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Biosynthesis and characterization of gold nanoparticles produced by laccase from Paraconiothyrium variabile
Colloids and Surfaces B: Biointerfaces 87 (2011) 23–27
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Biosynthesis and characterization of gold nanoparticles produced by laccase from Paraconiothyrium variabile Mohammad Ali Faramarzi a,∗ , Hamid Forootanfar a,b a Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 14174, Iran b Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Pharmaceutics Research Center, Kerman University of Medical Sciences, Kerman, Iran
a r t i c l e
i n f o
Article history: Received 2 January 2011 Received in revised form 19 March 2011 Accepted 13 April 2011 Available online 27 April 2011 Keywords: Gold nanoparticles Laccase Green synthesis Paraconiothyrium variabile
a b s t r a c t During recent years investigation on the development of eco-friendly processes for production of gold nanoparticles (GNPs) have received much attention due to hazardous effects of chemical compounds used for nanoparticle preparation. In the present study, the purified laccase from Paraconiothyrium variabile was applied for synthesis of Au nanoparticles (AuNPs) and the properties of produced nanoparticles were characterized. The UV–vis spectrum of formed AuNPs showed a peak at 530 nm related to surface plasmon absorbance of GNPs represented the formation of gold nanoparticles after 20 min incubation of HAuCl4 (0.6 mM) in the presence of 73 U laccase at 70 ◦ C. Transmission electron microscopy (TEM) image of AuNPs showed well dispersed nanoparticles in the range of 71–266 nm as determined by the laser light scattering method. The pattern of energy dispersive X-ray (EDX) of the prepared GNPs confirmed the structure of gold nanocrystals. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Due to unique physicochemical characteristics of gold nanoparticles and wide usages in different fields, a number of publications on the preparation and characterization of GNPs has extensively increased during recent decades [1,2]. For example, the recently recognized behavior of gold to act as a soft Lewis acid and large surface-to-volume ratio of nanogold as well as its inert property have widely enhanced the application of GNPs as a catalyst in the field of organic synthesis [1]. The ability of gold to produce heat after absorbing light provides a medicinal usage named as photothermal therapy [3]. All mentioned usages together with application of nanogold in gene and drug delivery increased studies on development of methods for GNPs production [4,5]. Although preparation of nanogold by physical procedures (such as laser ablation) provides AuNPs with narrow range of particle size, it needs expensive equipments and has low yield [6]. Hazardous effects of organic solvents, reducing agents and toxic reagents applied for synthesis of AuNPs on environment, encouraged researchers to develop eco-friendly methods for preparation of gold nanoparticles [7,8]. Biosynthesis of gold nanoparticles either intra- or extracellularly using bacterial [5], fungal [9] and microalgal [3] strains as well as herbal extracts [10,11] have been recently reported. Beside production of AuNPs using these living factories, the ability of
amine-containing molecules such as chitosan [4], peptides [12] and amino acids [13] to reduce aqueous chloroaurate ions to AuNPs was also reported. Kalishwaralal and co-workers [14] studied the ability of Bacillus licheniformis for AuNPs production and confirmed the role of its ␣-amylase as a reducing agent in the synthesis of nanogold after optimization and inhibitor studies. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) has been widely studied by many researchers because of diverse applications of this well-known multicopper oxidase. Decolorization ability of the purified laccases from fungal and bacterial strains was investigated [15–17]. Zhang et al. [18] reported degradation of chlorophenols by this oxidase. Aflatoxin B1 (a harmful mycotoxin) was found to be degraded using laccases from Trametes versicolor and some other fungal strains [19]. Recently, Paraconiothyrium variabile, a laccase producing ascomycete, was isolated from soil and a blue enzyme with laccase activity was purified and characterized [20]. In the present study, the ability of purified laccase from P. variabile for production of gold nanoparticles was investigated. Characterization of prepared AuNPs was also studied.
2. Materials and methods 2.1. Chemicals
∗ Corresponding author. Tel.: +98 21 66954712; fax: +98 21 66954712. E-mail address: [email protected] (M.A. Faramarzi). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.04.022
Chloroauric acid (HAuCl4 ) was purchased from Merck Chemical Co. (Darmstadt, Germany). 2,2 -Azinobis-(3-ethylbenzthiazoline-
24
M.A. Faramarzi, H. Forootanfar / Colloids and Surfaces B: Biointerfaces 87 (2011) 23–27
Fig. 1. Formation of gold nanoparticles by purified laccase in presence of different concentrations ((a), control; (b) 0.1; (c) 0.2; (d) 0.3; (e) 0.4; (f) 0.5 and (g) 0.6 mM) of HAuCl4 after 20 min incubation at 70 ◦ C.
6-sulphonate) (ABTS) was provided from Sigma–Aldrich (St. Louis, Mo, USA). All other chemicals were of analytical grades. 2.2. Purification of laccase Laccase of P. variabile (a newly isolated ascomycete) was purified as described previously [20] at four steps of ammonium sulphate precipitation, anion exchange chromatography on Q-Sepharose XL column and gel filtration chromatography by Sephadex G-100 column after ammonium sulphate precipitation of active fraction from anion exchange step. All purification steps were carried out at 4 ◦ C. The purified enzyme was applied for biosynthesis of gold nanoparticles.
Fig. 2. Spectrums of prepared nanogold after treatment of HAuCl4 (0.6 mM) by laccase at different temperatures ((a) control; (b) 30; (c) 50; (d) 60; (e) 80 and (f) 70 ◦ C).
Transmission electron microscopy was performed using a Zeiss 902A TEM operated at 80 kV. Inspection of surface morphology as well as the elemental composition analysis of gold nanoparticles was carried out by scanning electron microscope (SEM) equipped with an EDX (energy dispersive X-ray) microanalysis. Samples for SEM observation were prepared by mounting of AuNPs on specimen stubs and then coating them with gold in a sputter coater device (model SCD 005; Bal-Tec). The patterns of particle size distribution were determined using a Zetasizer MS2000 (Malvern Instruments). The FTIR spectrum of dried powder of gold nanoparticle (in KBr pellet) was recorded by a Perkin Elmer instrument at a resolution of 4 cm−1 .
2.3. Laccase assay and protein estimation
2.6. Temperature stability of the purified laccase
Oxidation of ABTS as laccase substrate was used for determination of the enzyme activity [19,21]. The reaction mixture consisted of 0.5 mL appropriate diluted enzyme and 0.5 mL ABTS (5 mM) dissolved in 100 mM citrate buffer pH 4.5. Change in absorbance at 420 nm was measured using a UV/Visible Spectrophotometer (UVD 2950, Labomed, Culver City, USA) after incubation at 37 ◦ C and 120 rpm for 10 min. The enzymatic activity was calculated using the molar extinction coefficient of ABTS (ε420 = 36,000 M−1 cm−1 ) [22]. One unit of enzyme activity was defined as the amount of enzyme that can oxidize 1 mol of substrate per minute [19]. Protein concentration was estimated using the Bradford dye-binding method [23].
To investigate the effect of temperature on the laccase stability, the purified laccase was incubated at different temperatures (30–80 ◦ C) for 2 h and the residual activities were determined every 30 min followed by calculating relative activities compared to untreated sample.
2.4. Preparation of gold nanoparticles Aqueous solution of purified laccase was prepared by dissolving 40 mg (equal to 730 U) of purified laccase in 5 mL of deionized water, thereafter 500 L of such solution (corresponding to 73 U laccase activity) was added to aqueous solution of HAuCl4 (2.5 mL) with concentrations range of 0.1–1 mM followed by incubation of reaction mixtures at different temperatures (30–80 ◦ C). Formation of a ruby red color indicated production of AuNPs [3]. 2.5. Characterization of gold nanoparticles A Labomed Model UVD-2950 UV-Vis Double Beam PC Scanning spectrophotometer was used to record the UV–visible spectrum of the reaction mixture (containing laccase and chloroauric acid as said above) in comparison to control sample (reaction mixture without enzyme) in the range of 200–700 nm. Samples for examination by transmission electron microscopy (TEM) were prepared by placing one drop of formed nanogold on carbon-coated copper TEM grids followed by slowly evaporating at room temperature.
3. Results 3.1. Reduction of HAuCl4 solution The absorption spectra of untreated and treated gold solution illustrated in Fig. 1 indicated formation of gold nanoparticles due to presence of the surface plasmon absorption of the Au nanoparticles at 520 nm. 3.2. Effect of HAuCl4 concentration on production of AuNPs As shown in Fig. 1, increasing concentration of chloroauric acid to 0.6 mM led to increase in absorbance of 520 nm corresponded to gold nanoparticle formation. More than such critical concentration aggregation of nanogold increases the particle size.
Table 1 Particle size distributions and average sizes of prepared nanogold after treatment of HAuCl4 (0.6 mM) by purified laccase of Paraconiothyrium variabile at different temperatures. Temperature (◦ C)
Particle size distribution (nm)
Average size (nm)
30 50 60 70 80
148–266, 478–995 46–478 53–308 71–266 128–641
162, 543 127 120 124 227
M.A. Faramarzi, H. Forootanfar / Colloids and Surfaces B: Biointerfaces 87 (2011) 23–27
25
Fig. 3. (a) Scanning electron microscopy (SEM); (b) energy dispersive X-ray (EDX); and (c) transmission electron microscopy (TEM) images of produced AuNPs by purified laccase from Paraconiothyrium variabile.
Fig. 4. FTIR spectrum of gold nanoparticles after treatment of chloroauric acid by purified laccase at 70 ◦ C.
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M.A. Faramarzi, H. Forootanfar / Colloids and Surfaces B: Biointerfaces 87 (2011) 23–27
30 40 50 60 70 80
100
Relative activity (%)
80 60 40 20 0 0
15
30
45
60 Time (min)
75
90
105
120
Fig. 5. Relative activities of the purified laccase after incubation at various temperatures for 120 min. Residual activity (compared to control) was determined each 30 min intervals.
3.3. Effect of temperature on production of AuNPs Fig. 2 represents the effect of temperature on the characteristics of AuNPs. The temperature of 70 ◦ C was the best temperature in which the gold nanoparticles were formed in the size range of 71–266 nm after 20 min incubation (Table 1). The produced nanoparticles at temperature of 30 ◦ C showed two ranges of particle size distributions including 148–266 and 478–995, respectively (Table 1). Formation of AuNPs at 30 ◦ C, 50 ◦ C and 60 ◦ C required more incubation times (5, 2 and 1 h, respectively) and at 80 ◦ C, AuNPs represented larger particle size distribution due to aggregation. According to the obtained results, the best temperature and HAuCl4 concentration for AuNPs production were found to be 70 ◦ C and 0.6 mM, respectively. 3.4. Characterization of AuNPs Scanning electron microscopy (SEM), energy dispersive Xray EDX and TEM image of gold nanoparticles are illustrated in Fig. 3a–c, respectively. Elemental analysis of treated samples by EDX microanalysis (Fig. 3c) confirmed the gold composition of prepared nanoparticles. The FTIR spectrum of AuNPs (Fig. 4) showed two major peaks at 3448.5 and 1642.8. The 3448.5 cm−1 corresponded to OH and/or NH functional groups and presence of carbonyl group could be ascribed to the peak of 1642.8 cm−1 . According to Shakibaie et al. [3] these functional groups could be used in bioconjugation and/or immobilization of various compounds. 3.5. Study on the thermal stability of purified laccase As shown in Fig. 5, at temperature above 60 ◦ C the laccase activity decreased sharply and at 80 ◦ C only 0.4% of initial activity remained after 30 min incubation. Relative activity of the enzyme at 50 ◦ C was found to be 50.4% after 2 h incubation. 4. Discussion Hazardous effects of solvents and chemical reagents used for synthesis of AuNPs on the environment encouraged researchers to study on preparation of gold nanoparticles via biological methods both using living microorganisms and biomolecules [5,14]. Shih and colleagues [4] studied the formation and characterization of AuNPs synthesized by alkaline solution of chitosan and demonstrated more concentration of such polysaccharide decrease
the particle size of prepared gold nanoparticles. Sun et al. [24] reported the mechanism of nanogold formation in presence of chitosan and ascribed this reduction to formation of open chain form of the used biopolymer in acidic condition. Beside investigations on the ability of chitosan suspension for production of gold nanoparticles, many reports have focused on the preparation of AuNPs by peptide sequences and proteins. In the study of Wang and co-workers [12], the biomimetic synthesis of nanogold using the peptide sequence of MS14 (MHGKTQATSGTIQS) was determined. Kalishwaralal et al. [14] showed that the amylase produced by B. licheniformis is the reductive agent responsible for biosynthesis of nanogold. Besides, Rangnekar et al. [25] showed that ␣-amylase and its thiol groups being away from catalytic site is responsible for synthesis of nanogold while other enzymes without such exposing group do not reduce chloroauric acid to Au nanoparticles. In the present study, a biopolymer, laccase from P. variabile, was used as the reductive agent for synthesis of gold nanoparticles. Incubation of the pure laccase in the presence of chloroaurate ions at different temperatures reduces Au3+ to Au◦ . Aggregation of nanoparticles above the critical HAuCl4 concentration (0.6) led to form larger gold particles. Increasing the incubation temperature from 30 ◦ C to 70 ◦ C decreased the time for formation of nanoparticles from 5 h to 20 min and at 80 ◦ C, the production of large nanoparticles was achieved within few minutes. It was also observed that the particle size and the distribution of the obtained nanoparticles were inversely dependent on the temperature within the range of 30–60 ◦ C. It is notable that at 70 ◦ C, the average particle size increased (124 nm) and the particle size distribution was narrowed in comparison with performing the process at 60 ◦ C (Table 1). Meanwhile, larger size of produced AuNPs at 80 ◦ C could be interpreted by the aggregation of the fine nanostructures which was in agreement with the report of Pal et al. [26] on the effect of Triton X-100 to avoid excessive gold agglomeration. Overall, the optimum temperature was found to be 70 ◦ C. Comparison of laccase stability profiles at different temperatures (Fig. 5) showed that the formation of nanogold was happened when the activity decreased. Therefore, exposing of reducing functional groups might be responsible to form gold nanostructures. This finding is in agreement with the study of Kalishwaralal and colleagues [14] demonstrated that denaturation of the purified amylase from B. licheniformis increased the rate of AuNPs biosynthesis because of exposing reductive groups (such as thiol group of cysteine and tertiary amine of histidine) to gold solution.
M.A. Faramarzi, H. Forootanfar / Colloids and Surfaces B: Biointerfaces 87 (2011) 23–27
5. Conclusions The purified laccase from P. variabile was applied for synthesis of gold nanoparticles and the produced nanostructures was characterized. Optimum temperature and HAuCl4 concentration for AuNPs (average particle size of 124 nm) were found to be 70 ◦ C and 0.6 mM, respectively. Acknowledgements This work was supported financially by the grant number 89-0433-12093 from Tehran University of Medical Sciences, Tehran, Iran and Pharmaceutical Research Center, Kerman University of Medical Sciences, Kerman, Iran. References [1] F.K. Alanazi, A.A. Radwan, I.A. Alsarra, Saudi Pharm. J. 18 (2010) 179. [2] E.J. Yoo, T. Li, H.G. Park, Y.K. Chang, Ultramicroscopy 108 (2008) 1273. [3] M. Shakibaie, H. Forootanfar, K. Mollazadeh-Moghaddam, Z. Bagherzadeh, N. Nafissi-Varcheh, A.R. Shahverdi, M.A. Faramarzi, Biotechnol. Appl. Biochem. 57 (2010) 71. [4] C.-M. Shih, Y.-T. Shieh, Y.-K. Twu, Carbohyd. Polym. 78 (2009) 309. [5] K. Kalishwaralal, V. Deepak, S.R.K. Pandian, S. Gurunathan, Bioresour. Technol. 100 (2009) 5356. [6] K. Kalishwaralal, V. Deepak, S.R.K. Pandian, M. Kottaisamy, S. BarathManiKanth, B. Kartikeyan, S. Gurunathan, Colloid Surf. B 77 (2010) 257. [7] I. Maliszewska, L. Aniszkiewicz, Z. Sadowski, Acta Phys. Pol. A 116 (2009) S–163. [8] P. Mukherjee, S. Senapati, D. Mandal, A. Ahmad, M.I. Khan, R. Kumar, M. Sastry, Chem. Biol. Chem. 3 (2002) 461.
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[9] D. Philip, Spectrochim. Acta A 73 (2009) 374. [10] A.N. Mishra, S. Bhadauria, M.S. Gaur, R. Pasricha, B.S. Kushwah, Int. J. Green Nanotechnol.: Phys. Chem. 1 (2010) P118. [11] N. Ramezani, Z. Ehsanfar, F. Shamsa, G. Amin, H.R. Shahverdid, H.R. MonsefEsfahani, A. Shamsaie, R. Dolatabadi Bazaz, A.R. Shahverdi, Z. Naturforsch. 63b (2008) 903. [12] Z. Wang, J. Chen, P. Yang, W. Yang, Appl. Organomet. Chem. 21 (2007) 645. [13] P.R. Selvakannan, S. Mandal, S. Phadtare, A. Gole, R. Pasricha, S.D. Adyanthaya, M. Sastry, J. Colloid Interf. Sci. 269 (2004) 97. [14] K. Kalishwaralal, S. Gopalram, R. Vaidyanathan, V. Deepak, S.R.K. Pandian, S. Gurunathan, Colloid Surf. B 77 (2010) 174. [15] P. Peralta-Zamora, C.M. Pereira, E.R.L. Tiburtius, S.G. Moraes, M.A. Rosa, R.C. Minussi, N. Duran, Appl. Catal. B: Environ. 42 (2003) 131. [16] A.P.M. Tavares, R.O. Cristovao, J.M. Loureiro, R.A.R. Boaventura, E.A. Macedo, J. Hazard. Mater. 162 (2009) 1255. [17] L. Pereira, A.V. Coelho, C.A. Viegas, M.M. Correia dos Santos, M.P. Robalo, L.O. Martins, J. Biotechnol. 139 (2009) 68. [18] J. Zhang, X. Liu, Z. Xu, H. Chen, Y. Yang, Int. Biodeter. Biodegr. 61 (2008) 351. [19] J.F. Alberts, W.C.A. Gelderblom, A. Botha, W.H. Vanzyl, Int. J. Food Microbiol. 135 (2009) 47. [20] H. Forootanfar, M.A. Faramarzi, A.R. Shahverdi, M. Tabatabaei Yazdi, Bioresour. Technol. 102 (2011) 1808. [21] L.H. Jonsson, M. Saloheimo, M. Pentilla, Curr. Genet. 32 (1997) 425. [22] L. Lu, M. Zhao, B.-B. Zhang, S.-Y. Yu, X.-J. Bian, W. Wang, Y. Wang, Appl. Microbiol. Biotechnol. 74 (2007) 1232. [23] R.J. Simpson, Proteins and Proteomics: A Laboratory Manual, first ed., CSHL Press, New York, 2003, pp. 857–859. [24] C. Sun, R. Qu, H. Chen, C. Ji, C. Wang, Y. Sun, B. Wang, Carbohyd. Res. 343 (2008) 2595. [25] A. Rangnekar, T.K. Sarma, A.K. Singh, J. Deka, A. Ramesh, A. Chattopadhyay, Langmuir 23 (2007) 5700. [26] A. Pal, S. Shah, S. Devi, Colloids Surf. A: Physicochem. Eng. Aspects 302 (2007) 483.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Biosynthesis and characterization of gold nanoparticles produced by laccase from Paraconiothyrium variabile Mohammad Ali Faramarzi a,∗ , Hamid Forootanfar a,b a Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 14174, Iran b Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Pharmaceutics Research Center, Kerman University of Medical Sciences, Kerman, Iran
a r t i c l e
i n f o
Article history: Received 2 January 2011 Received in revised form 19 March 2011 Accepted 13 April 2011 Available online 27 April 2011 Keywords: Gold nanoparticles Laccase Green synthesis Paraconiothyrium variabile
a b s t r a c t During recent years investigation on the development of eco-friendly processes for production of gold nanoparticles (GNPs) have received much attention due to hazardous effects of chemical compounds used for nanoparticle preparation. In the present study, the purified laccase from Paraconiothyrium variabile was applied for synthesis of Au nanoparticles (AuNPs) and the properties of produced nanoparticles were characterized. The UV–vis spectrum of formed AuNPs showed a peak at 530 nm related to surface plasmon absorbance of GNPs represented the formation of gold nanoparticles after 20 min incubation of HAuCl4 (0.6 mM) in the presence of 73 U laccase at 70 ◦ C. Transmission electron microscopy (TEM) image of AuNPs showed well dispersed nanoparticles in the range of 71–266 nm as determined by the laser light scattering method. The pattern of energy dispersive X-ray (EDX) of the prepared GNPs confirmed the structure of gold nanocrystals. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Due to unique physicochemical characteristics of gold nanoparticles and wide usages in different fields, a number of publications on the preparation and characterization of GNPs has extensively increased during recent decades [1,2]. For example, the recently recognized behavior of gold to act as a soft Lewis acid and large surface-to-volume ratio of nanogold as well as its inert property have widely enhanced the application of GNPs as a catalyst in the field of organic synthesis [1]. The ability of gold to produce heat after absorbing light provides a medicinal usage named as photothermal therapy [3]. All mentioned usages together with application of nanogold in gene and drug delivery increased studies on development of methods for GNPs production [4,5]. Although preparation of nanogold by physical procedures (such as laser ablation) provides AuNPs with narrow range of particle size, it needs expensive equipments and has low yield [6]. Hazardous effects of organic solvents, reducing agents and toxic reagents applied for synthesis of AuNPs on environment, encouraged researchers to develop eco-friendly methods for preparation of gold nanoparticles [7,8]. Biosynthesis of gold nanoparticles either intra- or extracellularly using bacterial [5], fungal [9] and microalgal [3] strains as well as herbal extracts [10,11] have been recently reported. Beside production of AuNPs using these living factories, the ability of
amine-containing molecules such as chitosan [4], peptides [12] and amino acids [13] to reduce aqueous chloroaurate ions to AuNPs was also reported. Kalishwaralal and co-workers [14] studied the ability of Bacillus licheniformis for AuNPs production and confirmed the role of its ␣-amylase as a reducing agent in the synthesis of nanogold after optimization and inhibitor studies. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) has been widely studied by many researchers because of diverse applications of this well-known multicopper oxidase. Decolorization ability of the purified laccases from fungal and bacterial strains was investigated [15–17]. Zhang et al. [18] reported degradation of chlorophenols by this oxidase. Aflatoxin B1 (a harmful mycotoxin) was found to be degraded using laccases from Trametes versicolor and some other fungal strains [19]. Recently, Paraconiothyrium variabile, a laccase producing ascomycete, was isolated from soil and a blue enzyme with laccase activity was purified and characterized [20]. In the present study, the ability of purified laccase from P. variabile for production of gold nanoparticles was investigated. Characterization of prepared AuNPs was also studied.
2. Materials and methods 2.1. Chemicals
∗ Corresponding author. Tel.: +98 21 66954712; fax: +98 21 66954712. E-mail address: [email protected] (M.A. Faramarzi). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.04.022
Chloroauric acid (HAuCl4 ) was purchased from Merck Chemical Co. (Darmstadt, Germany). 2,2 -Azinobis-(3-ethylbenzthiazoline-
24
M.A. Faramarzi, H. Forootanfar / Colloids and Surfaces B: Biointerfaces 87 (2011) 23–27
Fig. 1. Formation of gold nanoparticles by purified laccase in presence of different concentrations ((a), control; (b) 0.1; (c) 0.2; (d) 0.3; (e) 0.4; (f) 0.5 and (g) 0.6 mM) of HAuCl4 after 20 min incubation at 70 ◦ C.
6-sulphonate) (ABTS) was provided from Sigma–Aldrich (St. Louis, Mo, USA). All other chemicals were of analytical grades. 2.2. Purification of laccase Laccase of P. variabile (a newly isolated ascomycete) was purified as described previously [20] at four steps of ammonium sulphate precipitation, anion exchange chromatography on Q-Sepharose XL column and gel filtration chromatography by Sephadex G-100 column after ammonium sulphate precipitation of active fraction from anion exchange step. All purification steps were carried out at 4 ◦ C. The purified enzyme was applied for biosynthesis of gold nanoparticles.
Fig. 2. Spectrums of prepared nanogold after treatment of HAuCl4 (0.6 mM) by laccase at different temperatures ((a) control; (b) 30; (c) 50; (d) 60; (e) 80 and (f) 70 ◦ C).
Transmission electron microscopy was performed using a Zeiss 902A TEM operated at 80 kV. Inspection of surface morphology as well as the elemental composition analysis of gold nanoparticles was carried out by scanning electron microscope (SEM) equipped with an EDX (energy dispersive X-ray) microanalysis. Samples for SEM observation were prepared by mounting of AuNPs on specimen stubs and then coating them with gold in a sputter coater device (model SCD 005; Bal-Tec). The patterns of particle size distribution were determined using a Zetasizer MS2000 (Malvern Instruments). The FTIR spectrum of dried powder of gold nanoparticle (in KBr pellet) was recorded by a Perkin Elmer instrument at a resolution of 4 cm−1 .
2.3. Laccase assay and protein estimation
2.6. Temperature stability of the purified laccase
Oxidation of ABTS as laccase substrate was used for determination of the enzyme activity [19,21]. The reaction mixture consisted of 0.5 mL appropriate diluted enzyme and 0.5 mL ABTS (5 mM) dissolved in 100 mM citrate buffer pH 4.5. Change in absorbance at 420 nm was measured using a UV/Visible Spectrophotometer (UVD 2950, Labomed, Culver City, USA) after incubation at 37 ◦ C and 120 rpm for 10 min. The enzymatic activity was calculated using the molar extinction coefficient of ABTS (ε420 = 36,000 M−1 cm−1 ) [22]. One unit of enzyme activity was defined as the amount of enzyme that can oxidize 1 mol of substrate per minute [19]. Protein concentration was estimated using the Bradford dye-binding method [23].
To investigate the effect of temperature on the laccase stability, the purified laccase was incubated at different temperatures (30–80 ◦ C) for 2 h and the residual activities were determined every 30 min followed by calculating relative activities compared to untreated sample.
2.4. Preparation of gold nanoparticles Aqueous solution of purified laccase was prepared by dissolving 40 mg (equal to 730 U) of purified laccase in 5 mL of deionized water, thereafter 500 L of such solution (corresponding to 73 U laccase activity) was added to aqueous solution of HAuCl4 (2.5 mL) with concentrations range of 0.1–1 mM followed by incubation of reaction mixtures at different temperatures (30–80 ◦ C). Formation of a ruby red color indicated production of AuNPs [3]. 2.5. Characterization of gold nanoparticles A Labomed Model UVD-2950 UV-Vis Double Beam PC Scanning spectrophotometer was used to record the UV–visible spectrum of the reaction mixture (containing laccase and chloroauric acid as said above) in comparison to control sample (reaction mixture without enzyme) in the range of 200–700 nm. Samples for examination by transmission electron microscopy (TEM) were prepared by placing one drop of formed nanogold on carbon-coated copper TEM grids followed by slowly evaporating at room temperature.
3. Results 3.1. Reduction of HAuCl4 solution The absorption spectra of untreated and treated gold solution illustrated in Fig. 1 indicated formation of gold nanoparticles due to presence of the surface plasmon absorption of the Au nanoparticles at 520 nm. 3.2. Effect of HAuCl4 concentration on production of AuNPs As shown in Fig. 1, increasing concentration of chloroauric acid to 0.6 mM led to increase in absorbance of 520 nm corresponded to gold nanoparticle formation. More than such critical concentration aggregation of nanogold increases the particle size.
Table 1 Particle size distributions and average sizes of prepared nanogold after treatment of HAuCl4 (0.6 mM) by purified laccase of Paraconiothyrium variabile at different temperatures. Temperature (◦ C)
Particle size distribution (nm)
Average size (nm)
30 50 60 70 80
148–266, 478–995 46–478 53–308 71–266 128–641
162, 543 127 120 124 227
M.A. Faramarzi, H. Forootanfar / Colloids and Surfaces B: Biointerfaces 87 (2011) 23–27
25
Fig. 3. (a) Scanning electron microscopy (SEM); (b) energy dispersive X-ray (EDX); and (c) transmission electron microscopy (TEM) images of produced AuNPs by purified laccase from Paraconiothyrium variabile.
Fig. 4. FTIR spectrum of gold nanoparticles after treatment of chloroauric acid by purified laccase at 70 ◦ C.
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M.A. Faramarzi, H. Forootanfar / Colloids and Surfaces B: Biointerfaces 87 (2011) 23–27
30 40 50 60 70 80
100
Relative activity (%)
80 60 40 20 0 0
15
30
45
60 Time (min)
75
90
105
120
Fig. 5. Relative activities of the purified laccase after incubation at various temperatures for 120 min. Residual activity (compared to control) was determined each 30 min intervals.
3.3. Effect of temperature on production of AuNPs Fig. 2 represents the effect of temperature on the characteristics of AuNPs. The temperature of 70 ◦ C was the best temperature in which the gold nanoparticles were formed in the size range of 71–266 nm after 20 min incubation (Table 1). The produced nanoparticles at temperature of 30 ◦ C showed two ranges of particle size distributions including 148–266 and 478–995, respectively (Table 1). Formation of AuNPs at 30 ◦ C, 50 ◦ C and 60 ◦ C required more incubation times (5, 2 and 1 h, respectively) and at 80 ◦ C, AuNPs represented larger particle size distribution due to aggregation. According to the obtained results, the best temperature and HAuCl4 concentration for AuNPs production were found to be 70 ◦ C and 0.6 mM, respectively. 3.4. Characterization of AuNPs Scanning electron microscopy (SEM), energy dispersive Xray EDX and TEM image of gold nanoparticles are illustrated in Fig. 3a–c, respectively. Elemental analysis of treated samples by EDX microanalysis (Fig. 3c) confirmed the gold composition of prepared nanoparticles. The FTIR spectrum of AuNPs (Fig. 4) showed two major peaks at 3448.5 and 1642.8. The 3448.5 cm−1 corresponded to OH and/or NH functional groups and presence of carbonyl group could be ascribed to the peak of 1642.8 cm−1 . According to Shakibaie et al. [3] these functional groups could be used in bioconjugation and/or immobilization of various compounds. 3.5. Study on the thermal stability of purified laccase As shown in Fig. 5, at temperature above 60 ◦ C the laccase activity decreased sharply and at 80 ◦ C only 0.4% of initial activity remained after 30 min incubation. Relative activity of the enzyme at 50 ◦ C was found to be 50.4% after 2 h incubation. 4. Discussion Hazardous effects of solvents and chemical reagents used for synthesis of AuNPs on the environment encouraged researchers to study on preparation of gold nanoparticles via biological methods both using living microorganisms and biomolecules [5,14]. Shih and colleagues [4] studied the formation and characterization of AuNPs synthesized by alkaline solution of chitosan and demonstrated more concentration of such polysaccharide decrease
the particle size of prepared gold nanoparticles. Sun et al. [24] reported the mechanism of nanogold formation in presence of chitosan and ascribed this reduction to formation of open chain form of the used biopolymer in acidic condition. Beside investigations on the ability of chitosan suspension for production of gold nanoparticles, many reports have focused on the preparation of AuNPs by peptide sequences and proteins. In the study of Wang and co-workers [12], the biomimetic synthesis of nanogold using the peptide sequence of MS14 (MHGKTQATSGTIQS) was determined. Kalishwaralal et al. [14] showed that the amylase produced by B. licheniformis is the reductive agent responsible for biosynthesis of nanogold. Besides, Rangnekar et al. [25] showed that ␣-amylase and its thiol groups being away from catalytic site is responsible for synthesis of nanogold while other enzymes without such exposing group do not reduce chloroauric acid to Au nanoparticles. In the present study, a biopolymer, laccase from P. variabile, was used as the reductive agent for synthesis of gold nanoparticles. Incubation of the pure laccase in the presence of chloroaurate ions at different temperatures reduces Au3+ to Au◦ . Aggregation of nanoparticles above the critical HAuCl4 concentration (0.6) led to form larger gold particles. Increasing the incubation temperature from 30 ◦ C to 70 ◦ C decreased the time for formation of nanoparticles from 5 h to 20 min and at 80 ◦ C, the production of large nanoparticles was achieved within few minutes. It was also observed that the particle size and the distribution of the obtained nanoparticles were inversely dependent on the temperature within the range of 30–60 ◦ C. It is notable that at 70 ◦ C, the average particle size increased (124 nm) and the particle size distribution was narrowed in comparison with performing the process at 60 ◦ C (Table 1). Meanwhile, larger size of produced AuNPs at 80 ◦ C could be interpreted by the aggregation of the fine nanostructures which was in agreement with the report of Pal et al. [26] on the effect of Triton X-100 to avoid excessive gold agglomeration. Overall, the optimum temperature was found to be 70 ◦ C. Comparison of laccase stability profiles at different temperatures (Fig. 5) showed that the formation of nanogold was happened when the activity decreased. Therefore, exposing of reducing functional groups might be responsible to form gold nanostructures. This finding is in agreement with the study of Kalishwaralal and colleagues [14] demonstrated that denaturation of the purified amylase from B. licheniformis increased the rate of AuNPs biosynthesis because of exposing reductive groups (such as thiol group of cysteine and tertiary amine of histidine) to gold solution.
M.A. Faramarzi, H. Forootanfar / Colloids and Surfaces B: Biointerfaces 87 (2011) 23–27
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