Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles

Abstract

Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO₃) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet –visible (UV–Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV–Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405–420 nm for 1 mM metal precursor solution (AgNO₃) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO₃ with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO₃ concentration and the size of produced Ag NPs.

1 Introduction

Silver nanoparticles (Ag NPs) are defined as a collection of particles in the size range of 1–100 nm. Silver particles’ physical, chemical, and electrical properties change when these reach the size range (1–100 nm). These properties depend on the size of the silver particles [1]. Ag NPs are used in different areas: in medicine to combat viruses such as human immunodeficiency virus, in food industries as antibacterial agents in food packages, as a catalyst in chemical reactions, or in semiconductors [1, 2, 3, 4, 5].

There are several methods (physical, chemical, and biological) for fabrication of Ag NPs. Of all the chemical methods, the microemulsion method, chemical reduction techniques, and electrochemical approaches can be mentioned. One of the disadvantages of chemical methods is the use of chemical reducing agents that are potential risks to human health and environment and usually expensive [6]. Physical methods do not include toxic chemicals and usually act faster; however, the high cost of equipment to prepare nanoparticles is their main disadvantage. Physical vapour condensation and arc‐discharge are examples of physical methods [7]. Today, the important role of living organisms such as bacteria, fungi, plants etc. in the synthesis of metal nanoparticles has been recognised. In addition, since there is better control of the distribution of the obtained particles and also no environmental toxicity created in the biological synthesis method, this method is the most preferred method by scientists. The first synthesis of Ag NPs using bacteria was conducted in 2000 using Pseudomonas stutzeri AG259 bacteria [6, 8]. Among all the bacteria with the ability of synthesising Ag NPs, Staphylococcus aureus, B. licheniformis and Escherichia coli can be mentioned [8, 9]. Various types of fungi such as Verticillium and Fusarium Oxysporum can also be used in Ag NPs synthesis [6, 9, 10].

There are few reports on the synthesis of Ag NPs by yeast; the extracellular synthesis of Ag NPs by a silver‐tolerant yeast strain MKY3 may be stated as an example [11]. Microalga Chlorococcum humicola is also able to synthesise Ag NPs [12]. Plants such as Eucalyptus chapmaniana leaves [13], Callicarpa maingayi stem bark [14], Arbutus unedo leaves [15], and Carob leaves [16] have also the potential to synthesise Ag NPs.

Previous research works have shown that nitrate reductase enzyme released by micro‐organisms is the main factor in the synthesis of Ag NPs [10]. In addition, studies have indicated that nicotinamide adenine dinucleotide Hydrogen (NADH) and NADH‐dependent nitrate reductase enzyme are important factors in metal nanoparticles biosynthesis. Some bacteria contain NADH cofactors, NADH‐dependent enzymes, and particularly nitrate reductase that may justify silver ions reductions and sequential formations of nanoparticles [17]. The nitrate reductase enzyme is a large molecule that contains molybdenum and non‐haem iron [18, 19]. Nitrate reductase of E. coli is currently one of the enzymes with best features; it is also one of the best known membrane proteins [20]. Nitrate reductase obtained from E. coli is a membrane‐associated enzyme that can be obtained in large quantities, with the growth of organisms in anaerobic conditions and in the presence of nitrate [19].

The present paper is aimed to use nitrate reductase enzyme available in E. coli bacteria for the synthesis of Ag NPs. Thus, by changing the cultural conditions of the bacteria, nitrate reductase enzyme activity was calculated and its optimal condition was determined. Using the optimised condition, the Ag NPs were synthesised through changes in the concentration of silver nitrate (AgNO₃) and the amount of enzyme. Gurunathan et al. [21] could synthesise Ag NPs using E. coli; however, the main feature of these studies is measuring the nitrate reductase enzyme activity in order to reach the maximum activity of the enzyme and thereby increase the production of Ag NPs.

2 Materials and methods

2.1 Bacteria and chemicals

The used materials in the experiment were as follows: AgNO₃, polyvinylpyrrolidone (PVP), glucose, potassium nitrate (KNO₃), potassium dihydrogen phosphate, dipotassium phosphate, ethylenediamminetetraacetate, β ‐NADH, hydrochloric acid (HCl), sulphanilamide, N ‐(1‐naphthyl) ethylenediamine dihydrochloride, sodium nitrate provided by Merck Corporation and Tryptic Soy Broth (TSB) medium made in Spain. In addition, E. coli bacteria were purchased from the Veterinary School Laboratory of Tehran University.

2.2 Preparation of medium and bacteria

2.2.1 Medium preparation

TSB medium was prepared according to the instruction (suspend 30 g/l, then heat with frequent agitation and boil for 1 min to completely dissolve the medium; then autoclave at 121°C for 15 min). Then glucose and KNO₃ were added to the medium in different ratios. Five containers – each with different medium compositions – were prepared as follows.

Container A contained only TSB medium; container B contained TSB medium, 1.5 g glucose and 0.5 g KNO₃; container C contained TSB medium, 0.5 g glucose and 1.5 g KNO₃; container D contained TSB medium, 0.5 g glucose and 1 g KNO₃; and finally, container E contained 1 g glucose and 0.5 g KNO₃ (all ratios are based on TSB gram in 100 ml of distilled water). Then the media were put in an autoclave for 15 min; after cooling, the bacterial colonies were placed in them using loops. The flasks containing cultured E. coli bacteria were stored in an incubator for 1 day at 37°C.

2.3 Enzyme activity survey

2.3.1 Principles

In this method, the enzyme activity is determined using a spectrophotometer. A certain amount of each solution (in milliletres based on reference instruction) was poured into a cell and after imposing certain conditions, a spectrophotometer recorded the absorbance rate for Blank, Test and Standards at 540 nm wavelengths. Using the obtained values, the graph of A₅₄₀ nanometres in terms of micromole nitrite was drawn. This graph can be used to determine the enzyme activity in different samples [22, 23]

$$\mathrm{nitrate}+\beta -\mathrm{NADH}\stackrel{\mathrm{nitrate}\mathrm{reductase}}{--------\to }\mathrm{nitrite}+\beta -\mathrm{NAD}+{\mathrm{H}}_2\mathrm{O}$$

$$\mathrm{nitrite}+\mathrm{sulphanilamide}+\mathrm{NED}--------\to \mathrm{nitrite}\mathrm{colour}\mathrm{complex}$$

2.4 Synthesis of Ag NPs

By using a spectrophotometer and applying the enzyme activity survey method, the best medium combination was determined. Container C – which contained TSB medium, 0.5 g glucose and 1.5 g KNO₃ – showed maximum activity of the enzyme. The process of Ag NPs synthesis was conducted using this optimal combination.

2.4.1 Effect of enzyme amount on the Ag NPs formation

Four containers containing 100 cc of 0.001 M AgNO₃ and 1 cc of 1 M PVP (for stabilisation and non‐accumulation of nanoparticles) were provided. Bacterial suspension (the best medium) was centrifuged by a 4500 rpm centrifuge for 25 min and the obtained supernatant was used. The resulting supernatant was added to four containers with the values of 1, 5, 10, and 20 cc. The colourless solutions of AgNO₃ in all four containers turned brown in colour after a few minutes, indicating the possibility of Ag NPs formation.

2.4.2 Effect of AgNO₃ concentration on the formation on the Ag NPs formation

Two containers containing 100 cc of 0.01 M AgNO₃ and 100 cc of 0.001 M AgNO₃ along with 1 cc of PVP were prepared. About 20 cc of bacterial culture supernatant was added to both containers and after a few minutes the solutions’ colour changed into brown.

2.5 Characterisation of Ag NPs

To prove the existence of nanoparticles and measure the resulting samples efficacy and size, ultraviolet–visible (UV–Vis) spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS) analysis were applied.

3 Results and discussion

3.1 Analysis of Ag NPs synthesis using optical spectroscopy

One of the methods of nanoparticles analysis is to study their absorption spectrum in the range of visible light and UV wavelengths. This method is used in the study of invisible particles of semiconductors and metal nanoparticles such as copper, silver, and gold with plasmon resonance in the range of visible light wavelengths.

For evaluation of Ag NPs produced in the above‐mentioned experiments, the samples were studied regarding their absorption in the range of 350–600 nm wavelengths. Blank used in these experiments was distilled water containing 1 cc of PVP (PVP:20 g/l). Results of UV–Vis spectroscopy showed that surface plasmon resonance (absorbance peak) was centred at 420, 415, 410, and 405 nm for 1, 5, 10, and 20 cc supernatant, respectively, Figs. 1 a –d. For 0.01 M of AgNO₃, as the results in Fig. 1 e show, by increasing the concentration from 0.001 to 0.01 M (with the same amount of supernatant, 20 cc), the wavelength of peak (λ _max) has shifted from 405 to 435 nm. The increase of λ _max indicates the production of nanoparticles with a bigger size. It was also observed that the reduction of silver ions into Ag NPs began at the start of reaction.

Fig. 1.

UV–Vis spectra of Ag colloids

a Spectra recorded after adding 1 cc bacterial supernatant to 100 cc of 1 mM AgNO₃

b After adding 5 cc

c After adding 10 cc

d After adding 20 cc

e After adding 20 cc bacterial supernatant to 100 cc of 0.01 M AgNO₃

3.2 Analysis of synthesised Ag NPs using DLS

To accurately measure the particles size and distribution, DLS analysis was used. Accordingly, the obtained samples from the above experiments were analysed to determine the particles size and distribution through DLS analysis; these were then delivered to the laboratory. The results of DLS analysis showed the majority of the nanoparticles have a size of 93.24 nm for 0.001 M AgNO₃ with 1 cc supernatant. In addition, for 5, 10, and 20 cc of supernatant, the sizes of nanoparticles are 74.47, 46.49, and 45.73,respectively (Figs. 2 a –d. For the concentration of 0.01 M (with 20 cc supernatant), according to the size distribution deport by volume (figure not shown), micro‐sized particles are also formed. Therefore, this concentration (0.01 M) is not suitable for the synthesis of nano‐sized silver particles.

Fig. 2.

Curve of size distribution by number

a 1 mM AgNO₃ with 1 cc PVP and 1 cc bacterial culture supernatant

b With 5 cc bacterial culture supernatant

c With 10 cc bacterial culture supernatant

d With 20 cc bacterial culture supernatant

3.3 Analysis of synthesised Ag NPs using TEM

The properties of nanostructured materials depend on their shapes and sizes. TEM is one of the most important and widely used devices in studies related to the properties of nanostructured materials. The produced samples in the above experiments were observable by TEM. Images of the nanoparticles showed presence of spherical nanoparticles (Fig. 3).

Fig. 3.

TEM micrograph of silver particles synthesised by E. coli for 0.001 M AgNO₃ with 20 cc supernatant and 1 cc PVP

4 Conclusions

In this research, we studied the production of Ag NPs using bacterial supernatant of E. coli. The effects of AgNO₃ concentration and also bacterial supernatant on the size of Ag NPs were investigated.

The final produced Ag NPs were characterised and studied usingUV–Vis, DLS (DLS analysis) and TEM. As was observed with an increase in the supernatant from 1 to 20 cc, the maximum peak wavelength (λ _max) was decreased from 420 to 405 nm, which indicates a decrease in the size of produced nanoparticles. As seen by using UV–Vis, with the same amount of supernatant (20 cc), the wavelength of maximum absorption increases by increasing the concentration of nitrate. DLS analysis for different values indicates that by increasing supernatant concentration smaller particles can be achieved (the average size of nanoparticles increased). In addition, by increasing the concentration of AgNO₃ from 0.001 to 0.01 M, particles of micro‐size are formed. Thus, the low concentration of AgNO₃ is preferred to high concentration for synthesising Ag nanoparticles. TEM showed the synthesised Ag NPs; images confirmed Ag NPs production of nano‐size.

The probable mechanism involved in the synthesis of Ag NPs using nitrate reductase enzyme is that nitrate reductase in E. coli catalyses the reaction in which nitrate is converted into nitrite [24]

$$\begin{array}{r}\mathrm{N}{{\mathrm{O}}_3}^{-}+\mathrm{NAD}\left(\mathrm{P}\right)\mathrm{H}+2{\mathrm{e}}^{-}+{\mathrm{H}}^{+}\to \mathrm{N}{{\mathrm{O}}_2}^{-}+\mathrm{NAD}{\left(\mathrm{P}\right)}^{+}+{\mathrm{H}}_2\mathrm{O}\end{array}$$ (1)

By dissolving AgNO₃ in deionised water, it splits into (Ag⁺ and NO₃ ⁻). By using the free electron of reaction (1), Ag⁺ ions are converted into Ag.

By providing the suitable conditions, good control over the size and distribution of produced Ag NPs can be achieved and also the time duration for forming of nanoparticles can be reduced. In this case, biological method is superior to other methods (chemical and physical methods) as this method is cost effective and can be performed in ambient conditions without spending energy. However, more research is needed to achieve this goal.