Isolation,Purification And Identification Of Silver Nanoparticles-Synthesized Protein From Lysinibacillus Sphaericus

Metal nanomaterials are an important branch of nanomaterials.They use precious metals such as gold and silver.And copper are the representatives,among which silver nanoparticles（Ag NPs）has the most research results.With the popularization of green and environmental protection concepts,biological methods for the synthesis of Ag NPs using biological systems have attracted much attention.Under the protection of biomolecules,the stability and dispersibility of Ag NPs have been greatly improved.It has good biocompatibility,special physical and chemical properties,and excellent antibacterial,anti-inflammatory and low biological toxicity.The material field has unique advantages and a pivotal position.In-depth study of the synthetic mechanism of biological preparation of Ag NPs is of great significance for the preparation of more efficient,controllable morphology and stable antibacterial nano-silver products.Through pre-experiment,this paper found that the supernatant of Lysinibacillus sphaericus fermentation broth after centrifugal sterilization can induce the synthesis of Ag NPs.Because the main component of the supernatant of the fermentation broth is extracellular secreted protein,it is speculated that the synthesis mechanism of Ag NPs may be related to the synergy of certain proteins or certain types of proteins in the supernatant of L.sphaericus fermentation broth.In this study,L.sphaericus was first activated and fermented,and its fermentation broth was separated and purified by centrifugal sterilization,salting out,ion-exchange chromatography,etc.Finally,the purified protein（Ag NPs synthetic protein）with the ability to efficiently synthesize Ag NPs was successfully isolated from the supernatant of L.sphaericus fermentation broth.The protein size is about 115 k Da.The results of the Ag NPs synthesis ability experiment show that 5m L of the purified protein（50μg/m L,p H 11）and 17 mg Ag NO₃can synthesize high-purity nano-silver within 24 h under a shaker at 37℃and 200 rpm.In this study,Maldi-TOF sequencing of Ag NPs synthetic protein was performed.The results showed that the protein is a hypothetical protein[Lysinibacillus sphaericus],containing 1083amino acids.Through correlation analysis of its amino acid composition and protein domain,it was preliminarily presumed that the protein may have the function of biological catalytic reduction,and finally the Swiss-Model was used to simulate the possible three-dimensional structure of the protein.The successful isolation and subsequent identification studies of Ag NPs synthetic proteins are of great significance for revealing the synthesis mechanism of Ag NPs prepared by experimental strain L.sphaericus,and even micro-organisms.The Ag NPs synthetic protein from strain L.sphaericus confirmed its ability to synthesize Ag NPs efficiently through experiments,but the morphology and performance parameters of the prepared nano-silver need to be determined by various characterizations.In this study,Ag NPs prepared by Ag NPs synthesis protein was characterized by EDS energy spectrum,TEM,FTIR infrared spectrum and DLS particle size distribution.According to the characterization results,it is known that the bio-Ag NPs prepared in the experiment have a spherical shape,a small size,a uniform particle size distribution of 30 to 50 nm,and extracellular proteins may be attached to the surface,which has good biocompatibility.Then through the Oxford Cup antibacterial experiment and antioxidant activity experiment,it was found that the biological Ag NPs prepared by the experiment also has excellent antibacterial activity and antioxidant activity.After passing the Oxford Cup antibacterial experiment and antioxidant activity experiment,the results show that the biological Ag NPs has excellent antibacterial activity and antioxidant activity.In short,this thesis purified and discovered the key proteins that play a role in the synthesis of Ag NPs by Lysinibacillus sphaericus and predicted their functions by mass spectrometry identification,revealing the synthesis mechanism of this kind of bacteria to produce Ag NPs.On this basis,this study also successfully amplified the corresponding DNA（which can be called Ag NPs synthesis gene or target gene）of the Ag NPs synthesis protein by PCR technology and completed sequencing,which provided basics for subsequent experimental personnel to carry out prokaryotic expression work.Expect to be able to prepare a large number of bacteria with the ability to efficiently synthesize Ag NPs,which has extremely broad application prospects and industrial value.