ABSTRACT
The interaction of three proteins, viz. Bovine Serum Albumin (BSA), Human Serum Albumin (HSA) and Hen Egg White Lysozyme (HEWL) with gold nanoparticles (GNPs) is investigated using surface plasmon resonance (SPR) spectroscopy, fluorescence spectroscopy and circular dichroism (CD). Size and morphology of the samples was established using Transmission Electron Microscopy (TEM) and stability studies was established using zeta potential analysis. The stability of protein-GNP complex was found to be greater than that of individual protein as well as individual GNPs. Also HEWL-GNP complex was more stable compared to the other protein complexes. Absorbance of proteins increases with increase in gold nanoparticle concentration due to the extension of peptide strands of protein and decrease in hydrophobicity of gold nanoparticles. A ground state complex is also formed which is evident from the moderate shift observed in the absorbance peaks. Apparent association constant was also determined from the absorption spectra and was found to be maximum for HEWL and minimum for HSA. Gold nanoparticles were found to act as quenchers and reduced the protein fluorescence intensity. Binding constant and number of binding sites were found to be maximum for HEWL and minimum for HSA. The temperature dependent fluorescence studies were also performed to calculate the thermodynamic parameters and to determine the nature of interaction between the proteins and gold nanoparticles. The circular dichroism studies elucidate the reason behind the maximum binding for HEWL and minimum binding for HSA. TGA analysis determined the thermal stability of the samples. Fluorescence lifetime studies indicate static quenching of proteins. Antibacterial activity of protein-gold nanoparticles was studied against four pathogens, viz. Bacillus pumilus, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. HEWL exhibits a tunable antimicrobial activity against Pseudomonas aeruginosa due to the maximum binding of HEWL with gold nanoparticles. The study proposes a novel method for adjusting the antibacterial activity of HEWL against Pseudomonas aeruginosa when the resistance of this pathogen is a major issue in the chemotherapy of many infectious diseases. Thus the combination therapy of protein-gold nanoparticles could prove to be a new approach in medical field in the near future.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Gold/chemistry , Metal Nanoparticles/chemistry , Muramidase/chemistry , Serum Albumin, Bovine/chemistry , Serum Albumin, Human/chemistry , Animals , Bacillus pumilus/drug effects , Binding Sites , Cattle , Chickens , Circular Dichroism , Escherichia coli/drug effects , Humans , Muramidase/metabolism , Protein Binding , Pseudomonas aeruginosa/drug effects , Serum Albumin, Bovine/metabolism , Serum Albumin, Human/metabolism , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Staphylococcus aureus/drug effects , Surface Plasmon Resonance , Thermodynamics , ThermogravimetryABSTRACT
In the present study, we characterized bacteriocin BaCf3, isolated and purified from Bacillus amyloliquefaciens BTSS3, and demonstrated its inhibitory potential on growth and biofilm formation of certain food spoilage bacteria and pathogens. Purification was by gel filtration chromatography and its molecular weight was 3028.422 Da after MALDI-TOF MS. The bacteriocin was highly thermostable withstanding even autoclaving conditions and pH tolerant (2.0-13.0). The bacteriocin was sensitive to oxidizing agent (DMSO) and reducing agent (DTT). The de novo sequence of the bacteriocin BaCf3 was identified and was found to be novel. The sequence analysis shows the presence of a disulphide linkage between C6 and C13. The microtitre plate assay proved that BaCf3 could reduce up to 80% biofilm produced by strong biofilm producers from food samples. In addition, BaCf3 did not show cytotoxicity on 3-TL3 cell line.
ABSTRACT
The bacterial isolates from Centroscyllium fabricii (deep sea shark) gut were screened for antagonistic activity by cross-streak method and disc diffusion assay. This study focuses on strain BTSS-3, which showed antimicrobial activity against pathogenic bacteria including Salmonella Typhimurium, Proteus vulgaris, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Bacillus circulans, Bacillus macerans and Bacillus pumilus. BTSS3 was subjected to phenotypic characterization using biochemical tests, SEM imaging, exoenzyme profiling and antibiotic susceptibility tests. Comparative 16S rDNA gene sequence analysis indicated that this strain belonged to the genus Bacillus, with high (98%) similarity to 16S rDNA sequences of Bacillus amyloliquefaciens. The chemical nature of the antibacterial substance was identified by treatment with proteolytic enzymes. The antibacterial activity was reduced by the action of these enzymes pointing out its peptide nature. It was observed from the growth and production kinetics that the bacteriocin was produced in the eighth hour of incubation, i.e., during the mid-log growth phase of the bacteria.
