Abstract
The capping proteins involved in the biosynthesis of gold nanoparticles (AuNPs) have received little attention. The discovery and purification of these proteins is critical for predicting probable production pathways. Understanding the surface composition of biologically synthesized AuNPs can also help with their functionalization for biomedical applications. When compared to bulk gold, AuNPs have intriguing attributes, which ensures them to have a wide range of applications. The aim of this study was to isolate and analyse the capping proteins on AuNPs produced biologically by Enterobacter sp. Pb204. These capping proteins are involved in the biosynthesis of AuNPs. Bacterial cells were extracted by centrifugation for 5 minutes at 3000 g. Cell biomass was applied to HAuCl4 solution and the mixtures were incubated at 37 °C for 72 hours to ensure that Enterobacter sp. Pb204 was capable of synthesizing AuNPs. Following incubation, the colour of the mixture changed from light yellow to deep purple. To confirm the visual findings, ultraviolet-visible absorption spectrophotometry was used to determine the position of the full local surface plasmon resonance effect (LSPR) band. In the ultraviolet-visual (UV-Vis) spectrum, the appearance of a large plasmon peak revealed a heavy plasma resonance at 550 nm. Transmission electron microscope (TEM) was used to examine the average size and distribution, shape, and presence of the capping proteins of the produced AuNPs. The capping proteins associated with the synthesized AuNPs were isolated using a variety of methods in this research. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine all protein-bound AuNPs, or proteins isolated using the different modifications. The peptide bands of interest were excised from the gel and sent to the Centre for Proteomic and Genomic Research for peptide sequencing. The peptides were trypsinized before being analyzed with the Q-Exactive mass spectrometer using liquid chromatography coupled to mass spectrometry (LCMS). The Byonic software generated a list of top proteins that have/include a peptide sequence matches (PSM) that is also found in the three peptides sequenced for this study. The best identifications were chosen and are listed. From the findings, all three peptides showed PSMs with the DNA protection during starvation protein. A second type of protein to which PSMs matched with high frequency was ribosomal proteins. This was true for the 13 and 16 kDa peptides. While the data presented in the study is insufficient to definitively identify the capping proteins found in AuNPs produced by Enterobacter sp. Pb204, it does lead to a technique for isolating significant amounts of these proteins for SDS-PAGE and LC-MS investigations. The remaining larger peptides that were not sequenced due to the presumption iii that capping proteins are small in size should be sequenced in the future to ensure that the capping protein does not fold and function as a homodimer or heterodimer. Key words: AuNPs, Capping protein, SDS-PAGE, Peptide sequence matches (PSM).
M.Sc. (Biotechnology)