Green synthesis of protein capped silver nanoparticles from phytopathogenic fungus Macrophomina phaseolina (Tassi) Goid with antimicrobial properties against multidrug-resistant bacteria - PubMed (original) (raw)

Green synthesis of protein capped silver nanoparticles from phytopathogenic fungus Macrophomina phaseolina (Tassi) Goid with antimicrobial properties against multidrug-resistant bacteria

Supriyo Chowdhury et al. Nanoscale Res Lett. 2014.

Abstract

In recent years, green synthesis of nanoparticles, i.e., synthesizing nanoparticles using biological sources like bacteria, algae, fungus, or plant extracts have attracted much attention due to its environment-friendly and economic aspects. The present study demonstrates an eco-friendly and low-cost method of biosynthesis of silver nanoparticles using cell-free filtrate of phytopathogenic fungus Macrophomina phaseolina. UV-visible spectrum showed a peak at 450 nm corresponding to the plasmon absorbance of silver nanoparticles. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and transmission electron microscopy (TEM) revealed the presence of spherical silver nanoparticles of the size range 5 to 40 nm, most of these being 16 to 20 nm in diameter. X-ray diffraction (XRD) spectrum of the nanoparticles exhibited 2θ values corresponding to silver nanoparticles. These nanoparticles were found to be naturally protein coated. SDS-PAGE analysis showed the presence of an 85-kDa protein band responsible for capping and stabilization of the silver nanoparticles. Antimicrobial activities of the silver nanoparticles against human as well as plant pathogenic multidrug-resistant bacteria were assayed. The particles showed inhibitory effect on the growth kinetics of human and plant bacteria. Furthermore, the genotoxic potential of the silver nanoparticles with increasing concentrations was evaluated by DNA fragmentation studies using plasmid DNA.

Keywords: Antimicrobial; Capping; DNA fragmentation; Green synthesis; Macrophomina phaseolina; Silver nanoparticles.

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Figures

Figure 1

Figure 1

Synthesis of silver nanoparticles using cell-free filtrate of Macrophomina phaseolina and spectroscopic analysis. (a) Photograph of 1 mM AgNO3 solution without cell filtrate (1, control), mycelia-free cell filtrate of M. phaseolina (2), and 1 mM AgNO3 with cell filtrate after 24-h incubation at 28°C (3). (b) UV–vis spectra recorded as a function of time of reaction at 24, 48, and 72 h of incubation of an aqueous solution of 1 mM AgNO3 with the M. phaseolina cell filtrate showing absorption peak at 450 nm.

Figure 2

Figure 2

Electron micrographs of silver nanoparticles. (a) Scanning electron microscopy micrograph of silver nanoparticles produced with M. phaseolina at 50,000 magnification (bar = 1 μm). (b, c, d) Transmission electron micrograph of silver nanoparticles at different magnifications (bar = 100 nm). (e) Measurement of nanoparticles of different shapes. (f) Histogram showing particle size distribution of silver nanoparticles with majority of the particles showing 16 to 20 nm size range.

Figure 3

Figure 3

X-ray diffraction patterns of silver nanoparticles synthesized from cell-free filtrate of M. phaseolina showing characteristic peaks.

Figure 4

Figure 4

Antimicrobial effect of silver nanoparticles against normal and multidrug-resistant human bacteria E . coli by disc diffusion method. (a) Plate showing increasing inhibition zone of E. coli (DH5α) with increasing concentration of nanoparticles: clockwise from top 0.51, 1.02, 2.55, 3.57, and 5.1 μg in a total volume 100 μl in water. (b) Plate showing increasing inhibition zone of MDR E. coli (DH5α-MDR) with increasing concentration of nanoparticles: clockwise from top 0.51, 1.02, 2.55, 3.57, and 5.1 μg in a total volume 100 μl in water. (c) Graph of antimicrobial assay of the nanoparticles on E. coli (DH5α ) in which 10, 20, 50, 70, and 100% nanoparticle solution corresponds to 0.51, 1.02, 2.55, 3.57, and 5.1 μg of silver nanoparticles in 100 μl solution, respectively. (d) Graph of antimicrobial assay of the silver nanoparticles on MDR E. coli (DH5α-MDR). Vertical bars indicate mean of three experiments ± standard error of mean (SEM). Different letters on bars indicate significant differences among treatments (P = 0.05).

Figure 5

Figure 5

Antimicrobial effect of silver nanoparticles on normal and multidrug-resistant plant pathogenic bacteria A. tumefaciens by disc diffusion method. (a) Plate showing increasing inhibition zone of A. tumefaciens (LBA4404) with increasing concentrations of nanoparticles: clockwise from top 0.51, 1.02, 2.55, 3.57, and 5.1 μg in a total a volume 100 μl in water. (b) Plate showing increasing inhibition zone of MDR A. tumefaciens (LBA4404-MDR) with increasing concentration of nanoparticles: clockwise from top 0.51, 1.02, 2.55, 3.57, and 5.1 μg in a total volume of 100 μl in water. (c) Graph of antimicrobial assay of the nanoparticles on A. tumefaciens (LBA4404) in which 10, 20, 50, 70, and 100% nanoparticle solution corresponds to 0.51, 1.02, 2.55, 3.57, and 5.1 μg of silver nanoparticles in 100 μl solution. (d) Graph of antimicrobial assay of the silver nanoparticles on MDR A. tumefaciens (LBA4404-MDR). Vertical bars indicate mean of three experiments ± standard error of mean (SEM). Different letters on bars indicate significant differences among treatments (P = 0.05).

Figure 6

Figure 6

Inhibitory effect of silver nanoparticles on the growth kinetics of human and plant pathogenic bacteria. (a) Absorbance data for bacterial growth of plant pathogenic bacteria (Agrobacterium tumefaciens) LBA4404 without or with the nanoparticles for 0, 4, 6, 8, 12, and 24 h postinoculation. (b) Absorbance data for bacterial growth of human pathogenic bacteria (E. coli) DH5α without or with nanoparticles for 0, 4, 6, 8, 12, and 24 h postinoculation showing significant inhibitory effect on the growth kinetics of the bacteria.

Figure 7

Figure 7

SDS-PAGE analysis of capping protein around the silver nanoparticles. Lane 1, molecular size marker; lane 2, extracellular proteins in the cell filtrate; lane 3, nanoparticles loaded without boiling show no protein band; and lane 4, nanoparticles after boiling with 1% SDS loading buffer show a major 85-kDa capping protein.

Figure 8

Figure 8

Agarose gel electrophoresis of plasmid pZPY112 treated with different concentrations of the silver nanoparticles (μg/100 μl). Lane 1, DNA molecular weight marker. Lane 2, control plasmid without silver nanoparticles showing only supercoiled plasmid band that moves ahead of relaxed circular and linear plasmids. Lane 3, plasmid incubated with 0.51 μg nanoparticles showing disappearance of the supercoiled plasmid band and appearance of relaxed circular and linear plasmid bands along with smaller fragmented DNA. Lane 4, plasmid incubated with 1.02 μg nanoparticles. Lane 5, plasmid incubated with 2.55 μg nanoparticles. Lane 6, plasmid incubated with 3.57 μg nanoparticles showing gradual degradation of the fragmented DNA bands; and lane 7, plasmid incubated with 5.1 μg nanoparticles showing more degradation of DNA.

References

    1. Mohanpuria P, Nisha K, Rana NK, Yadav SK. Biosynthesis of nanoparticles: technological concepts and future applications. J Nanopart Res. 2008;9:507–517.
    1. Sharma VK, Yngard RE, Lin Y. Silver nanoparticles: green synthesis and their antimicrobial activities. Adv Colloid Interface Sci. 2009;9:83–96. -PubMed
    1. Knoll B, Keilmann F. Near-field probing of vibrational absorption for chemical microscopy. Nature. 1999;9:134–137.
    1. Gao G, Huang P, Zhang Y, Wang K, Qin W, Cui D. Gram scale synthesis of super paramagnetic Fe3O4 nanoparticles and fluid via a facile solvothermal route. Cryst Eng Comm. 2011;9:1782–1785.
    1. Gao G, Wang K, Huang P, Zhang Y, Zhi X, Bao C, Cui D. Superparamagnetic Fe3O4–Ag hybrid nanocrystals as a potential contrast agent for CT imaging. Cryst Eng Comm. 2012;9:7556–7559.

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