Antimicrobial Activity and Physical Characterization of Silver Nanoparticles Green Synthesized Using Nitrate Reductase from Fusarium oxysporum

Abstract

Nanostructures from natural sources have received major attention due to wide array of biological activities and less toxicity for humans, animals, and the environment. In the present study, silver nanoparticles were successfully synthesized using a fungal nitrate reductase, and their biological activity was assessed against human pathogenic fungi and bacteria. The enzyme was isolated from Fusarium oxysporum IRAN 31C after culturing on malt extract-glucose-yeast extract-peptone (MGYP) medium. The enzyme was purified by a combination of ultrafiltration and ion exchange chromatography on DEAE Sephadex and its molecular weight was estimated by gel filtration on Sephacryl S-300. The purified enzyme had a maximum yield of 50.84 % with a final purification of 70 folds. With a molecular weight of 214 KDa, it is composed of three subunits of 125, 60, and 25 KDa. The purified enzyme was successfully used for synthesis of silver nanoparticles in a way dependent upon NADPH using gelatin as a capping agent. The synthesized silver nanoparticles were characterized by X-ray diffraction, dynamic light scattering spectroscopy, and transmission and scanning electron microscopy. These stable nonaggregating nanoparticles were spherical in shape with an average size of 50 nm and a zeta potential of -34.3. Evaluation of the antimicrobial effects of synthesized nanoparticles by disk diffusion method showed strong growth inhibitory activity against all tested human pathogenic fungi and bacteria as evident from inhibition zones that ranged from 14 to 25 mm. Successful green synthesis of biologically active silver nanoparticles by a nitrate reductase from F. oxysporum in the present work not only reduces laborious downstream steps such as purification of nanoparticle from interfering cellular components, but also provides a constant source of safe biologically-active nanomaterials with potential application in agriculture and medicine.

Green Extracellular Synthesis of the Silver Nanoparticles Using Thermophilic Bacillus Sp. AZ1 and its Antimicrobial Activity Against Several Human Pathogenetic Bacteria

“…Therefore, biological method of synthesis could be introduced as an alternative method for elimination of such problems (10)(11)(12). Until now, the AgNPs were synthesized by bacteria (13), plants (14), fungi (15), Algae (16), and yeasts (17). Inorganic nanoparticles can be generated using micro-organisms through either extracellular or intracellular activities (18).…”

Bioinspired synthesis, characterization and antifungal activity of enzyme-mediated gold nanoparticles using a fungal oxidoreductase

“…The fungus was at initially grown for 5 days at 28 °C on Sabouraud dextrose agar. The mycelium was detached from culture medium and aseptically moved to a liquid culture medium containing (per liter) malt extract 3 g, yeast extract 3 g, peptone 5 g and glucose 10 g. The submerged cultures were incubated for 4 days at 26 °C with shaking 200 rpm [30]. Fungal biomass was washed thrice with sterile distilled water and subcultured to new cultures at the above conditions for extra 2 days.…”

“…The obtained supernatant (570 ml) was added to the culture filtrate (410 ml), concentrated by ultrafiltration (cutoff 100,000 Da) and used for purification of fungal sulfite oxidoreductase. The amount of protein was estimated by Lowry's way using bovine serum albumin as standard [25,31,32].…”

“…The active fractions No. 31-34 were pooled, dialyzed against phosphate buffer 0.2 M, pH 7.2 and eventually concentrated using Amicon filters (Millipore) [25,31,32].…”

“…A green system based on clean biological molecules such as enzyme and peptide has been used successfully for the production of nanoparticles and monitoring their properties. The recent progress in greener nanotechnology includes the enzymatic process of synthesis suggesting enzymes of fungal and bacterial sources to be responsible for the nanoparticle formation [23][24][25]. Employment of microbial enzymes for synthesis of nanoparticles is a new field with growing importance due to the reality that the enzymatically produced nanoparticles have the potential for usage in homogenized catalysts and they are suitable for nonlinear optics [26][27][28].…”