Accepted Manuscript Title: Enzymatic formation of gold nanoparticles by submerged culture of the basidiomycete Lentinus edodes Author: Elena P. Vetchinkina Ekaterina A. Loshchinina Andrey M. Burov Lev A. Dykman Valentina E. Nikitina PII: DOI: Reference:
S0168-1656(14)00199-0 http://dx.doi.org/doi:10.1016/j.jbiotec.2014.04.018 BIOTEC 6674
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
29-11-2013 22-2-2014 25-4-2014
Please cite this article as: Vetchinkina, E.P., Loshchinina, E.A., Burov, A.M., Dykman, L.A., Nikitina, V.E.,formation of gold nanoparticles by submerged culture of the basidiomycete Lentinus edodes, Journal of Biotechnology (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Enzymatic formation of gold nanoparticles by submerged culture of the
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basidiomycete Lentinus edodes
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Elena P. Vetchinkina, Ekaterina A. Loshchinina*, Andrey M. Burov,
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Lev A. Dykman, Valentina E. Nikitina
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Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of
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Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia
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*Corresponding author. Tel: #(845-2) 97-04-03; 97-03-27; fax: #(845-2) 97-03-83; 97-04-44.
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E-mail address: [email protected] (Ekaterina A. Loshchinina)
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• Lentinus edodes can reduce Au(III) from HAuCl4 to Au(0), forming nanoparticles
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Highlights
• Au(0) accumulated on the surface and within the hyphae as 5-50 nm
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nanospheres
• The fungal phenol oxidases were found to be involved in the Au reduction
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ABSTRACT
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We report for the first time that the medicinal basidiomycete Lentinus edodes can reduce Au(III)
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from chloroauric acid (HAuCl4) to elemental Au [Au(0)], forming nanoparticles. Several
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methods, including transmission electron microscopy, electron energy loss spectroscopy, X-ray
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fluorescence, and dynamic light scattering, were used to show that when the fungus was grown
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submerged, colloidal gold accumulated on the surface of and inside the mycelial hyphae as
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electron-dense particles mostly spherical in shape, with sizes ranging from 5 to 50 nm.
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Homogeneous proteins (the fungal enzymes laccase, tyrosinase, and Mn-peroxidase) were found
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for the first time to be involved in the reduction of Au(III) to Au(0) from HAuCl4. A possible
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mechanism forming Au nanoparticles is discussed.
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Keywords:
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Lentinus edodes
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Gold nanoparticles
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Extracellular and intracellular synthesis
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Reduction mechanisms
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Phenol oxidases
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1. Introduction
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Nanoparticles are finding wide application in medicine, biology, and technology in areas as
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wide apart as cancer therapy and information technology (Dreaden et al., 2012; Dykman and
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Khlebtsov, 2012; Dykman et al., 2008). Various physical and chemical methods have been
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developed to synthesize nanoparticles of desired sizes and shapes; however, these methods,
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though successfully used, can be costly and require the use of hazardous chemical compounds.
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Therefore, there is a growing interest in “green” (environmentally and human friendly)
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nanoparticle synthesis with the aid of microbial biotechnologies (Krumov et al., 2009;
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Narayanan and Sakthivel, 2010).
Au nanoparticles obtained from reduction of chloroaurates by bacteria, fungi, or plants have
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unique antimicrobial, antifungal, and antitumor properties (Ahmad et al., 2013; Das et al., 2009;
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Iravani, 2011). An active search is in progress to find novel effective biological agents for the
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preparation of nanoparticles of various chemical natures (Durán et al., 2007; Musarrat et al.,
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2011; Popescu et al., 2010; Rai and Duran, 2011). Several bacteria have been found able to
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reduce Au from auric compounds to form nanoparticles, both extracellularly (e.g., Lactobacillus
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sp., Rhodopseudomonas capsulata, Pseudomonas aeruginosa, and Bacillus flexus; He et al.,
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2007; Husseiny et al., 2007; Murugan et al., 2013; Nair and Pradeep, 2002) and intracellularly
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(e.g., Shewanella algae and Stenotrophomonas maltophilia; Konishi et al., 2004; Nangia et al.,
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2009). Synthesis of Au and Ag nanoparticles by several lower fungi (Mourato et al., 2011; Sastry
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et al., 2003) and by the basidiomycetes Volvariella volvacea, Coriolus versicolor, and Pleurotus
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sapidus (Philip et al., 2009; Sanghi and Verma, 2009; Sarkar et al., 2013) has also been
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documented; however, the higher fungi transformed compounds in their culture liquid and could
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not accumulate elemental particles intracellularly.
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There are still many open questions in this area of research. Biologically synthesized
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nanoparticles often have various shapes and sizes, and the synthesis itself is time-consuming.
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Nothing is known of which enzymes are involved in colloidal gold bioreduction, though recent
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data suggest that extracellular phenol oxidases might have a role (Sanghi et al., 2011). No
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experimental data are available concerning the biosynthesis of Au nanoparticles by
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homogeneous enzymes. Therefore, comprehensive studies are needed to overcome these
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problems and improve the synthetic rate and the properties of nanoparticles. Such studies should
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examine all factors of importance to the process of synthesis, such as methods of cultivation,
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extraction, and stabilization, as well as should decipher the cellular, biochemical, and molecular
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mechanisms of reduction involved.
There are very few data on the fungal transformation of Au compounds with release of
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elemental Au [Au(0)]. However, higher fungi constitute a very favorable object of study, as they
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can accumulate high concentrations of various elements. This is especially true of cultivated
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edible medicinal basidiomycetes, as they are nontoxic, have a high biomass yield, and can
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accumulate large amounts of reduced nanoparticles in their mycelium. One such fungus, the
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xylotrophic basidiomycete Lentinus edodes (shiitake), owing to the unique complex of
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biologically active compounds that it contains in its mycelium and fruit bodies, has been actively
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used as raw material to prepare a range of highly effective drugs and biologically active food
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supplements (Jones, 1995; Mizuno, 1995). The current interest in this fungus is also due to the
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high nutritive value and the taste of its fruit bodies.
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The aim of this study was twofold: (i) To examine the ability of Lentinus edodes to reduce
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Au(III) to Au(0) and accumulate nanoparticles in its mycelium. (ii) To identify the enzymes
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involved in this process.
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2. Materials and methods
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2.1. Fungus and culture conditions
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The xylotrophic medicinal basidiomycete Lentinus edodes (Berk.) Sing [Lentinus edodes
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(Berk.) Pegler] (shiitake), strain F-249, was obtained from the collection of higher fungi held by
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the Laboratory of Microbiology at this institute. The fungal culture was maintained on 4% beer-
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wort agar plates at 4°С.
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L. edodes was grown submerged in a synthetic medium of the following composition (g/L):
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D-glucose,
1; L-asparagine, 0.1; KH2PO4, 2; K2HPO4, 3; MgSO4 · 7H2O, 2.5; FeSO4 · 7H2O, 0.03
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(рН 5.8). Growth was conducted in 100-mL flasks containing 50 mL of medium at the mycelial
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growth temperature optimal for this species (26°С; Przybylowicz and Donoghue, 1991). For
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inoculation, we used a 14-day-old culture of L. edodes grown on agar-supplemented synthetic
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medium at 26°С.
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The effect of auric compounds on L. edodes growth and the xylotroph’s reduction ability
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were examined by adding 10 to 500 µM of aqueous chloroauric acid (HAuCl4; Sigma-Aldrich,
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USA) to the liquid synthetic medium. The solutions of the compounds were added under sterile
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conditions to each of the growth flasks individually just before seeding. L. edodes growth was
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characterized by the accumulation of dry biomass. The mycelium was passed through filters that
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had been preweighed on an analytical balance, and then it was dried to a constant weight and
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weighed again. The increment in biomass was compared between control (3-h-old culture) and
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experimental trials. Experiments to measure fungal growth characteristics and biomass
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accumulation were done in 5 to 10 replicates.
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2.2. Enzyme isolation and purification
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Crude enzyme extracts were obtained by growing the fungus at 26°С for 14 days in a
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synthetic medium of the above composition. The mycelium was then separated from the culture
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medium, rinsed in distilled water, and mechanically ground in a porcelain mortar with quartz
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sand to destroy the cell envelope. Extraction was done by using 20 mg of homogenized
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mycelium per 2 mL of 20 mM Tris–HCl buffer (pH 7.5). The homogenate was spun down at
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12,000 g for 15 min, separated from the sediment, filtered, and desalted on a column of
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Sephadex G-25 (Pharmacia, Sweden) equilibrated with extraction buffer. Further purification
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was done by high-performance liquid chromatography (HPLC) on a TSK Bioassist Super Q
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(Sweden) anion-exchange column equilibrated with a pH 7.5 20 mМ Tris–HCl. Proteins were
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eluted with an NaCl gradient of 0.01 to 1.0 M, and elution was recorded with a Uvicord S-II
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apparatus (LKB, Sweden) at 280 nm. The fractions with Mn-peroxidase, laccase, and tyrosinase
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activities were dialyzed against water and used in the experiment.
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2.3. Polyacrylamide gel electrophoresis
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Protein composition was examined by nondenaturing electrophoresis in a 7.5%
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polyacrylamide gel (PAG; Laemmli, 1970). For visualization of phenol oxidases, PAG was
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specifically stained in reaction mixtures of 1% acetic acid, 0.2% o-dianisidine (Sigma, USA),
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and 50 mM Na tartrate (pH 4.5). Red-brown staining of the protein bands corresponding to
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laccase developed within 15 min of gel incubation in the reaction mixture (Gaal’ et al., 1982).
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For staining of PAG for Mn-peroxidase activity, 0.1 mM H2O2 and 0.2 mM MnSO4 were added
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to the above reaction mixture. Red-brown staining of the protein bands corresponding to Mn-
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peroxidase developed within 20 min of gel incubation (Glenn and Gold, 1985). For staining of
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PAG for tyrosinase activity, a reaction mixture of 2 mM 3-(3,4-dihydroxyphenyl)-L-alanine (L-
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DOPA; Serva, Germany) and 50 mM Tris–HCl (pH 7.5) was used. Brown bands corresponding
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to tyrosinase developed within 10 min (Pomerantz and Murthy, 1974). The molecular masses of
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the proteins were determined as described by Weber and Osborn (1969).
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2.4. Enzyme assays
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The substrate specificities of laccase, tyrosinase, and Mn-peroxidase were determined
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qualitatively by the ability of these enzymes to oxidize specific phenolic substrates, such as 2,2′-
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azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS; Niku-Paavola et al.,
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1988), syringaldazine (Leonowicz and Crzywnowicz, 1981), 2.6-dimethoxyphenol (DMOP;
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Sigma) (Slomczynski et al., 1995), and L-DOPA (Serva; Pomerantz and Murthy, 1974).
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The effect of inhibitors on laccase activity was studied in a mixture containing 50 mM Na/K-
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phosphate buffer (pH 6.0), the enzyme, the substrate (syringaldazine), and an inhibitor. The
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reaction was performed at 25°C. The following inhibitors were tested: 0.1 to 100 mM EDTA,
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0.001 to 0.1 mM β-mercaptoethanol, and 0.1 to 4% SDS (Fluka, Switzerland; Pozdnyakova et
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al., 2006).
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Enzyme activities were determined with a Specord M40 spectrophotometer (Carl Zeiss, Jena,
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Germany) in 1-cm quartz cuvettes at 18°C. Laccase activity was measured by the oxidation rate
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for 0.2 mM ABTS in 15 mM Na tartrate (pH 4.5). The oxidation of ABTS to a stable cation
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radical was measured by the increase in absorbance at 436 nm (ε436 = 29300 M-1 cm-1)
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(Slomczynski et al., 1995). Mn-peroxidase activity was determined by the oxidation rate for 0.2
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mM ABTS in 50 mM Na tartrate (pH 4.5) supplemented with 0.1 mM H2O2 and 0.2 mM Mn2+.
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The oxidation of ABTS to a stable cation radical was measured by the increase in absorbance at
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436 nm (ε436 = 29300 M-1 cm-1) (Glenn and Gold, 1985). Tyrosinase activity was determined by
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the oxidation rate for 2 mM L-DOPA in 50 mM Tris–HCl (pH 7.5). The oxidation of L-DOPA to
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DOPAquinone was measured by the increase in absorbance at 475 nm (ε475 = 3700 M-1 cm-1)
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(Pomerantz and Murthy, 1974).
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The reaction time was 5 min in all experiments. One unit of enzyme activity was defined as
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the amount required to convert 1 μmol of substrate to product per minute, or to catalyze the
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formation of 1 μmol of product per minute, and is expressed as micromoles per minute per
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milligram of protein. Protein was estimated by the Bradford method (Bradford, 1976).
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2.5. Transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS), and
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electron spectroscopic imaging (ESI)
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The mycelium and the culture liquid were studied by negative contrasting. To this end, the
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material was mounted on nickel grids coated with 1% formvar in dichloroethane. After the
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sample had dried, the cells were contrasted with 1% aqueous uranyl acetate (Tandler, 1990).
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Ultrathin sections were obtained by fixing the fungal hyphae in epoxy resin, as follows. The
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mycelial hyphae were collected by centrifugation (13,000 × g for 20 min) at room temperature,
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and the material was then transferred to 2-mL polypropylene test tubes and fixed in 0.5%
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glutaraldehyde for 3 h. Subsequent fixation was done for 12 h in a 2.5% glutaraldehyde solution
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made with phosphate buffer (0.1 M, pH 7.2). The material was then held in a 0.1% OsO4
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solution made with phosphate buffer supplemented with 34 mg/mL sucrose (postfixation).
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Samples were dehydrated in increasing concentrations of alcohols and in absolute acetone.
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After dehydration, the samples were held in propylene oxide for 45 min, and then they were
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embedded in Epon 812 and propylene oxide (each time for 24 h) at ratios of 1:2, 1:1, and 2:1.
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Next, the material was embedded in pure epoxy resin. Polymerization was done at 37, 45, and
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57°С for 24 h each. Ultrathin sections were cut on an LKB-III microtome (Sweden), mounted on
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nickel grids, and stained with lead citrate (Reynolds, 1963).
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For nanoparticle studies, the fungal mycelium was grown in the presence of 50 μM HAuCl4
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for 14 days, washed free of the cultivation medium with water, collected by centrifugation, and
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lyophilized. The fungal hyphae were then mechanically disrupted in a porcelain mortar with
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quartz sand, resuspended in MilliQ water, and separated from elemental particles through a 0.22-
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μm-pore-size membrane filter (Millipore). The cell debris and nanoparticle aggregations
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remained on the filter. The culture liquid, containing reduced Au nanoparticles, was filtered
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through the usual paper filter to remove the hyphal debris and was dialyzed against MilliQ water
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for 24 h, with several changes of water. The nanoparticles were mounted on nickel grids coated
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with 1% formvar in dichloroethane. A Libra 120 electron microscope (Carl Zeiss, Germany)
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operating at 120 keV was used to take photomicrographs.
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2.6. X-ray fluorescence analysis
Elemental Au in the fungal mycelium was detected by an X-ray fluorescence study of
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mycelial samples. To this end, L. edodes was grown in the synthetic medium without (control)
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and with (experiment) 50 μM HAuCl4. The grown mycelial samples were washed free of the
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growth medium with distilled water, collected by centrifugation, and lyophilized. The fungal
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hyphae were then mechanically disrupted and were separated from elemental particles through a
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Millipore membrane filter (0.22 μm pore size). The nanoparticles were resuspended in minimal
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distilled water and were air dried at 20°С. The content of Au0 was analyzed with an ED 2000
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energy-dispersive spectrometer (Oxford Instruments, UK). The measuring conditions were as
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follows: element detection range, Na–U; X-ray tube, silver anode; tube voltage, 35 keV (medium
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elements); primary X-ray beam filter, thin Ag; exposure time, 600 s; spectral path, air. The
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content of Au0 was evaluated by the basic parameters method included in the instrument’s
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software.
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2.7. Nanoparticle characterization
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Absorbance spectra were measured with a Specord 250 UV–vis spectrophotometer (Analytik
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Jena, Germany). The size, shape, and relative number of electron-dense nanoformations were
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evaluated from TEM images obtained on a Libra 120 electron microscope (Carl Zeiss). The ζ-
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potential, average size, and size distribution of the synthesized Au0 particles were measured by
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dynamic light scattering with a Zetasizer Nano ZS instrument (Malvern, UK).
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2.8. Statistical analysis
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There were five independent experiments, each having no less than five replications. Data
were processed with Microsoft Excel software (Microsoft Office XP; Microsoft Corp., USA).
3. Results and discussion
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3.1. Effect of HAuCl4 on the growth of L. edodes F-249
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We first tested how sensitive L. edodes F-249 was to HAuCl4 and how this compound
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affected the growth characteristics of the fungal mycelium under submerged cultivation. The
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minimum growth-inhibiting concentration was found to range from 60 to 80 µM. At 300 to 500
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µM, growth was absent, and at 10 µM, it did not change relative to the control.
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When L. edodes was grown with 10 to 80 µM HAuCl4 in liquid synthetic medium, the
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culture liquid and mycelium acquired a reddish-lilac coloration. The control culture, grown
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under the same conditions but without HAuCl4, was cream white throughout the cultivation time.
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The change in the color of microbial colonies growing in the presence of auric compounds from
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various hues of lilac to red is the first sign of Au reduction to the elemental state. Such coloration
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is characteristic of bacteria, algae, and lower fungi growing with auric compounds and indicates
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that the culture has accumulated Au nanoparticles (Philip, 2009).
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Because the accumulation of Au(0) in the mycelium correlated with increased contents of
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HAuCl4 in the growth medium, it can be assumed that in L. edodes, as in many microorganisms
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(Garbisu et al., 1996), the reduction of this compound to Au(0) relieves the toxicity of high Au
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concentrations in the fungus.
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To detect electron-dense nanoparticles, we examined the fungal hyphae grown with 50 µM
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HAuCl4 and the culture liquid by negative contrasting. As seen in Fig. 1, spherical electron-
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dense formations were observed on the surface of the hyphae and also near them, as L. edodes
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mycelium had not been washed free of the culture medium. No such formations were present in
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With 50 µM HAuCl4, the culture grew well and the mycelial colonies had an intense lilac-red
coloration. Hence, 50 µM was used in all subsequent experiments.
3.2. TEM, EELS, and ESI
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the control. The particle diameter ranged from 5 to 50 nm, with most particles being of ~ 20 nm
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diameter. It has been found in the past few years that a number of lower fungi (Mourato et al., 2011;
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Sastry et al., 2003) and the basidiomycetes Volvariella volvacea (Philip, 2009) and Coriolus
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versicolor (Sanghi and Verma, 2009) can synthesize Au nanoparticles. However, reduction was
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observed in the culture liquid, without intracellular accumulation of elemental particles (by
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contrast, in Verticillium sp. and Trichothecium sp., nanoparticles have been observed in the cell
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wall and in the cytoplasmic membrane; Ahmad et al., 2005; Senapati et al., 2004). No
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information is currently available on Au nanoparticles resulting from L. edodes reduction of
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auric compounds, and neither is there any published evidence that fungi in general and
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basidiomycetes in particular can reduce Au to the elemental state and accumulate Au
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nanoparticles in the hyphal interior. To confirm that the elemental particles were localized
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intracellularly and that they had accumulated in the interior of the fungal mycelium, we
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embedded hyphae in epoxy resin to obtain transverse and longitudinal ultrathin sections of the
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cells. TEM confirmed that the hyphal cytoplasm contained electron-dense formations, of mostly
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spherical shape (Fig. 2). Most nanoparticles had diameters of 5 to 15 nm; however, some were of
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30 to 50 nm diameter. There were almost no elemental particles within the cell walls, suggesting
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that the particles had not penetrated through the cell wall but had been formed directly in the
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cytoplasm.
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The longitudinal and transverse ultrathin sections of L. edodes hyphae were examined by ESI
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analysis and by EELS on a Libra 120 electron microscope. An Au distribution map was obtained
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that confirmed these electron-dense formations to be intracellular Au nanoparticles (Fig. 3).
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For nanoparticle studies, the mycelium containing reduced Au was mechanically disrupted
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and was separated from the particles. The particles were resuspended in distilled water and were
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mounted on formvar-coated nickel grids. Fig. 4 shows these particles to be similarly sized (~ 20
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nm) nanospheres of a regular shape. The Au nanoparticles isolated from the culture liquid and
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fungal cells were relatively stable, did not precipitate, and did not show any signs of aggregation,
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at least for several weeks.
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3.3. Nanoparticle study by X-ray fluorescence
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To additionally confirm that the nanoparticles were indeed reduced Au, we examined them
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by X-ray fluorescence. The resulting spectra showed intense emission lines typical of Au (Fig.
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5A). The presence of Au was determined by the lines at 9.713 (Lα1), 11.443 (Lβ1), 11.585
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(Lβ2), 10.308 (Ln), and 13.382 (Ly1) keV. The spectra taken for the control culture, grown
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without HAuCl4, showed absence of Au (Fig. 5B).
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3.4. Involvement of L. edodes phenol-oxidizing enzymes in the biosynthesis of Au nanoparticles
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It is known that various bacteria can reduce Au both intra- and extracellularly (Narayanan
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and Sakthivel, 2010). The growth of L. edodes in the presence of HAuCl4 changed the color of
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both the fungal culture and the growth medium. We found that no components of the synthetic
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medium were involved in Au reduction but that the culture liquid, which contained various
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products of the fungus’s vital functions, including extracellular proteins, could reduce HAuCl4 to
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Au(0). TEM confirmed our assumption that the synthesis of Au nanoparticles was associated
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directly with L. edodes mycelium. We showed that the (mostly spherical) particles were
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localized not only extracellularly but also inside the cells, where they accumulated in large
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quantities. These facts indicated that the biochemical reduction was directly related to L. edodes
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metabolism. Work conducted in the past decade has shown that some bacteria (e.g., Shewanella sp.;
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Konishi et al., 2004) can reduce Au ions by using self-synthesized proteins, e.g., NADH-
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dependent enzymes (Tikariha et al., 2012). In Rhodopseudomonas capsulata, for example, the
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involvement of NADH-dependent nitratereductase was shown (He et al., 2007). The
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bioreduction of auric compounds in the culture medium has also been suggested to involve
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oxidoreductases (in lower fungi) (Ramezani et al., 2010) and laccases and ligninases (in the
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basidiomycete Phanerochaete сhrysosporium) (Sanghi et al., 2011). This work is the first to
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establish that the basidiomycete L. edodes can reduce HAuCl4 to the elemental state both
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extracellularly (as does P. сhrysosporium; Sanghi et al., 2011) and inside its hyphae. It has also
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been found that both the intracellular extract and the culture liquid of L. edodes have phenol
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oxidase activity; that is, the culture liquid also contains enzymes able to oxidize a number of
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phenolic substrates. One can speculate that the reductive mechanism involves phenol-oxidizing
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enzymes, as L. edodes phenol oxidases are present both intra- and extracellularly (Vetchinkina et
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al., 2008).
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To support this speculation, we isolated phenol oxidases, including intracellular laccases,
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tyrosinases, and Mn-peroxidase, from a submerged culture of L. edodes and purified them by
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HPLC. Nondenaturing polyacrylamide gel electrophoresis and specific gel staining for Mn-
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peroxidase, laccase, and tyrosinase activities made it possible to visualize the protein bands
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corresponding to these enzymes. Three proteins, eluting from the column at 0.25 to 0.33 M
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NaCl, displayed laccase activity, with the specific enzyme activity ranging from 5.8 to 10.2
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U/mg. The single-subunit enzymes had approximately the same molecular masses (100 to 120
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kDa). The intracellular Mn-peroxidase was a protein with a specific activity of 24.7 U/mg that
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consisted of two subunits (100 to 110 kDa) and was eluted with 0.3 M NaCl. Two 140-kDa
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tyrosinases were eluted with 0.35 to 0.40 M NaCl, and one tyrosinase, of approximately 300
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kDa, was eluted with 0.5 M NaCl. The specific activities of the tyrosinases were 2.1, 3.3, and 0.9
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U/mg, respectively.
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The enzyme types were determined conventionally by the ability to oxidize specific phenolic
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substrates. At pH 4.0, the laccases oxidized ABTS and DMOP; this pH optimum is characteristic
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of other fungal laccases as well (Niku-Paavola et al., 1988; Pozdnyakova et al., 2006). The
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enzymes also oxidized syringaldazine, a known test substrate for laccase activity (Slomczynski
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et al., 1995). In addition, laccase interactions with different concentrations of SDS, EDTA, and
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β-mercaptoethanol partly or completely inhibited the activity of the enzymes, again pointing to
11
their laccase nature (Leonowicz and Crzywnowicz, 1981; Pozdnyakova et al., 2006). The Mn-
12
peroxidase could oxidize ABTS and DMOP only in the presence of H2O2, which catalyzes
13
substrate oxidation for all peroxidases, and Mn2+, which does so only for Mn-dependent
14
peroxidases (Glenn and Gold, 1985; Kamitsuji et al., 2004). The tyrosinases could not oxidize
15
the substrates mentioned above; however, they degraded L-DOPA to DOPAquinone. Unlike
16
laccases, tyrosinases are of narrow substrate specificity, and L-DOPA serves as a test substrate
17
for tyrosinase activity (Pomerantz and Murthy, 1974).
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Aqueous solutions of the purified enzymes were incubated with 50 µM HAuCl4 at room
19
temperature. As early as 2 h later, the solutions acquired a lilac or red coloration, indicative of
20
the synthesis and accumulation of Au(0). The other isolated proteins, without phenol oxidase
21
activity, and the control proteins (bovine serum albumin and several fungal lectins) did not
22
reduce Au to the elemental state.
Page 15 of 35
16 1
To confirm that the nanoparticle suspension was indeed Au, we took absorbance spectra of
2
the samples. The spectra had an absorption peak at 540 nm, corresponding to colloidal Au (Fig.
3
6).
5
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4
3.5. Characterization of the nanoparticles reduced by L. edodes phenol oxidases
cr
6
The suspension of the particles reduced by the laccases and tyrosinases had a lilac hue and
8
that of the particles reduced by Mn-peroxidase had a red hue. The suspension color is often
9
enough to assume what shape and size the reduced particles would have. The lilac color
10
corresponds to particles that are mostly 50 nm in size and often variously shaped, while the red
11
color is associated with smaller spherical particles of 10 to 20 nm diameter.
M
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This speculation was confirmed by TEM. As seen in Fig. 7A, the particles synthesized by
13
Mn-peroxidase were regular spheres of 5 to 20 nm diameter. Those synthesized by the laccases
14
and tyrosinases were irregular spheres, with a small amount of triangles and tetrahedrons present.
15
The size of these particles ranged from 5 to 120 nm, with 30-nm particles predominating (Fig.
16
7B, C).
te
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17
d
12
The size of the Au particles was also evaluated by dynamic light scattering (Fig. 8A, B), and
18
the results confirmed the electron microscopic data. The ζ-potentials were 19.1, 22.7, and 23.8
19
mV for the particles synthesized by tyrosinase, laccase, and Mn-peroxidase, respectively. The ζ-
20
potential determines the extent and character of interparticle interactions in a disperse system,
21
and the greater it is, the more stable is the colloid. If the ζ-potential (positive or negative) is ≥ 30
22
mV, the dispersion will be stable to aggregation (Khlebtsov et al., 2013). In this study, the ζ-
23
potential of colloidal Au solutions was close to 30 mV, indicating that these solutions were
24
electrostatically stabilized and were not prone to coagulation or flocculation. The colloidal
Page 16 of 35
17 1
solutions of the particles synthesized by the purified phenol oxidases were more stable than those
2
of the particles formed in the culture liquid or by whole fungal cells, and they did not aggregate
3
for several months at 4ºC, possibly owing to nanoparticle stabilization by the protein molecules. Recent years have seen an increasing number of reports in which microorganisms have been
5
shown able to produce nanoparticles. This notwithstanding, there have been very few
6
comparisons between particles synthesized by living microbial cultures and particles formed by
7
metabolites isolated from these cultures. The typical focus of those studies has been on
8
intracellular nanoparticle formation as compared with synthesis in the presence of the cell-free
9
extract in which the microorganism was grown. Many investigators have pointed out that the size
10
and shape of microbially synthesized nanoparticles depend strongly on the culture conditions
11
(temperature, pH, lighting level, shaking; Ahmad, 2005; Mohanpuria et al., 2008; Sanghi et al.,
12
2011; Thakkar et al., 2010).
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4
The parameters inside the cell may differ greatly from those outside in the culture liquid. It
14
has also been speculated that the cell wall and the culture age might have a role in the
15
intracellular formation of Au nanoparticles. Gericke and Pinches (2006), using a cell-free extract
16
of the fungus Verticillium luteoalbum, observed changes in the nanoparticle morphology and the
17
appearance of a great number of large platelike structures, mostly triangular and hexagonal.
18
Those authors pointed out that as the cell interior contains many enzymes and peptides, which
19
may influence reduction and particle formation, synthesis in the presence of cell-free extracts is
20
preferable.
Ac ce p
te
d
13
21
Riddin et al. (2006) demonstrated that the fungus Fusarium oxysporum is capable of
22
producing nanoparticles of different shapes (hexagons, pentagons, circles, squares, rectangles)
23
and sizes (10–100 nm) by both inter- and extracellular processes; however, only the extracellular
24
production proved statistically significant. Govender et al. (2010) reported that platinum
Page 17 of 35
18
nanoparticles produced by a cell-free extract from F. oxysporum were all irregular in shape, with
2
sizes ranging from 1 to 180 nm. Most of the particles were between 20 and 60 nm. In
3
comparison, the nanoparticles produced by hydrogenase at pH 9 and 65°C were circular,
4
triangular, pentagonal and hexagonal, often appearing as nanoplates over a wide size
5
distribution, with most of them being between 20 and 80 nm.
ip t
1
Our results indicate that the difference between extracellular and intracellular synthesis is a
7
slight one. It might be that in addition to nanospheres, there were a small number of larger-sized
8
nanorods and nanotriangles inside the cells, as compared with the cell-free culture liquid.
9
However, the particles differed in size and shape, depending on which type of enzyme was
10
involved. The particles synthesized by Mn-peroxidase were regular spheres of 5 to 20 nm
11
diameter, as also were particles formed extracellularly in the culture liquid. The particles
12
synthesized by the laccases and tyrosinases were irregular spheres, with a small amount of
13
triangles and tetrahedrons present. These particles ranged from 5 to 120 nm, with 30-nm
14
particles predominating. Intracellularly, all these particle types were synthesized. These
15
differences may be related to the predominance and activity of this or other enzyme inside the
16
cells and to its release into the growth medium, as well as to different mechanisms of particle
17
synthesis by the enzymes.
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Possibly, laccase reduces Au ions indirectly through forming exogenous H2O2. When laccase
19
interacts with molecular oxygen in the absence of a reduced substrate, H2O2 forms in one of the
20
enzyme’s four active centers and becomes involved in Au reduction (Kupryashina et al., 2013).
21
Mn-peroxidase performs reduction through bypassing its major catalytic cycle at the cost of its
22
prosthetic group, containing protoporphyrin IX with an Fe3+ atom and able to be reduced to Fe2+.
23
To confirm this assumption, we did not supplement the reaction mixture with Mn2+ ions or with
Page 18 of 35
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H2O2, both being the principal Mn-peroxidase substrates. The redox reaction gives rise to
2
elemental Au, and the enzyme changes to a “quiescent,” fully oxidized form.
3
5
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4. Conclusions
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6
Using X-ray fluorescence, TEM, and dynamic light scattering, we have established that a
8
submerged culture of L. edodes F-249 can reduce Au(III) to Au(0) from HAuCl4, forming Au0
9
nanoparticles. TEM showed the presence of mostly spherical Au nanoparticles of 5 to 50 nm
10
diameter in the culture liquid, on the surface, and inside the hyphae of the fungus. This is the first
11
time that accumulation of spherical Au(0) nanoparticles inside the mycelial cells of higher
12
basidiomycetes has been found. This is also the first time that fungal intracellular phenol-
13
oxidizing enzymes (laccases, tyrosinases, and Mn-peroxidases) have been shown to be involved
14
in Au reduction to give electrostatically stabilized colloidal solutions. The biosynthesis of Au
15
nanoparticles by the edible medicinal basidiomycete L. edodes is of particular interest because it
16
is simple, accessible, and environmentally benign.
18 19 20 21 22
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Acknowledgments
This work was supported by a grant of OPTEK Co. We thank Mr. D.N. Tychinin (this institute) for his help in preparation of the manuscript.
23
Page 19 of 35
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Figure legends
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Fig. 1. Negative contrasting (TEM) of hyphae of L. edodes F-249 grown in liquid synthetic
4
medium in the presence of 50 µM HAuCl4. Bar markers = 1 μm (A), 500 nm (B).
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Fig. 2. TEM image of a transverse section of L. edodes F-249 hyphae embedded in epoxy resin
7
after Au reduction. Bar marker = 500 nm.
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Fig. 3. TEM images of the transverse and longitudinal sections of L. edodes F-249 hyphae (A, C,
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control) and an Au distribution map created by EELS (B, D). Bar marker = 500 nm.
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Fig. 4. TEM image of the Au nanospheres obtained after L. edodes F-249 mycelium was
13
disrupted. Bar marker = 200 nm.
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Fig. 5. X-ray fluorescence analysis of dry biomass of L. edodes F-249 mycelium grown in liquid
16
synthetic medium in the absence (A, control) and presence of 50 µM HAuCl4 (B). The emission
17
lines at 9.713, 11.443, 11.585, 10.308, and 13.382 keV (Lα1, Lβ1, Lβ2, Ln, and Ly1 lines,
18
respectively) correspond to Au.
19
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Fig. 6. Absorbance spectra of Au nanoparticles, obtained by reduction of Au(0) from HAuCl4
21
and synthesized by isolated and purified Mn-peroxidase (a), laccases (b), and tyrosinases (c) of
22
L. edodes F-249.
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Fig. 7. TEM images of Au nanoparticles obtained by reduction of Au(0) from HAuCl4 and
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synthesized by isolated and purified Mn-peroxidase (А), laccases (В), and tyrosinases (С) of L.
3
edodes F-249.
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Fig. 8. Size distribution by intensity (А) and size distribution by number (В) of Au(0) particles
6
synthesized from HAuCl4 by the Mn-peroxidase (a), laccases (b), and tyrosinases (c) of L.
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edodes F-249.
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S0168-1656(14)00199-0 http://dx.doi.org/doi:10.1016/j.jbiotec.2014.04.018 BIOTEC 6674
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
29-11-2013 22-2-2014 25-4-2014
Please cite this article as: Vetchinkina, E.P., Loshchinina, E.A., Burov, A.M., Dykman, L.A., Nikitina, V.E.,formation of gold nanoparticles by submerged culture of the basidiomycete Lentinus edodes, Journal of Biotechnology (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Enzymatic formation of gold nanoparticles by submerged culture of the
2
basidiomycete Lentinus edodes
3
Elena P. Vetchinkina, Ekaterina A. Loshchinina*, Andrey M. Burov,
5
Lev A. Dykman, Valentina E. Nikitina
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Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of
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Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia
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*Corresponding author. Tel: #(845-2) 97-04-03; 97-03-27; fax: #(845-2) 97-03-83; 97-04-44.
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E-mail address: [email protected] (Ekaterina A. Loshchinina)
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• Lentinus edodes can reduce Au(III) from HAuCl4 to Au(0), forming nanoparticles
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Highlights
• Au(0) accumulated on the surface and within the hyphae as 5-50 nm
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nanospheres
• The fungal phenol oxidases were found to be involved in the Au reduction
19
ABSTRACT
20
We report for the first time that the medicinal basidiomycete Lentinus edodes can reduce Au(III)
21
from chloroauric acid (HAuCl4) to elemental Au [Au(0)], forming nanoparticles. Several
22
methods, including transmission electron microscopy, electron energy loss spectroscopy, X-ray
23
fluorescence, and dynamic light scattering, were used to show that when the fungus was grown
24
submerged, colloidal gold accumulated on the surface of and inside the mycelial hyphae as
Page 1 of 35
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electron-dense particles mostly spherical in shape, with sizes ranging from 5 to 50 nm.
2
Homogeneous proteins (the fungal enzymes laccase, tyrosinase, and Mn-peroxidase) were found
3
for the first time to be involved in the reduction of Au(III) to Au(0) from HAuCl4. A possible
4
mechanism forming Au nanoparticles is discussed.
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Keywords:
7
Lentinus edodes
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Gold nanoparticles
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Extracellular and intracellular synthesis
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Reduction mechanisms
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Phenol oxidases
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1. Introduction
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Nanoparticles are finding wide application in medicine, biology, and technology in areas as
4
wide apart as cancer therapy and information technology (Dreaden et al., 2012; Dykman and
5
Khlebtsov, 2012; Dykman et al., 2008). Various physical and chemical methods have been
6
developed to synthesize nanoparticles of desired sizes and shapes; however, these methods,
7
though successfully used, can be costly and require the use of hazardous chemical compounds.
8
Therefore, there is a growing interest in “green” (environmentally and human friendly)
9
nanoparticle synthesis with the aid of microbial biotechnologies (Krumov et al., 2009;
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Narayanan and Sakthivel, 2010).
Au nanoparticles obtained from reduction of chloroaurates by bacteria, fungi, or plants have
12
unique antimicrobial, antifungal, and antitumor properties (Ahmad et al., 2013; Das et al., 2009;
13
Iravani, 2011). An active search is in progress to find novel effective biological agents for the
14
preparation of nanoparticles of various chemical natures (Durán et al., 2007; Musarrat et al.,
15
2011; Popescu et al., 2010; Rai and Duran, 2011). Several bacteria have been found able to
16
reduce Au from auric compounds to form nanoparticles, both extracellularly (e.g., Lactobacillus
17
sp., Rhodopseudomonas capsulata, Pseudomonas aeruginosa, and Bacillus flexus; He et al.,
18
2007; Husseiny et al., 2007; Murugan et al., 2013; Nair and Pradeep, 2002) and intracellularly
19
(e.g., Shewanella algae and Stenotrophomonas maltophilia; Konishi et al., 2004; Nangia et al.,
20
2009). Synthesis of Au and Ag nanoparticles by several lower fungi (Mourato et al., 2011; Sastry
21
et al., 2003) and by the basidiomycetes Volvariella volvacea, Coriolus versicolor, and Pleurotus
22
sapidus (Philip et al., 2009; Sanghi and Verma, 2009; Sarkar et al., 2013) has also been
23
documented; however, the higher fungi transformed compounds in their culture liquid and could
24
not accumulate elemental particles intracellularly.
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There are still many open questions in this area of research. Biologically synthesized
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nanoparticles often have various shapes and sizes, and the synthesis itself is time-consuming.
3
Nothing is known of which enzymes are involved in colloidal gold bioreduction, though recent
4
data suggest that extracellular phenol oxidases might have a role (Sanghi et al., 2011). No
5
experimental data are available concerning the biosynthesis of Au nanoparticles by
6
homogeneous enzymes. Therefore, comprehensive studies are needed to overcome these
7
problems and improve the synthetic rate and the properties of nanoparticles. Such studies should
8
examine all factors of importance to the process of synthesis, such as methods of cultivation,
9
extraction, and stabilization, as well as should decipher the cellular, biochemical, and molecular
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mechanisms of reduction involved.
There are very few data on the fungal transformation of Au compounds with release of
12
elemental Au [Au(0)]. However, higher fungi constitute a very favorable object of study, as they
13
can accumulate high concentrations of various elements. This is especially true of cultivated
14
edible medicinal basidiomycetes, as they are nontoxic, have a high biomass yield, and can
15
accumulate large amounts of reduced nanoparticles in their mycelium. One such fungus, the
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xylotrophic basidiomycete Lentinus edodes (shiitake), owing to the unique complex of
17
biologically active compounds that it contains in its mycelium and fruit bodies, has been actively
18
used as raw material to prepare a range of highly effective drugs and biologically active food
19
supplements (Jones, 1995; Mizuno, 1995). The current interest in this fungus is also due to the
20
high nutritive value and the taste of its fruit bodies.
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The aim of this study was twofold: (i) To examine the ability of Lentinus edodes to reduce
22
Au(III) to Au(0) and accumulate nanoparticles in its mycelium. (ii) To identify the enzymes
23
involved in this process.
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2. Materials and methods
2 3
2.1. Fungus and culture conditions
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The xylotrophic medicinal basidiomycete Lentinus edodes (Berk.) Sing [Lentinus edodes
6
(Berk.) Pegler] (shiitake), strain F-249, was obtained from the collection of higher fungi held by
7
the Laboratory of Microbiology at this institute. The fungal culture was maintained on 4% beer-
8
wort agar plates at 4°С.
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L. edodes was grown submerged in a synthetic medium of the following composition (g/L):
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D-glucose,
1; L-asparagine, 0.1; KH2PO4, 2; K2HPO4, 3; MgSO4 · 7H2O, 2.5; FeSO4 · 7H2O, 0.03
11
(рН 5.8). Growth was conducted in 100-mL flasks containing 50 mL of medium at the mycelial
12
growth temperature optimal for this species (26°С; Przybylowicz and Donoghue, 1991). For
13
inoculation, we used a 14-day-old culture of L. edodes grown on agar-supplemented synthetic
14
medium at 26°С.
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The effect of auric compounds on L. edodes growth and the xylotroph’s reduction ability
16
were examined by adding 10 to 500 µM of aqueous chloroauric acid (HAuCl4; Sigma-Aldrich,
17
USA) to the liquid synthetic medium. The solutions of the compounds were added under sterile
18
conditions to each of the growth flasks individually just before seeding. L. edodes growth was
19
characterized by the accumulation of dry biomass. The mycelium was passed through filters that
20
had been preweighed on an analytical balance, and then it was dried to a constant weight and
21
weighed again. The increment in biomass was compared between control (3-h-old culture) and
22
experimental trials. Experiments to measure fungal growth characteristics and biomass
23
accumulation were done in 5 to 10 replicates.
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2.2. Enzyme isolation and purification
2
Crude enzyme extracts were obtained by growing the fungus at 26°С for 14 days in a
4
synthetic medium of the above composition. The mycelium was then separated from the culture
5
medium, rinsed in distilled water, and mechanically ground in a porcelain mortar with quartz
6
sand to destroy the cell envelope. Extraction was done by using 20 mg of homogenized
7
mycelium per 2 mL of 20 mM Tris–HCl buffer (pH 7.5). The homogenate was spun down at
8
12,000 g for 15 min, separated from the sediment, filtered, and desalted on a column of
9
Sephadex G-25 (Pharmacia, Sweden) equilibrated with extraction buffer. Further purification
10
was done by high-performance liquid chromatography (HPLC) on a TSK Bioassist Super Q
11
(Sweden) anion-exchange column equilibrated with a pH 7.5 20 mМ Tris–HCl. Proteins were
12
eluted with an NaCl gradient of 0.01 to 1.0 M, and elution was recorded with a Uvicord S-II
13
apparatus (LKB, Sweden) at 280 nm. The fractions with Mn-peroxidase, laccase, and tyrosinase
14
activities were dialyzed against water and used in the experiment.
16
2.3. Polyacrylamide gel electrophoresis
17 18
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Protein composition was examined by nondenaturing electrophoresis in a 7.5%
19
polyacrylamide gel (PAG; Laemmli, 1970). For visualization of phenol oxidases, PAG was
20
specifically stained in reaction mixtures of 1% acetic acid, 0.2% o-dianisidine (Sigma, USA),
21
and 50 mM Na tartrate (pH 4.5). Red-brown staining of the protein bands corresponding to
22
laccase developed within 15 min of gel incubation in the reaction mixture (Gaal’ et al., 1982).
23
For staining of PAG for Mn-peroxidase activity, 0.1 mM H2O2 and 0.2 mM MnSO4 were added
24
to the above reaction mixture. Red-brown staining of the protein bands corresponding to Mn-
Page 6 of 35
7
peroxidase developed within 20 min of gel incubation (Glenn and Gold, 1985). For staining of
2
PAG for tyrosinase activity, a reaction mixture of 2 mM 3-(3,4-dihydroxyphenyl)-L-alanine (L-
3
DOPA; Serva, Germany) and 50 mM Tris–HCl (pH 7.5) was used. Brown bands corresponding
4
to tyrosinase developed within 10 min (Pomerantz and Murthy, 1974). The molecular masses of
5
the proteins were determined as described by Weber and Osborn (1969).
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2.4. Enzyme assays
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The substrate specificities of laccase, tyrosinase, and Mn-peroxidase were determined
10
qualitatively by the ability of these enzymes to oxidize specific phenolic substrates, such as 2,2′-
11
azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS; Niku-Paavola et al.,
12
1988), syringaldazine (Leonowicz and Crzywnowicz, 1981), 2.6-dimethoxyphenol (DMOP;
13
Sigma) (Slomczynski et al., 1995), and L-DOPA (Serva; Pomerantz and Murthy, 1974).
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The effect of inhibitors on laccase activity was studied in a mixture containing 50 mM Na/K-
15
phosphate buffer (pH 6.0), the enzyme, the substrate (syringaldazine), and an inhibitor. The
16
reaction was performed at 25°C. The following inhibitors were tested: 0.1 to 100 mM EDTA,
17
0.001 to 0.1 mM β-mercaptoethanol, and 0.1 to 4% SDS (Fluka, Switzerland; Pozdnyakova et
18
al., 2006).
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Enzyme activities were determined with a Specord M40 spectrophotometer (Carl Zeiss, Jena,
20
Germany) in 1-cm quartz cuvettes at 18°C. Laccase activity was measured by the oxidation rate
21
for 0.2 mM ABTS in 15 mM Na tartrate (pH 4.5). The oxidation of ABTS to a stable cation
22
radical was measured by the increase in absorbance at 436 nm (ε436 = 29300 M-1 cm-1)
23
(Slomczynski et al., 1995). Mn-peroxidase activity was determined by the oxidation rate for 0.2
24
mM ABTS in 50 mM Na tartrate (pH 4.5) supplemented with 0.1 mM H2O2 and 0.2 mM Mn2+.
Page 7 of 35
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The oxidation of ABTS to a stable cation radical was measured by the increase in absorbance at
2
436 nm (ε436 = 29300 M-1 cm-1) (Glenn and Gold, 1985). Tyrosinase activity was determined by
3
the oxidation rate for 2 mM L-DOPA in 50 mM Tris–HCl (pH 7.5). The oxidation of L-DOPA to
4
DOPAquinone was measured by the increase in absorbance at 475 nm (ε475 = 3700 M-1 cm-1)
5
(Pomerantz and Murthy, 1974).
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The reaction time was 5 min in all experiments. One unit of enzyme activity was defined as
7
the amount required to convert 1 μmol of substrate to product per minute, or to catalyze the
8
formation of 1 μmol of product per minute, and is expressed as micromoles per minute per
9
milligram of protein. Protein was estimated by the Bradford method (Bradford, 1976).
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2.5. Transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS), and
12
electron spectroscopic imaging (ESI)
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The mycelium and the culture liquid were studied by negative contrasting. To this end, the
15
material was mounted on nickel grids coated with 1% formvar in dichloroethane. After the
16
sample had dried, the cells were contrasted with 1% aqueous uranyl acetate (Tandler, 1990).
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Ultrathin sections were obtained by fixing the fungal hyphae in epoxy resin, as follows. The
18
mycelial hyphae were collected by centrifugation (13,000 × g for 20 min) at room temperature,
19
and the material was then transferred to 2-mL polypropylene test tubes and fixed in 0.5%
20
glutaraldehyde for 3 h. Subsequent fixation was done for 12 h in a 2.5% glutaraldehyde solution
21
made with phosphate buffer (0.1 M, pH 7.2). The material was then held in a 0.1% OsO4
22
solution made with phosphate buffer supplemented with 34 mg/mL sucrose (postfixation).
23
Samples were dehydrated in increasing concentrations of alcohols and in absolute acetone.
24
After dehydration, the samples were held in propylene oxide for 45 min, and then they were
Page 8 of 35
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embedded in Epon 812 and propylene oxide (each time for 24 h) at ratios of 1:2, 1:1, and 2:1.
2
Next, the material was embedded in pure epoxy resin. Polymerization was done at 37, 45, and
3
57°С for 24 h each. Ultrathin sections were cut on an LKB-III microtome (Sweden), mounted on
4
nickel grids, and stained with lead citrate (Reynolds, 1963).
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For nanoparticle studies, the fungal mycelium was grown in the presence of 50 μM HAuCl4
6
for 14 days, washed free of the cultivation medium with water, collected by centrifugation, and
7
lyophilized. The fungal hyphae were then mechanically disrupted in a porcelain mortar with
8
quartz sand, resuspended in MilliQ water, and separated from elemental particles through a 0.22-
9
μm-pore-size membrane filter (Millipore). The cell debris and nanoparticle aggregations
10
remained on the filter. The culture liquid, containing reduced Au nanoparticles, was filtered
11
through the usual paper filter to remove the hyphal debris and was dialyzed against MilliQ water
12
for 24 h, with several changes of water. The nanoparticles were mounted on nickel grids coated
13
with 1% formvar in dichloroethane. A Libra 120 electron microscope (Carl Zeiss, Germany)
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operating at 120 keV was used to take photomicrographs.
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2.6. X-ray fluorescence analysis
Elemental Au in the fungal mycelium was detected by an X-ray fluorescence study of
19
mycelial samples. To this end, L. edodes was grown in the synthetic medium without (control)
20
and with (experiment) 50 μM HAuCl4. The grown mycelial samples were washed free of the
21
growth medium with distilled water, collected by centrifugation, and lyophilized. The fungal
22
hyphae were then mechanically disrupted and were separated from elemental particles through a
23
Millipore membrane filter (0.22 μm pore size). The nanoparticles were resuspended in minimal
24
distilled water and were air dried at 20°С. The content of Au0 was analyzed with an ED 2000
Page 9 of 35
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energy-dispersive spectrometer (Oxford Instruments, UK). The measuring conditions were as
2
follows: element detection range, Na–U; X-ray tube, silver anode; tube voltage, 35 keV (medium
3
elements); primary X-ray beam filter, thin Ag; exposure time, 600 s; spectral path, air. The
4
content of Au0 was evaluated by the basic parameters method included in the instrument’s
5
software.
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2.7. Nanoparticle characterization
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Absorbance spectra were measured with a Specord 250 UV–vis spectrophotometer (Analytik
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Jena, Germany). The size, shape, and relative number of electron-dense nanoformations were
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evaluated from TEM images obtained on a Libra 120 electron microscope (Carl Zeiss). The ζ-
12
potential, average size, and size distribution of the synthesized Au0 particles were measured by
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dynamic light scattering with a Zetasizer Nano ZS instrument (Malvern, UK).
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2.8. Statistical analysis
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There were five independent experiments, each having no less than five replications. Data
were processed with Microsoft Excel software (Microsoft Office XP; Microsoft Corp., USA).
3. Results and discussion
22 23
3.1. Effect of HAuCl4 on the growth of L. edodes F-249
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We first tested how sensitive L. edodes F-249 was to HAuCl4 and how this compound
2
affected the growth characteristics of the fungal mycelium under submerged cultivation. The
3
minimum growth-inhibiting concentration was found to range from 60 to 80 µM. At 300 to 500
4
µM, growth was absent, and at 10 µM, it did not change relative to the control.
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When L. edodes was grown with 10 to 80 µM HAuCl4 in liquid synthetic medium, the
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culture liquid and mycelium acquired a reddish-lilac coloration. The control culture, grown
7
under the same conditions but without HAuCl4, was cream white throughout the cultivation time.
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The change in the color of microbial colonies growing in the presence of auric compounds from
9
various hues of lilac to red is the first sign of Au reduction to the elemental state. Such coloration
10
is characteristic of bacteria, algae, and lower fungi growing with auric compounds and indicates
11
that the culture has accumulated Au nanoparticles (Philip, 2009).
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Because the accumulation of Au(0) in the mycelium correlated with increased contents of
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HAuCl4 in the growth medium, it can be assumed that in L. edodes, as in many microorganisms
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(Garbisu et al., 1996), the reduction of this compound to Au(0) relieves the toxicity of high Au
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concentrations in the fungus.
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To detect electron-dense nanoparticles, we examined the fungal hyphae grown with 50 µM
22
HAuCl4 and the culture liquid by negative contrasting. As seen in Fig. 1, spherical electron-
23
dense formations were observed on the surface of the hyphae and also near them, as L. edodes
24
mycelium had not been washed free of the culture medium. No such formations were present in
16 17 18 19
With 50 µM HAuCl4, the culture grew well and the mycelial colonies had an intense lilac-red
coloration. Hence, 50 µM was used in all subsequent experiments.
3.2. TEM, EELS, and ESI
Page 11 of 35
12 1
the control. The particle diameter ranged from 5 to 50 nm, with most particles being of ~ 20 nm
2
diameter. It has been found in the past few years that a number of lower fungi (Mourato et al., 2011;
4
Sastry et al., 2003) and the basidiomycetes Volvariella volvacea (Philip, 2009) and Coriolus
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versicolor (Sanghi and Verma, 2009) can synthesize Au nanoparticles. However, reduction was
6
observed in the culture liquid, without intracellular accumulation of elemental particles (by
7
contrast, in Verticillium sp. and Trichothecium sp., nanoparticles have been observed in the cell
8
wall and in the cytoplasmic membrane; Ahmad et al., 2005; Senapati et al., 2004). No
9
information is currently available on Au nanoparticles resulting from L. edodes reduction of
10
auric compounds, and neither is there any published evidence that fungi in general and
11
basidiomycetes in particular can reduce Au to the elemental state and accumulate Au
12
nanoparticles in the hyphal interior. To confirm that the elemental particles were localized
13
intracellularly and that they had accumulated in the interior of the fungal mycelium, we
14
embedded hyphae in epoxy resin to obtain transverse and longitudinal ultrathin sections of the
15
cells. TEM confirmed that the hyphal cytoplasm contained electron-dense formations, of mostly
16
spherical shape (Fig. 2). Most nanoparticles had diameters of 5 to 15 nm; however, some were of
17
30 to 50 nm diameter. There were almost no elemental particles within the cell walls, suggesting
18
that the particles had not penetrated through the cell wall but had been formed directly in the
19
cytoplasm.
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The longitudinal and transverse ultrathin sections of L. edodes hyphae were examined by ESI
21
analysis and by EELS on a Libra 120 electron microscope. An Au distribution map was obtained
22
that confirmed these electron-dense formations to be intracellular Au nanoparticles (Fig. 3).
23
For nanoparticle studies, the mycelium containing reduced Au was mechanically disrupted
24
and was separated from the particles. The particles were resuspended in distilled water and were
Page 12 of 35
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mounted on formvar-coated nickel grids. Fig. 4 shows these particles to be similarly sized (~ 20
2
nm) nanospheres of a regular shape. The Au nanoparticles isolated from the culture liquid and
3
fungal cells were relatively stable, did not precipitate, and did not show any signs of aggregation,
4
at least for several weeks.
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3.3. Nanoparticle study by X-ray fluorescence
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To additionally confirm that the nanoparticles were indeed reduced Au, we examined them
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by X-ray fluorescence. The resulting spectra showed intense emission lines typical of Au (Fig.
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5A). The presence of Au was determined by the lines at 9.713 (Lα1), 11.443 (Lβ1), 11.585
11
(Lβ2), 10.308 (Ln), and 13.382 (Ly1) keV. The spectra taken for the control culture, grown
12
without HAuCl4, showed absence of Au (Fig. 5B).
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3.4. Involvement of L. edodes phenol-oxidizing enzymes in the biosynthesis of Au nanoparticles
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It is known that various bacteria can reduce Au both intra- and extracellularly (Narayanan
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and Sakthivel, 2010). The growth of L. edodes in the presence of HAuCl4 changed the color of
18
both the fungal culture and the growth medium. We found that no components of the synthetic
19
medium were involved in Au reduction but that the culture liquid, which contained various
20
products of the fungus’s vital functions, including extracellular proteins, could reduce HAuCl4 to
21
Au(0). TEM confirmed our assumption that the synthesis of Au nanoparticles was associated
22
directly with L. edodes mycelium. We showed that the (mostly spherical) particles were
23
localized not only extracellularly but also inside the cells, where they accumulated in large
Page 13 of 35
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quantities. These facts indicated that the biochemical reduction was directly related to L. edodes
2
metabolism. Work conducted in the past decade has shown that some bacteria (e.g., Shewanella sp.;
4
Konishi et al., 2004) can reduce Au ions by using self-synthesized proteins, e.g., NADH-
5
dependent enzymes (Tikariha et al., 2012). In Rhodopseudomonas capsulata, for example, the
6
involvement of NADH-dependent nitratereductase was shown (He et al., 2007). The
7
bioreduction of auric compounds in the culture medium has also been suggested to involve
8
oxidoreductases (in lower fungi) (Ramezani et al., 2010) and laccases and ligninases (in the
9
basidiomycete Phanerochaete сhrysosporium) (Sanghi et al., 2011). This work is the first to
10
establish that the basidiomycete L. edodes can reduce HAuCl4 to the elemental state both
11
extracellularly (as does P. сhrysosporium; Sanghi et al., 2011) and inside its hyphae. It has also
12
been found that both the intracellular extract and the culture liquid of L. edodes have phenol
13
oxidase activity; that is, the culture liquid also contains enzymes able to oxidize a number of
14
phenolic substrates. One can speculate that the reductive mechanism involves phenol-oxidizing
15
enzymes, as L. edodes phenol oxidases are present both intra- and extracellularly (Vetchinkina et
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al., 2008).
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To support this speculation, we isolated phenol oxidases, including intracellular laccases,
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tyrosinases, and Mn-peroxidase, from a submerged culture of L. edodes and purified them by
19
HPLC. Nondenaturing polyacrylamide gel electrophoresis and specific gel staining for Mn-
20
peroxidase, laccase, and tyrosinase activities made it possible to visualize the protein bands
21
corresponding to these enzymes. Three proteins, eluting from the column at 0.25 to 0.33 M
22
NaCl, displayed laccase activity, with the specific enzyme activity ranging from 5.8 to 10.2
23
U/mg. The single-subunit enzymes had approximately the same molecular masses (100 to 120
24
kDa). The intracellular Mn-peroxidase was a protein with a specific activity of 24.7 U/mg that
Page 14 of 35
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consisted of two subunits (100 to 110 kDa) and was eluted with 0.3 M NaCl. Two 140-kDa
2
tyrosinases were eluted with 0.35 to 0.40 M NaCl, and one tyrosinase, of approximately 300
3
kDa, was eluted with 0.5 M NaCl. The specific activities of the tyrosinases were 2.1, 3.3, and 0.9
4
U/mg, respectively.
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The enzyme types were determined conventionally by the ability to oxidize specific phenolic
6
substrates. At pH 4.0, the laccases oxidized ABTS and DMOP; this pH optimum is characteristic
7
of other fungal laccases as well (Niku-Paavola et al., 1988; Pozdnyakova et al., 2006). The
8
enzymes also oxidized syringaldazine, a known test substrate for laccase activity (Slomczynski
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et al., 1995). In addition, laccase interactions with different concentrations of SDS, EDTA, and
10
β-mercaptoethanol partly or completely inhibited the activity of the enzymes, again pointing to
11
their laccase nature (Leonowicz and Crzywnowicz, 1981; Pozdnyakova et al., 2006). The Mn-
12
peroxidase could oxidize ABTS and DMOP only in the presence of H2O2, which catalyzes
13
substrate oxidation for all peroxidases, and Mn2+, which does so only for Mn-dependent
14
peroxidases (Glenn and Gold, 1985; Kamitsuji et al., 2004). The tyrosinases could not oxidize
15
the substrates mentioned above; however, they degraded L-DOPA to DOPAquinone. Unlike
16
laccases, tyrosinases are of narrow substrate specificity, and L-DOPA serves as a test substrate
17
for tyrosinase activity (Pomerantz and Murthy, 1974).
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Aqueous solutions of the purified enzymes were incubated with 50 µM HAuCl4 at room
19
temperature. As early as 2 h later, the solutions acquired a lilac or red coloration, indicative of
20
the synthesis and accumulation of Au(0). The other isolated proteins, without phenol oxidase
21
activity, and the control proteins (bovine serum albumin and several fungal lectins) did not
22
reduce Au to the elemental state.
Page 15 of 35
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To confirm that the nanoparticle suspension was indeed Au, we took absorbance spectra of
2
the samples. The spectra had an absorption peak at 540 nm, corresponding to colloidal Au (Fig.
3
6).
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3.5. Characterization of the nanoparticles reduced by L. edodes phenol oxidases
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The suspension of the particles reduced by the laccases and tyrosinases had a lilac hue and
8
that of the particles reduced by Mn-peroxidase had a red hue. The suspension color is often
9
enough to assume what shape and size the reduced particles would have. The lilac color
10
corresponds to particles that are mostly 50 nm in size and often variously shaped, while the red
11
color is associated with smaller spherical particles of 10 to 20 nm diameter.
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This speculation was confirmed by TEM. As seen in Fig. 7A, the particles synthesized by
13
Mn-peroxidase were regular spheres of 5 to 20 nm diameter. Those synthesized by the laccases
14
and tyrosinases were irregular spheres, with a small amount of triangles and tetrahedrons present.
15
The size of these particles ranged from 5 to 120 nm, with 30-nm particles predominating (Fig.
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7B, C).
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The size of the Au particles was also evaluated by dynamic light scattering (Fig. 8A, B), and
18
the results confirmed the electron microscopic data. The ζ-potentials were 19.1, 22.7, and 23.8
19
mV for the particles synthesized by tyrosinase, laccase, and Mn-peroxidase, respectively. The ζ-
20
potential determines the extent and character of interparticle interactions in a disperse system,
21
and the greater it is, the more stable is the colloid. If the ζ-potential (positive or negative) is ≥ 30
22
mV, the dispersion will be stable to aggregation (Khlebtsov et al., 2013). In this study, the ζ-
23
potential of colloidal Au solutions was close to 30 mV, indicating that these solutions were
24
electrostatically stabilized and were not prone to coagulation or flocculation. The colloidal
Page 16 of 35
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solutions of the particles synthesized by the purified phenol oxidases were more stable than those
2
of the particles formed in the culture liquid or by whole fungal cells, and they did not aggregate
3
for several months at 4ºC, possibly owing to nanoparticle stabilization by the protein molecules. Recent years have seen an increasing number of reports in which microorganisms have been
5
shown able to produce nanoparticles. This notwithstanding, there have been very few
6
comparisons between particles synthesized by living microbial cultures and particles formed by
7
metabolites isolated from these cultures. The typical focus of those studies has been on
8
intracellular nanoparticle formation as compared with synthesis in the presence of the cell-free
9
extract in which the microorganism was grown. Many investigators have pointed out that the size
10
and shape of microbially synthesized nanoparticles depend strongly on the culture conditions
11
(temperature, pH, lighting level, shaking; Ahmad, 2005; Mohanpuria et al., 2008; Sanghi et al.,
12
2011; Thakkar et al., 2010).
M
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4
The parameters inside the cell may differ greatly from those outside in the culture liquid. It
14
has also been speculated that the cell wall and the culture age might have a role in the
15
intracellular formation of Au nanoparticles. Gericke and Pinches (2006), using a cell-free extract
16
of the fungus Verticillium luteoalbum, observed changes in the nanoparticle morphology and the
17
appearance of a great number of large platelike structures, mostly triangular and hexagonal.
18
Those authors pointed out that as the cell interior contains many enzymes and peptides, which
19
may influence reduction and particle formation, synthesis in the presence of cell-free extracts is
20
preferable.
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13
21
Riddin et al. (2006) demonstrated that the fungus Fusarium oxysporum is capable of
22
producing nanoparticles of different shapes (hexagons, pentagons, circles, squares, rectangles)
23
and sizes (10–100 nm) by both inter- and extracellular processes; however, only the extracellular
24
production proved statistically significant. Govender et al. (2010) reported that platinum
Page 17 of 35
18
nanoparticles produced by a cell-free extract from F. oxysporum were all irregular in shape, with
2
sizes ranging from 1 to 180 nm. Most of the particles were between 20 and 60 nm. In
3
comparison, the nanoparticles produced by hydrogenase at pH 9 and 65°C were circular,
4
triangular, pentagonal and hexagonal, often appearing as nanoplates over a wide size
5
distribution, with most of them being between 20 and 80 nm.
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Our results indicate that the difference between extracellular and intracellular synthesis is a
7
slight one. It might be that in addition to nanospheres, there were a small number of larger-sized
8
nanorods and nanotriangles inside the cells, as compared with the cell-free culture liquid.
9
However, the particles differed in size and shape, depending on which type of enzyme was
10
involved. The particles synthesized by Mn-peroxidase were regular spheres of 5 to 20 nm
11
diameter, as also were particles formed extracellularly in the culture liquid. The particles
12
synthesized by the laccases and tyrosinases were irregular spheres, with a small amount of
13
triangles and tetrahedrons present. These particles ranged from 5 to 120 nm, with 30-nm
14
particles predominating. Intracellularly, all these particle types were synthesized. These
15
differences may be related to the predominance and activity of this or other enzyme inside the
16
cells and to its release into the growth medium, as well as to different mechanisms of particle
17
synthesis by the enzymes.
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Possibly, laccase reduces Au ions indirectly through forming exogenous H2O2. When laccase
19
interacts with molecular oxygen in the absence of a reduced substrate, H2O2 forms in one of the
20
enzyme’s four active centers and becomes involved in Au reduction (Kupryashina et al., 2013).
21
Mn-peroxidase performs reduction through bypassing its major catalytic cycle at the cost of its
22
prosthetic group, containing protoporphyrin IX with an Fe3+ atom and able to be reduced to Fe2+.
23
To confirm this assumption, we did not supplement the reaction mixture with Mn2+ ions or with
Page 18 of 35
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H2O2, both being the principal Mn-peroxidase substrates. The redox reaction gives rise to
2
elemental Au, and the enzyme changes to a “quiescent,” fully oxidized form.
3
5
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4. Conclusions
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6
Using X-ray fluorescence, TEM, and dynamic light scattering, we have established that a
8
submerged culture of L. edodes F-249 can reduce Au(III) to Au(0) from HAuCl4, forming Au0
9
nanoparticles. TEM showed the presence of mostly spherical Au nanoparticles of 5 to 50 nm
10
diameter in the culture liquid, on the surface, and inside the hyphae of the fungus. This is the first
11
time that accumulation of spherical Au(0) nanoparticles inside the mycelial cells of higher
12
basidiomycetes has been found. This is also the first time that fungal intracellular phenol-
13
oxidizing enzymes (laccases, tyrosinases, and Mn-peroxidases) have been shown to be involved
14
in Au reduction to give electrostatically stabilized colloidal solutions. The biosynthesis of Au
15
nanoparticles by the edible medicinal basidiomycete L. edodes is of particular interest because it
16
is simple, accessible, and environmentally benign.
18 19 20 21 22
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Acknowledgments
This work was supported by a grant of OPTEK Co. We thank Mr. D.N. Tychinin (this institute) for his help in preparation of the manuscript.
23
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Figure legends
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Fig. 1. Negative contrasting (TEM) of hyphae of L. edodes F-249 grown in liquid synthetic
4
medium in the presence of 50 µM HAuCl4. Bar markers = 1 μm (A), 500 nm (B).
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Fig. 2. TEM image of a transverse section of L. edodes F-249 hyphae embedded in epoxy resin
7
after Au reduction. Bar marker = 500 nm.
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Fig. 3. TEM images of the transverse and longitudinal sections of L. edodes F-249 hyphae (A, C,
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control) and an Au distribution map created by EELS (B, D). Bar marker = 500 nm.
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Fig. 4. TEM image of the Au nanospheres obtained after L. edodes F-249 mycelium was
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disrupted. Bar marker = 200 nm.
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Fig. 5. X-ray fluorescence analysis of dry biomass of L. edodes F-249 mycelium grown in liquid
16
synthetic medium in the absence (A, control) and presence of 50 µM HAuCl4 (B). The emission
17
lines at 9.713, 11.443, 11.585, 10.308, and 13.382 keV (Lα1, Lβ1, Lβ2, Ln, and Ly1 lines,
18
respectively) correspond to Au.
19
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Fig. 6. Absorbance spectra of Au nanoparticles, obtained by reduction of Au(0) from HAuCl4
21
and synthesized by isolated and purified Mn-peroxidase (a), laccases (b), and tyrosinases (c) of
22
L. edodes F-249.
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Fig. 7. TEM images of Au nanoparticles obtained by reduction of Au(0) from HAuCl4 and
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synthesized by isolated and purified Mn-peroxidase (А), laccases (В), and tyrosinases (С) of L.
3
edodes F-249.
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Fig. 8. Size distribution by intensity (А) and size distribution by number (В) of Au(0) particles
6
synthesized from HAuCl4 by the Mn-peroxidase (a), laccases (b), and tyrosinases (c) of L.
7
edodes F-249.
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