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Synergistic Effect of Reductase and Keratinase for Facile Synthesis of Protein-Coated Gold Nanoparticles
Synergistic Effect of Reductase and Keratinase for Facile Synthesis of Protein-Coated Gold Nanoparticles
Journal of Microbiology and Biotechnology. 2015. May, 25(5): 612-619
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : November 11, 2014
  • Accepted : December 01, 2014
  • Published : May 28, 2015
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About the Authors
Sonali Gupta
Amity Institute of Microbial Biotechnology, Amity University Uttar Pradesh, Noida 201303, U.P., India
Surinder P. Singh
National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012, India
Rajni Singh
Amity Institute of Microbial Biotechnology, Amity University Uttar Pradesh, Noida 201303, U.P., India
rsingh3@amity.edu

Abstract
We have synthesized gold nanoparticles (GNPs) using chicken feathers (poultry waste) and Bacillus subtilis RSE163. Disulfide reductase and keratinase produced by Bacillus subtilis during the degradation of chicken feather has been used to reduce Au 3+ from HAuCl 4 precursor to produce gold nanoparticles. The synthesized biogenic GNPs were characterized by UV-visible spectroscopy, transmission electron microscopy (TEM), and zeta potential measurements. Fourier transform infrared (FTIR) spectroscopy indicated the presence of protein capping on synthesized GNPs, imparting multifunctionality to the GNP surface. Furthermore, the nontoxic nature of biogenic GNPs was insured by interaction with Escherichia coli (ATCC11103), where TEM images and enhancement of growth rate of E. coli in log phase signified their nontoxic nature. The results indicate that the synthesis of biocompatible GNPs using poultry waste may find potential applications in drug delivery and sensing.
Keywords
Introduction
Nanotechnology, enabling to engineer matter on the atomic and molecular scale, has given new dimensions to the field of science and technology in the last decades [26 , 40] . Engineered nanoparticles have been used extensively in drug delivery, imaging, immunosensing, and nanomedicine owing to their extraordinary optical, catalytic, thermal, and physical properties [18] . These nanoparticles can interact with biomolecules both on the surface and within the cells, leading to knowledge about various extra- and intracellular mechanisms [25] . Gold nanoparticles (GNPs) have attracted a great deal of interest and are the subject of intensive studies in biology and medicine owing to their extraordinary physicochemical properties, such as atmospheric stability, resistance to oxidation, surface functionalization, and biocompatibility [39] . Functionalized GNPs have been studied extensively for bio-imaging, single molecule tracking, biosensing, drug delivery, transfection, and diagnostic applications [24] . Although the synthesis of GNPs is very easy, the use of toxic chemicals in the synthesis of specific shape and size requirements limits their applications in biology. Different chemical reduction techniques were used for the synthesis of gold nanoparticles by using reductants like citrate, tryptophan, PEG 4000, and amino acid derivatives [1 , 27] . Recently, biological synthesis of metal nanostructures has attracted significant attention owing to the environmentally friendly green chemistry approach entailing plant- and organism (ranging from bacteria to fungi)-mediated synthesis of nanoparticles [14] . Biosynthesis of nanoparticles with the help of microorganism has great potential to produce metal nanoparticles with similar physical features to chemically synthesized nanoparticles, along with probable biocompatible organic coatings [33] . Recent studies reported the biosynthesis of gold and silver nanoparticles from Alcaligenes faecalis [11] , Fusarium oxysporum [44] , Bacillus licheniformis [41] , Rhodopseudomonas capsulate [20] , Aspergillus niger [5] , and Pseudomonas fluorescens [32] .
Feather waste produced by the poultry industry is generally considered as recalcitrant because of the high degree of cross-linking by disulfide bonds, hydrogen bonding, and hydrophobic interactions of keratin [4] .Enzymatic degradation of the feather engrosses the mutual characteristic actions of two enzymes, disulfide reductase and keratinase [31] . Disulfide reductase breaks the disulfide bridges that are responsible for the stringency of keratin in feather and also functionalizes the antioxidant defense mechanisms of bacteria with maintenance of the intracellular redox balance by catalyzing NADPH reduction [22] . Thus, the oxidized keratin proteins become better substrates for proteolytic digestion. A correlation was established between increased hydrophobicity on the keratin protein surface and the recognition and proteolytic degradation of oxidatively modified proteins, besides the other variables [6] . Microbes seem to have specific proteinases (keratinase) that selectively degrade oxidized proteins in an ATP-independent pathway. These keratinases cleave the hydrogen bonds of β-pleated sheets and release different amino acids. Keratinolytic enzymes can hydrolyze both native and denatured keratins and may have vital utility in biotechnological processes through the extension of nonpolluting processes. These extracellular reductases and keratinase could potentially be useful in the reduction of metals by using NADH or NADPH as the electron donor [36] . Bacillus subtilis RSE163 is a nonpathogenic bacterium that produces both keratinase and reductase in the presence of keratin feather as substrate. We have used reductase and keratinase synergistically for the reduction of Au 3+ ions to synthesize protein-coated gold nanoparticles by Bacillus subtilis RSE163 using chicken feather. To ensure the biocompatibility of synthesized gold nanoparticles, we investigated their interaction with Escherichia coli (ATCC11103), which is generally considered as a model organism for various fundamental and applied aspects of medicine, life science, and biotechnology.
Materials and Methods
- Materials
Bacillus subtilis RSE163 (Accession No. JQ887983) previously isolated in the laboratory was cultured and maintained at -20℃ in 50% glycerol. Feather-degrading specific medium (glucose 1%, peptone 1%, KH 2 PO 4 0.9%, K 2 HPO 4 0.3%, feather 0.5%) was inoculated with 1% (v/v) of bacterial culture and kept in an incubator at 37℃ at 180 rpm for 3 days for degradation. The medium-degraded chicken feather solution was centrifuged at 10,000 rpm for 10 min to separate the solid mass. The supernatant was collected separately for further reaction and experimental use. Synthesis of gold nanoparticles was carried out by adding 1:1 (v/v) chloroauric acid (10 -3 M) in supernatant and keeping it at 37℃, 100 rpm, along with their controls (supernatant without substrate and substrate solution alone), for 16 h. Optical properties of the synthesized GNPs were studied using a UV-Vis spectrophotometer (Shimadzu model 1700 UV-Vis). The effects of pH and substrate concentration were also studied on the formation of nanoparticles. All the studies have been conducted in triplicates.
- Estimation of Disulfide Reductase and Keratinolytic Activity
The activity of disulfide reductase and keratinase produced from strain Bacillus subtilis in the presence of chicken feather was determined using cell free extract of the feather medium after 3 days of incubation at 37℃. For the estimation of disulfide reductase activity, the reaction mixture contained 2 ml of (50 mM) potassium phosphate, 3 ml of enzyme, and 5 ml of distilled water. Then, 20 µl of DTNB (5,5 –dithiobis (2-nitrobenzoic acid) was added to 3 ml of this mixture. Yellow-colored sulfide formed upon reduction of DTNB, which was measured spectrophometrically at 412 nm. One unit of disulfide bond-reducing activity was defined as the amount of enzyme that catalyzes the formation of 1 µmole of sulfide per minute. Keratinase activity was estimated by incubating the reaction mixture that contained 100 µl of enzyme preparation and 4.9 ml of glycine NaOH buffer (0.5 M pH 9) with 25 mg of feather as substrate at 60℃ for 1 h, followed with addition of 4 ml of trichloroacetic acid (5% (w/v)) to stop the reaction. The reaction mixture was filtered and measured spectrophotometrically at 280 nm. An increase of a 0.01 absorbance was considered as 1 Unit of keratinase per milliliter in 1 h at 280 nm under the assay conditions [17] . All the experiments were performed in triplicates.
- Structural and Size Characterization
For structural and size characterization, HRTEM (Philips, CM-10 model) was used with an operating voltage of 100 kV. The gold nanoparticles were dispersed on carbon-coated copper TEM grids for analysis. Dynamic light-scattering measurements and zeta potential of the sample were determined by Malvern Zetasizer Nanoseries (Malvern Instruments Ltd, Malvern, UK) to measure the hydrodynamic size and stability of gold nanoparticles in solution. Data obtained were analyzed using Zetasizer software. The FTIR analysis of the sample were carried out with a Jafco 410 Series spectrophotometer in the wave number range of 4,000-400 cm –1 to identify possible organic capping on the surface of synthesized gold nanoparticles. Energy dispersive X-ray analysis was carried out using Carl Zeiss EVO-40 at 20 KV to authenticate the presence of gold nanoparticles and its chemical composition in electron micrographs.
- Interaction of GNPs withE. coli
The experiment was performed in autoclaved 100 ml Erlenmeyer flasks containing 25 ml of the nutrient broth. One percent of E. coli culture (2.4 × 10 6 CFU/ml) was added to 100 µl of prepared gold nanoparticles in a flask and incubated at 37℃ in a rotor shaker at 120 rpm. Samples were withdrawn at different time intervals of 0, 4, 6, 12, 24, and 48 h under sterile condition and the absorbance was measured at 600 nm using a UV-Vis spectrophotometer (Shimadzu model 1700 UV-Vis). The experiments were carried out in triplicates and standard error was calculated [38] .
Results and Discussion
Biodegradation of chicken feather by Bacillus subtilis RSE163 is an enzymatic synchronized process for the production of both disulfide reductase and keratinase [17 , 34] . In the present study, 0.24 ± 0.05 U/ml and 366 ± 15.79 U of disulfide reductase and keratinase were produced after 3 days by Bacillus subtilis RSE163 in the presence of chicken feather [17] . For the mixture of disulfide reductase and keratinase incubated with 1 mM of HAuCl 4 for 16 h, a change in color of solution from pale yellow to purple was observed, indicating the formation of gold nanoparticles ( Fig. 1 ).
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Test tubes showing the formation of gold nanoparticles. (1) Substrate control, (2) supernatant control, and (3) purple color due to formation of gold nanoparticles.
The disulfide reductase obtained from extracellular secretion through Bacillus subtilis RSE163 in the presence of chicken feathers was capable for the reduction of Au 3+ ions to form gold nanoparticles in the experimental samples. Chicken feather has a beta pleated sheet structure cross linked by disulfide bridges. These disulfide bridges are cleaved by disulfide reductase and release thiol groups whereas keratinase hydrolyzes the beta sheet and liberates different amino acids [17] . Availability of dithiol substrate (β-keratin) in the media enhances the live-cell redox during the growth of Bacillus subtilis RSE163, leading to oxidization of reductase and reduction of auric ions simultaneously [33] . Microorganisms have developed complex systems to maintain the equanimity in thiol redox environment under normal physiological circumstances. The disulfide reductases catalyze the thiol-disulfide exchange reactions to endorse the formation or reduction of protein disulfide bonds within cells with simultaneous oxidation and reduction of NADP and NADPH ( Fig. 2 ) [12 , 19 , 37] .
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Schematic representation of mechanism of formation of gold nanoparticles using reductase and keratinase.
The appearance of a characteristic absorbance peak in UV-Vis spectra around 540 nm is attributed to surface plasmon resonance (SPR) absorbance of gold, and Fig. 3 A confirms the formation of gold nanoparticles from the degraded chicken feathers by Bacillus subtilis . The broadness of the peak indicates either the broad size dispersity of nanoparticles or the coating of nanoparticle surface with some organic moiety. We may also expect the protein coating from the degraded chicken feather will contribute to the broadness of the SPR peak. Similar SPR-related absorbance has been observed for gold nanoparticles synthesized using other bacterial systems, like Pseudomonas fluorescens [32] and E. coli DH5α [10] .
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Optical properties of synthesized gold nanoparticles. (A) UV-Vis spectra showing the synthesized gold nanoparticles. (B) UV-Vis spectra showing the effect of different pH on the synthesis of gold nanoparticles. (C) UV-Vis spectra showing the effect of HAuCl4 concentration ratio (supernatant: substrate) on the synthesis of gold nanoparticles.
The effects of pH on enzyme-producing media and Au precursor concentration on the formation of gold nanoparticles were monitored by UV-visible spectroscopy ( Figs. 3 B and 3 C). Broad SPR absorption bands were for gold nanoparticles synthesized at pH 8 and 10, but at pH 9 and 11 comparatively sharp absorption spectra were observed, indicating that a higher pH is not favorable for protein coating, and anisotropic growth is also expected as a result of co-adjuvant action of reductase and capping agents. However, the TEM picture did not signify this. Quester et al. [30] demonstrated the formation of quasi spherical and triangle shaped nanoparticles from the fungus Neurospora crassa , and showed that pH values played a crucial role in defining the shape and size of the particles. Zhang et al. [49] have shown the size variation of nanoparticles with the gradual increase in pH. Similarly, with increment in concentration of chloroauric acid (1:0.25, 1:0.5, 1:1, and 1:1.5), SPR bands were sharper, and there was consequent increase in peak area; that is, red shifting (620-780 nm) of plasmon ( Fig. 3 C) [43] . This may be because of the higher concentration of chloroauric acid, which promotes the competition between cauric ions and functional groups present in cell-free supernatant, resulting in the large size and/or aggregated gold nanoparticles [45] . Young et al. [48] also reported different sizes of gold nanoparticles of 60 and 40 nm with respect to 0.01 and 0.03 mM concentrations of choloroauric acid. Other researchers have also demonstrated the synthesis of different shaped (spherical, triangles, hexagons, spheres) nanoparticles by controlling the key parameters like pH, reaction time, and substrate concentration [9 , 15] . The dissimilarity in shape of the nanopartcles might also be due to different reductases associated with the nanoparticle biosynthesis [8] . Identification of the nanoparticle size and charge was confirmed by HRTEM studies, DLS, and zeta potential measurements. The representative HRTEM image showed well-dispersed spherical-shaped gold nanoparticles ranging between 50 and 80 nm ( Fig. 4 ). Dynamic light scattering revealed the hydrodynamic size of gold nanoparticles of about 100 nm ( Fig. 5 A), which is comparable to the size obtained from HRTEM imaging, indicating well-dispersed nanoparticles. There was increase of 20 to 30 nm in size, endorsed by the presence of the protein capping attached to the nanoparticle surface along with the bound water molecules [42] . Zeta potential indicates the degree of repulsion for adjacent similarly charged particles and stability. A minimum zeta potential of more than -30 mV is essential for good stability and of more than -60 mV for excellent stability [7] . The value of the surface charge of gold nanoparticles was -44.1 mV, using ζ potential measurements, indicating good stability of the synthesized nanoparticles ( Fig. 5 B). FTIR spectroscopy was used to characterize the surface functionalization of synthesized gold nanoparticles and is shown in Fig. 6 . The absorption bands located at 3,850 cm -1 and 3,464 cm -1 are assigned as the absorption peak of the N-H group and O-H group. The band at 2,078 cm -1 is related to aromatic C-H stretching [21] , and characteristic bands at 1,640 cm -1 and 1,449 cm -1 are for amide-conjugated carbonyl group and methylene scissoring vibrations indicating the presence of organic capping, probably the protein capping [32] .The capping of protein on nanoparticles engross through the thiol group released via sulfytolysis, which facilitates the binding of other peptides and free amine groups hydrolyzed by keratinase. Our results are also in agreement with Gajbhiye et al. [13] , who reported the coating of protein on nanoparticles either through a free amine group or cysteine residues or electrostatic attraction of negatively charged carboxylate groups present in extracellular and intracellular enzymes of microorganism. The protein coating is attributed to the enhanced stability of the gold nanoparticles in solution, as well as biocompatibility.
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HRTEM image of gold nanoparticles synthesized by reduction of HAuCl4 (10−3 M) solution.
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Identification of gold nanoparticle size and charge. (A) Graph of dynamic light scattering measurements of enzymatically synthesized gold nanoparticles. (B) Distribution graph of Zeta potential of enzymatically synthesized gold nanoparticles.
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FTIR spectrum of gold nanoparticles synthesized from keratinase.
An EDX spectrum of the scanned sample was recorded in the spot profile mode by selecting the densely populated gold nanoparticle regions on the surface of the film. Strong peaks located from 2 to 4 KeV divulge the presence of pure metallic gold nanoparticles, while peaks of carbon and oxygen were also recorded on the left side ( Fig. 7 ). The EDX spectrum specified that the reaction product was composed of high-purity gold nanoparticles [9] . The carbon and oxygen spots in the scrutinized sample are attributed to carboxyl and amine groups present in protein capped on the gold nanoparticles.
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Spot profile EDX spectrum of enzymatically synthesized gold nanoparticles.
- Interaction of Gold Nanoparticles withE. coli
The synthesized GNPs were incubated with E. coli to explore the biocompatibility and internalization efficacy of biogenic GNPs in living cells. Fig. 8 shows the variation in optical density (OD), revealing the effect of gold nanoparticles on the growth of E. coli through a comparative growth curve. It was observed that at the initial phase, both the GNPs-treated and non-treated cultures represented the same lag phase until 2 h. After 2 h, there was a gradual increase in the exponential phase of gold nanoparticletreated culture. It could be attributed to the protein-capped gold nanoparticles, which are supposed to inactivate ROS (reactive oxygen species) generation, leading to higher bacterial cell proliferation [16] . Barath et al. [3] have also suggested GNPs as an anti-oxidative agent by reducing the formation of ROS and scavenging free radicals to maintain control over hyperglycemic conditions.
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Bacterial growth in the presence of gold nanoparticles.
The transmission electron microscopic studies of ultrathin sections of E. coli cells treated with GNPs clearly revealed the presence of GNPs within the cytoplasm ( Fig. 9 A). Movement of GNPs inside the cell membrane barriers is presented in Fig. 9 B, whereas Fig. 9 C confirms their accumulation inside the cell without affecting the structural integrity and shape of the cells. The outer membrane of E. coli cells contains porins, which act like pores and make the outer membrane permeable [23] . The interaction between the bacterial cell and gold nanoparticles could also be possible owing to Van der Waals, electrostatic, and H-bonding forces between negatively charged bacterial surfaces and positively charged gold nanoparticles [29 , 39] . The GNPs bind with a negatively charged group present on bacterial surfaces, which helps them in membrane penetration and cellular internalization [46] . Vigderman and Zubarev [47] reported the endosomal cellular uptake of gold nanoparticles coated with a chemotherapeutic agent for prevarication of multidrug resistance within the tumor cell by detour of the efflux pump in the bacterial cell. Therefore, gold nanoparticles offer a platform as synthetic carriers for drug, gene, and growth factors delivery inside the cell owing to their non-complexity and surface functionality [2 , 35] .Stability and distribution of nanoparticles at different levels of organization in a biological system depend on their size and morphology.
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TEM images of movement of enzymatically synthesized gold nanoparticles inside Escherichia coli. (A) Gold nanoparticles outside the cell membrane. (B) Gold nanoparticles move inside the cell, crossing the cell membrane barriers. (C) Accumulation of gold nanoparticles inside the cell.
In conclusion, we demonstrated a safe, economic, and ecofriendly method for enzyme-mediated extracellular synthesis of spherical-shaped, protein-coated gold nanoparticles (size 25-50 nm), using Bacillus subtilis RSE163 in the presence of chicken feather. The optimized process of chicken feather degradation using Bacillus subtilis can further be utilized as biofactories for nanoparticle production. However, a systematic study is still required to understand the exact mechanism and biochemical reactions responsible for the synthesis of GNPs. The synthesized biogenic protein-coated gold nanoparticles may be considered as an excellent contender for the delivery of drugs, genetic material, and metabolites inside the cells in the form of nano biotransducers.
Acknowledgements
We acknowledge the Department of Anatomy, AIIMS, New Delhi and AIRF (JNU) Delhi, India for helping in HRTEM, FTIR studies, DLS, zeta potential measurements, and EDX analysis. We also extend our sincere thanks to the Ministry of Environment and Forest (19-118/2008-RE), Government of India for providing a senior research fellowship to Ms. Sonali Gupta.
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