In the recent decades, a high level of interests of science and technology is focused on the production of nanoparticles. Integration of nanotechnology and medicine has created significant advances in diagnosis and treatment, molecular biology and biological engineering (1, 2). Among the nanoparticles, metal nanoparticles which have wide applications in molecular engineering, nano medicine and nano electronics are most commonly used (3). Several physical, chemical and biological methods for production of gold nanoparticles were investigated. High levels of pollution due to using toxic chemicals, high costs, and unstable non-uniform particle size after building are some of the problems that encouraged scientists to find green synthesis methods to produce nanoparticles (4). Microbial cells and enzymes are the highly organized factories which can do bio transformation in metals and make metal nanoparticles easily, cheaply, and environmentally friendly (5).
Gold nanoparticle, because of stability in atmospheric conditions, resistance to oxidation, and environmental compatibility is known to have a particular importance in medicine and biotechnology. Also, they are used as biosensors in nanoelectronics and molecular engineering (6, 7). The first biological system in synthesis of gold nanoparticles was recognized in Pedomicrobium-like bacterium. Gold nanoparticle synthesis in other microorganism such as iron reducing and metal-like bacteria such as Cuperiavidous necator and Cuperiavidous metallidorans also was reported (8). Bacterial deposition of gold and production of gold nanoparticles by microorganisms has created a new way to recycle this valuable metal from the environment.
Regarding to the importance of biological production and the wide applications of gold nanoparticles, attention of scientist was drawn to efficient, easy, low-cost, and effective production of nanoparticles (3).
In our previous study, the role of dipicolinic acid extracted from Bacillus spores in silver nanoparticle synthesis was demonstrated and the results revealed that only silver nanoparticles were greatly produced under the experimental conditions (9). In order to optimize synthesis of other metal nanoparticles especially gold nanoparticles by spores, genetically engineered spores were used. In this study, spore displayed tyrosinase is presented a new rapid approach to green synthesis of gold nanoparticles.
Material and method
Spore Preparation and Detection of Enzymatic Activity: Bacillus subtilis DB104 (pSDJH-cotE-tyr) which was prepared in our previous research (10), was inoculated into Difco sporulation medium (DSM) containing 0.8 % (w/v) Difco Nutrient Broth, 0.1 % (w/v) KCl, 0.025 % (w/v) MgSO4·7H2O, 1 mM Ca(NO3)2, 0.01 mM MnCl2, and 0.01 mM FeSO4, pH 7 and incubated at 37 °C for 24 h on a shaker (200rpm) (11). Renografin (sodium diatrizoate, S-4506, Sigma) gradient method was used for spore purification.
Tyrosinase activity was assayed using L-tyrosine as a substrate, and Bacillus subtilis DB104 (pSDJH-cotE-tyr) spore solution as the tyrosinase sources (12). Purified tyrosinase from Bacillus megaterium DSM319 and Streptomyces also were used as confirmatory samples. The reaction mixture without tyrosinase was used as control.
Analysis of Gold Nanoparticles Biosynthesis: Two types of Bacillus subtilis spores with displayed tyrosinase and without displayed enzyme were studied. 100 µl of spore solutions containing 1.2 x 107 spores was added into 1 mL of aqueous solution of 1 mM AuCl3. The interaction of the spores and Au ions was completed in room temperature in 2 hours. Also, gold nanoparticle synthesis was studied using the 0.25 mM of purchased standard DPA (2, 6-Pyridinedicarboxylic acid, P63808 Sigma). The biosynthesized AuNps were characterized by XRD and TEM.
XRD Analysis: X-ray diffraction (XRD) analysis of the gold nanoparticles was done by X-ray Diffractometer, D8ADVANCE (Bruker, Germany). X-rays were made by a copper X-ray tube with wavelength 1.5406 Ǻ (Cu Kα) and Ni as a filter. Measurements were performed between 30° and 70° 2θ (9).
TEM (transmission electron microscopy): To prepare the samples for TEM analysis, 5 μL of biosynthesized Au nanoparticle solutions were dropped on carbon-coated copper grids. The grids were observed in a JEM-2100 Electron Microscope (JEOL, Japan) operated at 120 kV.
Production of Gold nanoparticles by Spore Displayed Tyrosinase: After a few hours of adding 1mM AuCl3 in spore suspension, sediments containing AuNPs were demonstrated (Figure1). As it is shown in Figure 1, the brown precipitate was appeared in samples of Bacillus megaterium tyrosinase, Streptomyces tyrosinase and spore displayed tyrosinase. Production of AuNPs did not demonstrate in the samples with dipicolinic acid and Bacillus subtilis spores without tyrosinase during this short period of time.
XRD pattern:The synthesized AuNPs were characterized using XRD technique. The XRD pattern of the nanoparticles produced by Bacillus subtilis DB104 (pSDJH-cotE-tyr) spores is presented in Figure 2, where there were three sharp peaks in the whole pattern of 2θ value ranging from 30 to 70. It actually is similar to the spectra took by the previous reports of AuNPs.
TEM analysis: AuNPs also were characterized by TEM. Figure 3 shows TEM images of AuNPs. The size of the biogenic AuNPs was from 2.5 to 35 nm. As shown, the TEM micrographs showed the presence of diverse morphology (cubic, triangular, spherical, and hexagonal structures) of AuNPs.
Fig. 1- The formation of AuNPs precipitate. Control: AuCl3 solution. BMT: Bacillus megaterium tyrosinase, ST: Streptomyces tyrosinase, SDT: Spore displayed tyrosinase, DPA: dipicolinic acid.
Fig. 2- XRD pattern of synthesized AuNPs.
Fig. 3- TEM images of biogenic AuNPs.
Discussion and Conclusion
Our previous research revealed that the extracted dipicolinic acid from Bacillus spores can quickly synthesis Ag nanoparticles (9). However a question about the role of dipicolinic acid in the production of other metal nanoparticles had remained. Our results showed that the production of AuNPs did not occur obviously by dipicolonic acid in a short period of time. Previous researches revealed that different enzymes have various patterns for synthesis nanoparticles so finding suitable enzymes for synthesis each metal nanoparticles is interesting. In order to improve spore properties to change trivalent metal ions to nanoparticles, the role of enzymes was studied. According to this phenomenon, in this research, the role of tyrosinase in making AuNPs by using spore displayed tyrosinase was considered.
The results revealed that spore displayed tyrosinase changes Au3+ to Au0. Furthermore, purified Bacillus megaterium tyrosinase and Streptomyces tyrosinase also produced AuNPs. The role of enzymes in the production of nanoparticles has been proven. Das et al., showed the role of (NADPH) oxidoreductasesin the production of AuNPs inRhizopus oryzae (13). Also, other reductases, hydrogenases, hydrolases, desulfhydrase and syntases were reported before (14). Li et al. used laccase as a reducing agent for green synthesis of gold nanoparticles (15).
Sanghi et al. also presented the role of laccase in extracellular synthesis of AuNPs and the role of ligninase in intracellular production of AuNPs in Phanerochaete Chrysosporium (16).
The supposed mechanism for the production of gold nanoparticles by Tyrosinase is electron transferring from copper ions (the cofactors of tyrosinase) to Au3+. Transformation of Au3+ to Au0 is a kind of reducing reaction. As it is mentioned before, several reductase enzymes reduce ions and make nanoparticles. Regarding the reduction of potential elements in aqueous solution, Cu give electron to Au3+. According to the mechanism of tyrosinase activity, during the production of L-DOPA from L-tyrosine a reducing agent is produced that generates AuNPs (Figure 4) (17).
Fig. 4- Mechanism of AuNPs synthesis by tyrosinase