp-AuNPs and Drp-AgNPs measured at 24 h were 3.99% and 18.20%, respectively. During the synthesis process, Au(III) was reduced to Au(I) and further to Au(0) and Ag(I) was reduced to Ag(0) by interactions with the hydroxyl, amine, carboxyl, phospho or sulfhydryl groups of proteins and subsequently stabilized by these groups. Some characteristics of Drp-AuNPs were different from those of Drp-AgNPs, which could be attributed to the interaction of the NPs with different binding groups of proteins. The Drp-AgNPs could be further formed into Au–Ag bimetallic NPs via galvanic replacement reaction. Drp-AuNPs and Au–Ag bimetallic NPs showed low cytotoxicity against MCF-10A cells due to the lower level of intracellular reactive oxygen species (ROS) generation than that of Drp-AgNPs. Conclusions These results are crucial to understand the biosynthetic mechanism and properties of noble metallic NPs using the protein extracts of bacteria. The biocompatible Au or Au–Ag bimetallic NPs are applicable in biosensing, bioimaging and biomedicine.

Jiulong Li,1,* Qinghao Li,1,* Xiaoqiong Ma,2,* Bing Tian,1 Tao Li,1 Jiangliu Yu,1 Shang Dai,1 Yulan Weng,1 Yuejin Hua1 1Key Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Institute of Nuclear-Agricultural Sciences, Zhejiang University, 2Central Laboratory, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou,China *These authors contributed equally to this work Abstract: Deinococcus radiodurans is an extreme bacterium known for its high resistance to stresses including radiation and oxidants. The ability of D. radiodurans to reduce Au(III) and biosynthesize gold nanoparticles (AuNPs) was investigated in aqueous solution by ultraviolet and visible (UV/Vis) absorption spectroscopy, electron microscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). D. radiodurans efficiently synthesized AuNPs from 1mM Au(III) solution in 8h. The AuNPs were of spherical, triangular and irregular shapes with an average size of 43.75nm and a polydispersity index of 0.23 as measured by DLS. AuNPs were distributed in the cell envelope, across the cytosol and in the extracellular space. XRD analysis confirmed the crystallite nature of the AuNPs from the cell supernatant. Data from the FTIR and XPS showed that upon binding to proteins or compounds through interactions with carboxyl, amine, phospho and hydroxyl groups, Au(III) may be reduced to Au(I), and further reduced to Au(0) with the capping groups to stabilize the AuNPs. Biosynthesis of AuNPs was optimized with respect to the initial concentration of gold salt, bacterial growth period, solution pH and temperature. The purified AuNPs exhibited significant antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria by damaging their cytoplasmic membrane. Therefore, the extreme bacterium D. radiodurans can be used as a novel bacterial candidate for efficient biosynthesis of AuNPs, which exhibited potential in biomedical application as an antibacterial agent. Keywords: Deinococcus radiodurans, gold nanoparticles, gold ions, reduction, antibacterial&nbsp