Citation: MR Obiedallah, MB Aboul-Nasr, SS Mohamed (2019) Optimizing Factors Affecting Silver Nanoparticles Genesis using Aspergillus oryzae filtrate and Identification of its Extracellular Secreted Proteins by Mass Spectrometry. SciEnvironm 2: 137.
Received date: July 01, 2019; Accepted date: July 16, 2019; Published date: July 19, 2019.
In this study, 53 marine fungal isolates were obtained from Padina sp. sample. Isolates were identified and screened for silver nanoparticles (SNPs) generation extracellularly. Thirty-seven species were SNPs+, from which fungal filtrate of a non-pathogen and an economically important isolate (Aspergillus oryzae (Ahlb.) Cohn) was selected for further investigation. Silver nanoparticles synthesized by A. oryzae filtrate were characterized by transmission electron microscope, showing spherical particles with an average particles size of 12.31 ±0.62. Selected area electron diffraction confirmed the presence of elemental silver in the sample. Mycological synthesized SNPs had an inhibitory effect against Bacillus cereus SBTBC, Enterococcus faecalis 8J, Escherichia coli and Salmonilla with EC50 values of 1.97, 1.64, 0.26 and 0.25, respectively, whereas Listeria monocytogenes 10403S and Staphylococcus aureus 7A were resistant. Optimized conditions for SNPs generation using this species were found to be, reaction pH (pH 9), mycelium levels at 0.1 g/ml, YMPG medium, raising reaction temperature, using undiluted fungal filtrate and AgNO3 concentration at 1.5 mM. Extracellular protein profile of A. oryzae was identified using MALDI-TOF Mass spectrometry to determine reductase protein(s) that is responsible for reducing silver ions into silver metal. FAD-dependent oxidoreductase was one of the eight most prominent protein bands. Here, we suggest that this protein plays an essential role in this reductive reaction that generates SNPs, as it is the only identified protein with a reductase activity in the fungal filtrate. Our findings addressed the optimized conditions for SNPs conditions using filtrate of A. oryzae for the first time, thus this large-scale and stabilized SNPs production can be achieved easily. Besides, reporting the presence of FAD-dependent oxidoreductase protein for the first time in A. oryzae filtrate, which might be responsible for SNPs generation.
Nanoparticle research has recently received considerable attention and is now an intense field of study. Nanoparticles usually range from 1-100 nm and have unique properties such as, chemical , electrical , magnetic, mechanical, and optical ; that are distinct from those of large materials . Nanoparticles are preferred in various applications over bulk materials, as they have a large surface area to volume ratio which enhance their properties . Hundreds of publications have reported the synthesis and control of nanoparticle formation (size and shape) within a solution using different methods (chemical, physical or biological), and by adjusting controllable parameters such as temperature, supersaturation, time and surface energy . Now, there is an increasing interest in producing silver nanoparticles (SNPs) using biological sources instead of using chemical and physical methods  in order, to replace the use of environmentally-hazardous compounds with natural, eco-friendly alternatives . Extracellular synthesis of SNPs by microorganisms has been widely reported for Fusarium oxysporum , Bacillus licheniformis , Bacillus amyloliquefaciens and subtilis , Escherichia coli , Aspergillus fumigatus , Aspergillus niger , Aspergillus flavus , Apergillus terreus , Trichoderma harzianum  and many others. In the current study, Aspergillus oryzae (Ahlb.) Cohn was isolated from an algal sample (Padina sp.) and its filtrate was used for SNPs generation. A. oryzae is widely used in East Asia in many fermentation industries such as shoyu (soy sauce), saké (rice wine), bean curd seasoning and vinegar production, due to its potential on producing a variety of hydrolytic enzymes . Also, it is a source of pharmaceutical grade enzymes, such as taka-amylase and protease . It belongs to the A. flavus group  and is considered as a non-pathogenic fungus . Also, the US Food and Drug Administration categorize A. oryzae as ‘generally recognized as safe’ . It is a fast-growing fungus, with high extracellular enzyme activity and exhibits high competitiveness against other fungal species . For these reasons, the Padina A. oryzae isolate was selected for the experimental analysis of its SNPs forming properties and optimization. Silver nanaoparticles generation using A. oryzae was reported by other researchers [23-25], but the exact reaction mechanism of SNPs formation is yet to be elucidated. An important aim of this research was to explore the extracellular protein profile of A. oryzae in order to determine the potential role of its extracellular proteins in SNPs production.
Results and discussion
A marine sample (Padina sp.; brown algae) was targeted for fungal isolation as microorganisms (halophilic) in such habitat are capable of reducing many inorganic elements such as, cadmium, gold, lead, silicon and gold , which may provide them with an advantage in SNPs production processes over their terrestrial counterparts. Fifty-three fungal isolates included 5 genera (Alternaria, Aspergillus, Fusarium, Penicillium and Trichoderma spp.) belong to Hyphomycetes group, sixteen species and one variety were obtained from Padina sp. sample. All isolates were morphologically identified and screened for SNPs potential. Thirty-one out of fifty-three isolates were SNPs+ with various activity (Figure 1). Results of screening Ascomycota group, showed that A. flavus isolates had the highest activity for generating SNPs (OD= 1.73), followed by A. oryzae (OD= 0.88). Whereas, for Penicillium sp., P. citriunum showed a moderate activity (OD=0.55). Generally, A. aegyptiacus, A. niger, P. corylophilum and P. oxalicum, were all negative for synthesizing SNPs and no peak was detected from 400-450 nm (Fig. 1). Section Falvi includes two main species, A. oryzae and A. parasiticus, A. oryzae is considered as a non-pathogenic fungus, while A. parasiticus most strains are known as potent aflatoxin producers . None of the A. oryzae cultures has been reported for aflatoxin production [27-29], so it was assumed that the aflatoxin gene homolog cluster is not functionally working in this species. Hence, little is known about its extracellular proteins and nanoparticles (SNPs) production by this fungus, it was selected for the experimental analysis of its extracellular secreted proteins, searching for reductant proteins that may contribute in reducing Ag+ into SNPs.
Silver NPs generation using A. oryzae was reported by other researchers [23-25], but the main factor(s) that plays an essential role in reducing Ag+ into Ag0 is yet to be isolated and identified. An important aim of this study is a proteome study of A. oryzae in order to determine the potential role of its extracellular proteins in SNPs production.
Silver nanoparticles production by A. oryzae filtrate
Aspergillus oryzae (Ahlb.) Cohn filtrate was used to generate SNPs as described above. A change of colour of the solution was detected at 24 h incubation (inset Fig. 2). A peak, characteristic of SNPs formation due to surface plasmon resonance (SPR), was detected at 435 nm (Figure 2).\
Figure 3 (a & b) shows a TEM micrograph of spherical monodispersed nanoparticles synthesized by A. oryzaeFigure 3 (a & b) shows a TEM micrograph of spherical monodispersed nanoparticles synthesized by A. oryzae filtrate at 24 h. For size measurements, Scandium image analysis software was used (arbitrary line tool) to determine measurements of 80 SNPs within the micrograph. These data were later analysed by Origin 2018b for data analysis and graphing software (histogram distribution), average particles size was 12.31 nm ±0.62, with minimum and maximum particles size of 4.03 and 23.80 nm, respectively (Fig. 3 (c)). Selected are electron diffraction (SAED) was used to determine the elemental composition of the generated SNP, showing peaks of elemental silver in the sample (Figure 3 (d)).
The antibacterial effect of generated SNPs
The EC50 values obtained for B. cereus SBTBC, Enterococcus faecalis 8J, E. coli and Salmonilla were 1.97, 1.64, 0.26 and 0.25, respectively (Table 1 & Figure 4). No inhibitory effect against Listeria monocytogenes 10403S and Staphylococcus aureus 7A was detected. This was in contrast with Binupriya et al.  who reported that synthesized SNPs using live and dead cell filtrates of A. oryzae var. viridis (5-50 nm) had an antibacterial effect against Staphylococcus aureus KCCM 12256 strain of MBC= 40 mg/L. That was the only study about the effect of synthesized SNPs by A. oryzae against bacterial strains up to date. More studies need to be carried out to clarify the effect of synthesized SNPs by A. oryzae against pathogenic bacteria.
Table 1: Measured EC50 and EC90 for tested bacterial strains against synthesized SNPs by A. oryzae filtrate.
Bacillus cereus SBTBC
Enterococcus faecalis 8J
This gives an indication the Gram-negative bacteria, Salmonilla is the most susceptible one to the inhibitory effect of SNPs than other tested bacteria. While, SNPs had no inhibitory effect against the Gram-positive bacteria, Listeria monocytogenes 10403S and Staphylococcus aureus 7A. The explanation for this inhibitory effect is probably due to the different bacterial cell-wall structures between Gram-positive and Gram-negative bacteria, as SNPs are basically interacting electrostatically . The Gram-negative bacteria is characterized by a porous outer membrane and a periplasmic membrane that would allow the passage of SNPs inside the bacterial cell and their intracellular accumulation .
Tenover  explained three possible mechanisms for the antibacterial effect of SNPs. One is due to the high surface area to volume ratio that acquire NPs with better penetration properties than bulk materials and accumulate in the cytoplasmic membrane which disturb permeability and respiration , ending with the cell death. A second explanation is possibly due to SNPs interaction with sulfur- and phosphorus- compounds such as protein and DNA and eventually damage the cell . The third proposed mechanism suggests that SNPs can release Ag+ ions into cytoplasmic components that has an essential role in the bactericidal effect .
Our results show that mycological synthesized SNPs are promising compounds as antibacterial agents that can be used in medical dressings, however the exact mechanism of their bactericidal effect needs to be elucidated, and the safety of using them commercially still need to be more confirmed.
The effect of growth medium, AgNO3 and fungal filtrate concentration on SNPs synthesis
Since there are not previous reports about optimizing controllable conditions for SNPs synthesis using A. oryzae filtrates, a main target in this research was to address the optimized conditions of several factors, including type of growth medium, AgNO3 and fungal filtrate concentration, pH, temperature and time. Three types of media (MM, PDB and YMPG) were tested for their impact on the fungal filtrate metabolites of A. oryzae for SNPs production. Figure 5, shows that YMPG gave the highest productivity of SNPs production (absorbance of 0.89 at 435 nm). The activity of the A. oryzae filtrate obtained from minimal medium was enhanced by nearly 4-fold weaker than that obtained using PDB. However, greatest SNPs production activity was obtained with YMPG medium (~nine-fold higher than MM). This is presumed to be due to the nutrients richness in YMPG medium, as it contains yeast extract as a source of B-complex vitamins, malt extract which acts as source of energy (nutrients and glucose), peptone which is a source of nitrogen and amino acids and all these components promote the fungus for secreting more extracellular enzymes. Our results are in accordance with previous results with filtrates of Fusarium oxysporum , where ten different media were tested and YMPG resulted in the highest production of SNPs with OD 1.7 at Absorbance 440 nm.
The effect of diluting the fungal filtrates on SNPs production was tested. This showed that the production of SNPs is directly proportional to the concentration of the fungal filtrate (Figure 6). This result clearly shows a quantitative relationship between SNPs production and the concentration of the fungal filtrate.
Fungal filtrate was treated with different concentrations of AgNO3 ranging from 0.5–5 mM. The resulting SNPs-dependent absorbance peaks showed a broadening and a red-shift for AgNO3 at ≥ 2mM (Figure 7). This is an indication that particle size increases at increasing AgNO3 concentration [35,36]. The most intense peak was formed with 1.5 mM AgNO3 with an absorbance of 1.1.
Effect of pH and temperature on SNPs synthesis with A. oryzae
The cell-free filtrates obtained from mycelia cultured in YMPG at pH 6.5 were adjusted to different pH values (5, 6, 7, 8 and 9), this resulted in clear differences in SNPs production with an increase in activity from pH 7 to 9. Thus, maximum activity was achieved at pH 9 (Figure 8). A study on SNPs production by F. oxysporum filtrates reported that optimum pH values are 9 and 11 ; this effect was considered to be caused by the enhanced availability of OH- ions that can provide electrons for reducing Ag+ into Ag0. It was also reported that OH- ions play an essential role in adsorbing SNPs and maintain their stability, preventing their aggregation . Birla et al.  suggested that mycogenesis of SNPS is faster in alkaline than in acidic conditions because proteins in fungal filtrates may bind with Ag+ more readily at higher pH.
Effect of temperature was studied in two ways. Firstly, the SNPs reaction was performed at a range of temperatures (Figure 9 (a)). At 100 °C, the appearance of the characteristic brown colour occurred by 10 min. This strong SNPs formation activity is presumed to occur during the warming period that happens before any labile components (as required for SNPs formation) become heat denatured at high temperature. Thus, it can be expected that at lower temperatures the time required for initial SNPs formation is increased and that labile components required for the reaction are maintained without denaturing for a longer period.
Secondly, effect of temperature was explored by pre-treating fungal filtrate only at 30-100 °C for 20 min. This resulted in a noticeable decrease (40-50%) in SNPs synthesis for those filtrates incubated at 100 °C, while the highest level of SNPs formation was obtained for fungal filtrates pre-incubated at 60 °C (Fig. 9 (b)). This suggests that the fungal filtrate contains labile components (e.g. proteins) that contribute to SNPs production that are inactivated at temperatures of > 60 °C. However, it should be noted that preheating only diminished SNPs production activity by ~50% indicating a role for both heat-labile and -stable factors in SNPs production.
Time required for the reaction to reach a saturation
Generation of SNPs was monitored from 1 to 214 h (Figure 10) to determine the time required for this reaction to reach a saturation. Figure 8 shows that a clear peak formed at 24 h and increased by time up to 190 h and almost no change occurred after that. This result indicates that the reaction can reach a saturation within a week and the formed SNPs still showing sharp peaks without broadening or having a blue or red- shifts, this means that increasing time did not affect shape, dispersity or stability of the formed SNPs.
Extracellular protein profile
To identify the extracellular proteins that might be responsible for SNPs production, protein concentration from two independent cultures (100 mL fungal filtrates) of A. oryzae were measured by Bradford assay (253.7 and 350.8 µg/mL), followed by ammonium sulphate precipitation, and then dialysed (against acetate buffer (50 mM sodium acetate trihydrate and 1 mM PMSF/1 L), pH 5.0) and concentrated by ultrafiltration where the volume was reduced 5-fold from 5 to 1 mL. Protein concentration was 2- and 3-fold increased from 456.7 and 596.3 to 912 and 1,790 µg/mL. The concentrated protein (retentate) promoted SNPs formation by ~2.5-fold increase from OD=0.9 to 2.2 at A435, while the unconcentrated protein showed no activity and no peak was formed. The 2,000 kDa retentate suggests molecules of 2,000 kDa also contribute to SNPs generation. These observations are consistent with the view that secreted proteins are essential components in the SNPs production capacity of fungal filtrates.
SDS-PAGE analysis showed a similar pattern of distinct bands in each of the concentrated samples, however levels of protein in the pre-concentrated samples were too low to be observed (Figure 11).
Protein identification by mass spectrometry
The SDS-PAGE of the extracellular protein sample indicated a series of distinct protein bands. The eight most prominent protein bands (along with a positive and negative control) were excised and subjected to identification by tryptic-digestion coupled mass-spectrometry (Figure 12). The identified proteins are listed in Table 1. From Mascot report, each GenInfo Identifier (gi) was used to search through NCBI website and full amino acid sequences were downloaded in .fasta* format for each protein. FASTA files were used to perform a search through SignalP 4.0 website and signal peptide for each protein has been determined . Signal peptides provide information about the protein’s transport system and its destination, however some proteins with no signalling regions are maintained in the cytoplasm. Search parameters were set to provide information about Eukaryotes as the organism group, D-cuttoff values, was set to default, graphics output was set to PNG, output format was set to standard and method was set to input sequences may include TM regions. Using the sequence information of band number 6, the DeepLoc-1.0 server could provide information (Figure 13,14) about the subcellular localization of Eukaryotics proteins using Neural Networks algorithm trained on Uniprot proteins with experimental evidence of subcellular localization, showing that it is an extracellular soluble protein.
Figure 13: Hierarchical tree about band number six provided by DeepLoc-1.0 server.
This protein’s structure (FAD dependent oxidoreductase gi|169774077 [Aspergillus oryzae RIB40]) is yet to be elucidated, so to define its structure a BLASTP was done through the NCBI website using data deposited from the protein data bank (PDB). It was found that the closest protein’s structure to gi|169774077 is the chain A, cyclohexylamine oxidase from Brevibacterium oxydans IH-35a  (Figure 15) with E-value: 3.75e-04, bit-score: 43.51, aligned-length: 48 and Identity to query: 48% (Appendix 15). Figures 5.16 and 5.17, were designed using Cn3D software . Table 2.
Figure 15: 3D structure of Cyclohexylamine Oxidase from Brevibacterium Oxydans Ih-35a generated by Cn3D software. 3D structure of Cyclohexylamine Oxidase, front view, coloured chains (A, B, C and D) from Brevibacterium Oxydans Ih-35a.
Table 2: Proteins identified in water aqueous filtrate of A. oryzae by mass spectrometry (MALDI-TOF), including sample number, approximate MW before cutting, best match from NCBI database, MW, species, hit score, matches, peptide and signal peptide.
An interesting study about the extracellular protein profile of A. oryzae  reported that extracellular protein profile is always dependent on the type of nutrients found in the growth medium. Before that, Bentley  also reported that fungal metabolites are always dependent on the medium used for the initial growth. According to this, it can be concluded that malt and yeast extracts, beside peptone that were used in the initial source of medium (YMPG) in this study were degraded by oryzin into polypeptides , then polypeptides can further be degraded into amino acids and dipeptides by leucine aminopeptidase . Thus, some of the produced amino acids would have reducing properties and help in reducing Ag+ into Ag0. A study about SNPs production by A. oryzae, reported the presence of nitrate reductase in the fungal filtrate using a nitrate reductase assay , explain that it is implicated in SNPs synthesis, however the exact mechanism of this reaction still need to be fully addressed. Another two studies reported that NADH-dependant nitrate reductase plays an essential role in reducing Ag+ into Ag0 in Fusarium oxysporum  and Bacillus licheniformis . Our results showed that, Band number 6 was identified as a FAD dependent oxidoreductase [Aspergillus oryzae RIB40] which is part of the family of proteins contains FAD dependent oxidoreductases and related proteins, thus the reduction activity of this fungal filtrate can be referred to the presence of this protein in its filtrate, while NADH-dependant nitrate reductase was not identified among extracellular proteins found in the fungal filtrate in this study. Although, synthesis of various types of nanoparticles using filtrate of A. oryzae was previously reported ; for silver nanoparticles ; for gold nanoparticles , for iron nanoparticles , but this is the first report linking its extracellular protein profile with SNPs generation and report the optimized conditions for SNPs synthesis using filtrates of this economically important species.
In conclusion, we have demonstrated that 37 marine fungal isolates are capable of SNPs production with various activities. Aspergillus oryzae filtrate was one of the most efficient sources for SNPs generation in comparison to all other screened isolates. Generated SNPs had an inhibitory effect against several bacterial strains (Bacillus cereus SBTBC, Enterococcus faecalis 8J, Escherichia coli and Salmonilla) expect for Listeria monocytogenes 10403S and Staphylococcus aureus 7A. In addition, optimized conditions for SNPs generation using this species were reported, including reaction pH (pH 9), longer reaction time (up to 214 h), mycelium levels at 0.1 g/mL, use of YMPG medium, high temperature, undiluted fungal filtrate and AgNO3 concentration at 1.5 mM. After MALDI-TOF identification of extracellular proteins secreted with this strain, (FAD dependent oxidoreductase) with a reductase activity is firstly reported in this study and SNPs formation is suggested to be due to the presence of this protein. Our findings will have some important implications for using A. oryzae in metal nanoparticles formation by directing future researchers for investigating its proteinaceous components found in the fungal filtrate and exploring their function to present a deep understanding of the mechanism by which metal nanoparticles are formed by molecules found in the filtrate of this species.
All chemicals were of analytical grade or higher and were purchased from Sigma, Fisher Scientific, Oxoid, Bio-Rad, Fluka or Melford, unless otherwise stated.
Origin of isolate
Aspergillus oryzae (Ahlb.) Cohn was isolated from a Padina sp. (brown algae) sample that was collected from the Sea Gull resort 35 km from El Gouna, Hurghada, Egypt. Morphological identification was achieved at Assiut University mycological centre (AUMC), Egypt, using appropriate identification books based on morphological characteristics and observing key features [45-47].
Culture preparation for silver nanoparticles production
Isolates were inoculated into yeast extract, malt extract, peptone, and glucose (YMPG) broth (3 g/L yeast extract, 3g/L malt extract, 5g/L peptone and 10g/L glucose) medium in duplicates. A new simple-method was employed to save time and media. This method will allow the detection of SNPs formation for many samples at one shot. Where the required volume of total reaction mixture was reduced to 700 μL only.
Liquid cultures were incubated at 25 °C for 96 h and agitated at 120 rpm. The mycelial mat was separated by sterile muslin filtration, then washed several times with sterile Milli-Q water, until a clear filtrate was obtained, and no medium components were detected. The mycelial mat was squeezed gently to eliminate the water content as much as possible, then 1 g (wet weight) was taken and suspended in 10 mL sterile Milli-Q water, in a sterile 20 universal containers (Thermo ScientificTM SterilinTM). The re-suspended mycelium was incubated for 72 h at 25 °C and agitated at 120 rpm. To obtain a clear and uncontaminated supernatant from the fungus mycelium, two further filtration steps were performed, the first using sterile muslin, followed by 0.22 µm membrane (Whatman). The collected fungal filtrate was used for reducing Ag+ on the day of collection. The total reaction volume was 700 μL (sufficient for 3 readings), contains 693 μL of fungal filtrate (99%, final volume), 7 μL AgNO3 (100 mM). The reaction mixture was kept in sterile microfuges and incubated at 25°C for 48 h, in dark.
A Spectramax® 190 microplate reader (operated by SoftMax® Pro 7 software version 7.0.2) was used to record the UV-visible spectra of 200 µL sample were loaded as technical triplicates into sterile microplates (96 wells, clear, flat bottom, Thermo ScientificTM). For each scan, absorbance values at 435 nm were averaged then plotted with the wavelength (nm) values and standard error determined.
Characterization of formed silver nanoparticles by TEM and SAED
After 24 h incubation, the SNPs reaction mixture (200 µL) was transferred into a sterile microfuge tube after shaking the sample vigorously, then 5 µL of the suspension were loaded onto carbon coated copper grids (Agar Scientific) of 0.3 mm diameter and left on bench to dry for over 24 h at room temperature. Micrographs were obtained at the Electron Microscopy Laboratory (EMLab), University of Reading, using JEM 2100 plus TEM, operating at 200 kV. An analytical transmission electron microscope fitted with a selected area electron diffraction (SAED), was used to determine the elemental composition of the generated SNPs.
Antibacterial effect of generated SNPs
One hundred and sixty mg/mL SNPs retained from 100 mL total reaction volume after centrifugation. Six pathogenic bacterial strains were used to study the effect of SNPs on Gram-positive (Bacillus cereus SBTBC, Enterococcus faecalis 8J, Listeria monocytogenes 10403S and Staphylococcus aureus 7A) and Gram-negative (Escherichia coli and Salmonilla) bacteria, that were obtained from School of Biological Sciences, University of Reading. Bacterial strains were recovered from frozen (-80 °C) glycerol (15% v/v) stocks on Luria Bertani (LB) agar plates at 37°C for 24 h. Andrews protocol  for determination of half maximal inhibitory concentration (EC50 or IC50) was followed, where the EC50 is the concentration of SNPs that would reduce bacterial population into the half. An overnight (16-18 h) cultures (1:10) were prepared by picking single colonies to be inoculated in 9 mL LB broth, 225 rpm, at 37 °C. Formed cultures were diluted into 1:100 by sub-cultured 1 mL into 9 mL LB broth, 250 rpm, at 37 °C for 2-5 h to exponential phase (OD600 0.4-0.6). Cultures were adjusted to OD600 = 0.3 and diluted in LB (1:50) prior to use in microbiological assays. In a 96-well flat-bottom transparent plate (Greiner), blank wells were loaded with 200 µL LB and negative control wells were loaded with 200 µL bacterial cells without SNPs, other wells were loaded with 100 µL of SNPs (stock solution concentration, 46.7 mg/mL; dilution factor 0.5) with various concentrations (26.7, 1.3, 6.7, 3.3, 1.7, 0.8, 0.42, 0.21, 0.1 and 0.05 mg/mL). Prepared bacterial cultures as described earlier were added (100 µL; equivalent to, ~5x105 bacteria/well) in technical duplicates, with two biological replicates for each strain. Microplates were incubated at 37°C for 16 h or 24 h, and absorbance measurements was taken at 600 nm.
The optical density of each individual culture at 16 h or 24 h was plotted and analyzed using Origin software for data analysis and graphing, version 2018b, using the Analysis tool, fitting option for Non-linear curve fit, Growth/Sigmoidal category and DoseResp function to calculate each of EC50 and EC90 concentrations for each affected bacterial strain.
Effect of different factors on SNPs synthesis
Silver nanoparticles synthesis was performed with A. oryzae filtrates obtained by culturing in YMPG medium as described above. Spectrum of generated SNPs was measured at 24 h using Spectramax® spectrophotometer. During each experiment two controls were used, fungal filtrate and AgNO3 solutions to distinguish the characteristic peak of formed SNPs in the reaction mixture other than the control solutions. The effect on SNPs production of growth using three types of media (minimal medium, MM; potato dextrose broth, PDB; and yeast extract malt extract peptone glucose, YMPG) was tested. The minimal medium is the modified standard Czapek-Dox medium by Correll et al. , it was prepared by adding 2 g/L NaNO3 as the sole nitrogen source and 30 g/L sucrose prior to autoclaving, with 1 g/L KH2PO4, 0.5 g/L MgSO4.7H2O, 0.5 g/L KCl, 0.01 g/L FeSO4.7H2O (as trace element) and final pH was adjusted to 6.5. Potato dextrose broth (PDB) medium contained a potato extract infusion of 200 g /L potatoes and 20g/L dextrose (purchased from Sigma Aldrich and prepared as manufacturer’s instruction). The YMPG medium components were detailed previously.
In addition, the effect of diluting the fungal filtrate to 10-99% concentration with of sterile Milli-Q water was also tested, as was the effect of using AgNO3 at different concentrations (0.5-5 mM). To test the effect of pH, the fungal filtrate was adjusted to pH 5, 6, 7, 8 or 9 using concentrated HCl or 3N NaOH before the SNPs reaction was initiated. The effect of temperature was tested by pre-heating the fungal filtrate at 100 °C for 20 min, followed by rapid cooling on ice, before SNPs synthesis was initiated. The effect of time on SNPs production was tested for periods of up to one week, using standard conditions.
Aspergillus oryzae extracellular protein analysis
For extracellular protein extraction, a submerged culture (4 days) of A. oryzae was prepared as described above, using 0.1 g/mL of mycelium. This was then suspended in Milli-Q water for 48 h at 25 °C and agitated at 120 rpm. The mycelium was then removed by filtration using a sterile muslin cloth, and ammonium sulphate ((NH4)2 SO4) was added to 100 mL this fungal filtrate in H2O to a concentration of 85% saturation. The solution was kept overnight at 4 °C on a magnetic stirrer. Using a sterile 50 mL Falcon tubes, the solution was centrifuged at 12000 ×g for 30 min. The precipitate was dissolved in 2 mL 50 mM acetate buffer (pH 5.0) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and (2 µL/mL) HaltTM Protease Inhibitor Cocktail (100X), to avoid proteolytic degradation during protein extraction. Snakeskin® Dialysis tubing (Product number 68035), 3,500 MWCO was soaked for 1 h in acetate buffer (50 mM sodium acetate trihydrate and 1 mM PMSF/1 L, pH 5.0) before use. The ammonium sulphate precipitated proteins were then applied to the tubing and dialysis was performed overnight at 4 °C on a stirrer against 1000-fold excess of the same buffer.
Five mL of dialysed protein were transferred into a 2,000 MWCO Vivaspin™ 20 mL sample concentrators (Fisher Scientific) and centrifuged for 30 min at 4 °C and 5,000 ×g. The protein concentration was estimated using a Bradford standard assay .
The retentate and filtrate resulted from the Vivaspin concentrators were tested for SNPs synthesis using a total volume reaction of 700 μL as previously explained to check their ability on SNPs generation after concentration.
Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
A vertical electrophoresis system which is a Mini-PROTEAN gel apparatus (Bio-Rad) was used to resolve the protein samples using SDS-PAGE . Sample (40 µL) were mixed with 40 µL of 2X sample-loading buffer (20% staking gel buffer, 2.5% glycerol, 4% SDS, 0.2% bromophenol blue, 0.2 M dithiothereitol (DTT)) and boiled for 5 minutes at Ëƒ95°C. The tubes were centrifuged at high speed for 1 minute, then 10 µL of each sample was loaded into the staking gel along with 10 µL of the protein marker (PageRulerTM Broad Range Unstained Protein Ladder, Thermo Scientific, 5-250 kDa). Gel concentration was 15% resolving gel (for 10 mL gel: 2.3 mL H2O, 5 mL Acrylamide (30%), 2.5 mL Tris (1.5 M, pH 8.8), 0.1 mL SDS (10%), 0.1 mL Ammonium persulphate (10%), 0.004 mL TEMED) and 5% staking gel (for 4mL gel: 2.7 mL H2O, 0.67 mL Acrylamide (30%), 0.5 mL Tris (1 M, pH 6.8), 0.04 mL SDS (10%), 0.04 mL Ammonium persulphate (10%), 0.004 mL TEMED). Used gels were 0.75 mm thick and were run at 60 mA for 50 min with freshly prepared SDS-PAGE running buffer (0.025 M Tris-Base, 0.192 M Glycine pH 8.3, 1% SDS). Gel was stained with Commassie Blue for 1 hour, followed by several washes using ultra-pure water, then de-stained overnight with de-stain solution. A SynGene (G-box) system was used to image the gel.
Protein identification by tryptic peptide mass spectroscopy
The concentrated, precipitated extracellular proteins of A. oryzae were subjected to SDS-PAGE and the resulting Coomassie blue stained protein bands were excised from the gel, diced into small pieces using a sterile scalpel under aseptic conditions, and were then placed into 0.5 mL microfuge tubes filled with sterile Milli-Q water. The Gundry et al.  protocol for protein sample preparation (with modification) was then followed for further processing. A negative control (empty lane) and a positive control (protein marker) were also included. Gel pieces were kept in 50 µL sterile water at -20 °C. The gel slices were de-stained with 200 µL 50% acetonitrile (MeCN) (Rathburn, UK) / 50% 10 mM triethylammonium bicarbonate (TEAB) (Sigma, UK) overnight. Next day, gels were dehydrated with 400 µL MeCN. Then, vortexed and kept for 1 h at 4 °C to further remove stain and salts. The MeCN was then removed using a SpeedVac Concentrator (SavantTM, Thermo Scientific) for 10 min. Gel pieces were hydrated again by adding 400 µL of 10 mM TEAB. The TEAB was then removed and substituted with 400 µL MeCN. The mixture was vortexed and incubated at room temperature. After 10 min, MeCN was removed by dehydration with a SpeedVac Concentrator for 10 min. the gel pieces were reduced using 300 µL of 10 mM dithiothreitol (DTT) (Sigma, UK) in 10 mM TEAB for 30 min at 50 °C. DTT was removed and the samples were left for a minute to cool down. Then, the gel pieces were alkylated with 200 µL of 50 mM iodoacetamide (IAA) in 10 mM TEAB for 30 min at room temperature in the dark. The IAA was removed, and the gel pieces were incubated with 10 mM DTT for 10 min to reduce N-terminal peptide alkylation. DTT was removed and the gel pieces were washed thrice with 200 µL 10 mM TEAB and once using 200 µL MeCN. The gel pieces were dehydrated in a SpeedVac Concentrator for 10 min followed by washing with MeCN and TEAB.
Porcine trypsin (Promega, UK) was resuspended in 10 mM TEAB on ice. Then, 10 µL containing 50-200 ng of trypsin were added to re-swell the dehydrated gel pieces on ice and left for 20 min. Depending on the amount of the gel for each sample, 30-50 µL of 10 mM TEAB were added to fully cover the gel. Samples were left on ice for 5 min, then incubated overnight at 25 °C in the dark. Gel digestion was performed on dry ice for 5 min, the gel slice was then allowed the gel to thaw and the contents were transferred to a fresh 0.2 mL PCR tube (Bioquote, UK). A total of 30 µL of 10% ACN/5% formic acid was added and the samples were sonicated (Jencons, UK) for 15 min. Addition of 10% ACN/5% formic acid (30 µL) was repeated and extracts were concentrated in a SpeedVac Concentrator. Thirty microliters of HPLC grade water (Rathburn, UK) were added to the samples, these were dried down, this was repeated and finally the dried samples were stored at -20 °C prior to analysis. Samples were analysed by mass spectrometer at the Chemical Analysis Facility (CAF), University of Reading.
Dried samples were reconstituted in 12 µL of 1% formic acid in water, vortexed, centrifuged and left for 20 min. After that, samples were transferred into low volume plastic HPLC vials. Ten microliters of each sample were injected onto an ACE 5-C18-300 HPLC column, 150 mm × 2.1 mm ID, 5 µM particles with 300 A pore size, using an Agilent 1100 HPLC. Buffer A was 0.1% formic acid in water and Buffer B was 0.1% formic acid in acetonitrile. The HPLC conditions were as follows: flow rate of 200 µL/min; column oven 40 °C; 0 min, 2% B; 45 min, 45% B, 50 min, 90% B; 60 min, 2% B; 70 min, 2% B. The column flow was directed to a Bruker Micro TOF-QII (QTOF instrument) operating in a positive ion mode. A data-dependent acquisition (DDA) experiment was performed. The mass range was 50-3000 m/z. Ions that were double, triple or quadruple charged and in the 400-2000 m/z range with counts Ëƒ4000 were isolated and fragmented by Collision-Induced Disassociation (CID). For data analysis, a Bruker Data Analysis software was used to generate Mascot Generic Format files which were searched against an in-house copy of the Mascot database using Mascot Daemon.
This research is financially supported by the Egyptian cultural affairs and missions sector, Ministry of higher education, Egypt. Thanks for Prof. Simon Andrews for editing this manuscript. Thanks for Dr. Peter Harris for EMLab facilities and for Micheal Nicholas staff at CAF laboratory for MALDI-TOF analysis
Kumar A, Mandal S, Selvakannan PR, Pasricha R, Mandale AB, et al. (2003) Investigation into the Interaction between Surface-Bound Alkylamines and Gold Nanoparticles. Langmuir 19: 6277-6282. [crossref]
Peto G, Molnar GL, Paszti Z, Geszti O, Beck A, et al. (2002) Mater Sci Eng C 19: 95-99.
Chandrasekharan N, Kamat PVJ (2002) Phys Chem Biophys 104: 10851-10857.
Odum L (2007) Effect of Silver nanoparticles on tomato plants and Development of a Plant Monitoring System (PMS). M.Sc. Thesis, University of Auburn, Alabama, United States.
Thanh NT, Maclean N, Mahiddine S (2014) Mechanisms of nucleation and growth of nanoparticles in solution. Chem Rev 114: 7610-7630. [crossref]
Klaus T, Joerger R, Olsson E, Granqvist C (1999) Silver-based crystalline nanoparticles, microbially fabricated. In Proceedings of the National Academy of Sciences, USA, 96: pp 13611-13614.
Mandal D, Bolander ME, Mukhopadhyay D, Sarkar G, Mukherjee P (2006) The use of microorganisms for the formation of metal nanoparticles and their application. Appl Microbiol Biotechnol 69: 485-492. [crossref]
Birla SS, Gaikwad SC, Gade AK, Rai MK (2013) Rapid synthesis of silver nanoparticles from Fusarium oxysporum by optimizing physicocultural conditions. ScientificWorldJournal 2013: 796018. [crossref]
Kalimuthu K, Suresh Babu R, Venkataraman D, Bilal M, Gurunathan S (2008) Biosynthesis of silver nanocrystals by Bacillus licheniformis. Colloids Surf B Biointerfaces 65: 150-153. [crossref]
Fouad H, Hongjie L, Yanmei D, Baoting Y, et al. (2017) Synthesis and characterization of silver nanoparticles using Bacillus amyloliquefaciens and Bacillus subtilis to control filarial vector Culex pipiens pallens and its antimicrobial activity. Artif Cells Nanomed Biotechnol 45: 1369-1378. [crossref]
Gurunathan S, Kalishwaralal K, Vaidyanathan R, Venkataraman D, Pandian SR, et al. (2009) Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli. Colloids Surf B Biointerfaces 74: 328-335. [crossref]
Bhainsa KC, D'Souza SF (2006) Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigatus. Colloids Surf B Biointerfaces 47: 160-164. [crossref]
Vigneshwaran N, Ashtaputre NM, Varadarajan PV, Nachane RP, Paralikar KM, et al. (2007). Mater Lett 61: 1413-1418.
Li G, He D, Qian Y, Guan B, Gao S, et al. (2012) Fungus-mediated green synthesis of silver nanoparticles using Aspergillus terreus. Int J Mol Sci 13: 466-476. [crossref]
Ahluwalia V, Kumar J, Sisodia R, Shakil NA, Walia S (2014) Ind Crops Prod. 55: 202-206.
Machida M, Yamada O, Gomi K (2008) Genomics of Aspergillus oryzae: learning from the history of Koji mold and exploration of its future. DNA Res 15: 173-183. [crossref]
Kusumoto KI, Matsushita-Morita M, Furukawa I, Suzuki S, Yamagata Y, et al. (2008) Efficient production and partial characterization of aspartyl aminopeptidase from Aspergillus oryzae. J Appl Microbiol 105: 1711-1719. [crossref]
Alvarez-Loayza P, White JF Jr, Giraldo CC (2008) First Report of Aspergillus flavus Colonizing Naturally Dispersed Seeds of Oxandra acuminata, Pseudomalmea diclina, and Unonopsis matthewsii in Peru. Plant Dis 92: 974. [crossref]
Domsch KH, Gams W, Anderson TH (1980) Compendium of soil fungi; Vol 1. Academic Press (London) Ltd.
Matsushita-Morita M, Furukawa I, Suzuki S, Yamagata Y, Koide Y, et al. (2010) J Appl Microbiol 109: 156-165.
Liang Y, Pan L, Lin Y (2009) Analysis of extracellular proteins of Aspergillus oryzae grown on soy sauce koji. Biosci Biotechnol Biochem 73: 192-195. [crossref]
Binupriya AR, Sathishkumar M, Yun SI (2009) Ind. Eng. Chem. Res. 49: 852-858.
Phanjom P, Ahmed GJ (2015) Nanosci. Nanotechnol 5: 14-21.
Bhimba BV, Gurung S, Nandhini SU (2015) Int J Chem Tech Res. 7: 68-72.
Asmathunisha N1, Kathiresan K (2013) A review on biosynthesis of nanoparticles by marine organisms. Colloids Surf B Biointerfaces 103: 283-287. [crossref]
Kusumoto, K.; Goto, T.; Manabe, M. Report of National Food Research Institute; Japan, 1990, 54, 14–17.
Manabe M, Matsuura S, Nakano M (1968) Nippon Shokuhin Kogyogakkaishi. 15: 341-346.
Murakami HJ (1971) Gen. Appl. Microbiol. 17: 281-309.
Ciobanu CS, Iconaru SL, Chifiriuc MC, Costescu A, Le Coustumer P, et al. (2013) Synthesis and antimicrobial activity of silver-doped hydroxyapatite nanoparticles. Biomed Res Int 2013: 916218. [crossref]
Tenover FC (2006) Mechanisms of antimicrobial resistance in bacteria. Am J Infect Control 34: S3-10. [crossref]
Murray Rg, Steed P, Elson He (1965) The Location of The Mucopeptide in Sections of The Cell Wall of Escherichia Coli and Other Gram-Negative Bacteria. Can J Microbiol 11: 547-560. [crossref]
Gibbins B, Warner L (2005) Med Device Diagnostic Indust Mag. 1: 1-2.
Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, et al. (2000) A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 52: 662-668. [crossref]
He B, Tan JJ, Liew KY, Liu HJ (2004) Mol Catal A Chem. 221: 121-126.
Zheng M, Gu M, Jin Y, Jin G (2001) MATER RES BULL. 36: 853-859.
Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8: 785-786. [crossref]
Mirza IA, Burk DL, Xiong B, Iwaki H, Hasegawa Y, et al. (2013) Structural analysis of a novel cyclohexylamine oxidase from Brevibacterium oxydans IH-35A. PLoS One 8: e60072. [crossref]
Wang Y, Geer LY, Chappey C, Kans JA, Bryant SH (2000) Cn3D: sequence and structure views for Entrez. Trends Biochem Sci 25: 300-302. [crossref]
Bentley R (2006) From miso, saké and shoyu to cosmetics: a century of science for kojic acid. Nat Prod Rep 23: 1046-1062. [crossref]
Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan MI, Kumar R, Sastry M. (2003) 28 (4), 313-318.
Kalimuthu K, Suresh Babu R, Venkataraman D, Bilal M, Gurunathan S (2008) Biosynthesis of silver nanocrystals by Bacillus licheniformis. Colloids Surf B Biointerfaces 65: 150-153. [crossref]
Binupriya AR, Sathishkumar M, Vijayaraghavan K, Yun SI (2010) Bioreduction of trivalent aurum to nano-crystalline gold particles by active and inactive cells and cell-free extract of Aspergillus oryzae var. viridis. J Hazard Mater 177: 539-545. [crossref]
Raliya R (2013) J Nanopart. 2013.
Kitch MA, Pitt JI (1992) A laboratory guide to the common Aspergillus species and their teleomorphs; Common Wealth Scientific and Industrial Research Organization, Division of Food Processing (CSIRO), North Ryde: Australia.
Pitt JI (1985) Laboratory Guide to common Penicillium species; CSRIO Division of Food Research, North Ryde. New South Waler: Australia.
Nelson PE, Toussoun TA, Marasas WF (1983) Fusarium species: an illustrated manual for identification. University Park, PA: Penn State University Press.
Andrews JM (2001) Journal of antimicrobial Chemotherapy 48: 5-16.