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Oxidation of anthracene by immobilized laccase from Trametes versicolor
Bioresource Technology 100 (2009) 4963–4968
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Oxidation of anthracene by immobilized laccase from Trametes versicolor Xiaoke Hu, Peng Wang, Huey-min Hwang * Department of Biology, Jackson State University, P.O. Box 18540, Jackson, MS 39217, USA
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Article history: Received 7 January 2008 Received in revised form 9 March 2009 Accepted 9 March 2009 Available online 28 June 2009 Keywords: Transformation Anthracene Trametes versicolor Laccase
a b s t r a c t The laccase of Trametes versicolor was immobilized on the functionalized nanoparticles SBA-15 with the average diameter less than 10 nm. Laccase mediated oxidations of anthracene (ANT) were investigated in the presence of two mediators, 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and 1-hydroxybenzotriazole (HBT). Oxidation of ANT was more efficiently enhanced by adding 1 mM of HBT than that by adding ABTS. After 48 h oxidation HBT group significantly oxidized ANT with residue 58% relative to 88% in the ABTS group. HPLC and GC/MS analyses indicated the main product of ANT oxidation was anthraquinone (ANQ). The fluorescein diacetate (FDA) uptake of two human cell lines was used to assess the cytotoxicity and genotoxicity of ANT and ANQ. Treatments with ANT and ANQ at 5 and 10 lM exhibited significant cytotoxicity to the HaCaT cells and the A3 lymphocytes and no significant genotoxicity was observed. The results illustrated that ANQ is less toxic than ANT as well. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Anthracene (ANT) is a tricyclic aromatic hydrocarbon compound that is found in high concentrations in polycyclic aromatic hydrocarbons (PAHs) – contaminated sediments, surface soils, and waste sites (Moody et al., 2001). There is evidence that it is absorbed following oral and dermal exposure. Targets for ANT toxicity could be the skin, hematopoietic system, lymphoid system, and gastrointestinal tract (Risk Assessment Information System, 2003). Concerning environmental hazards, some data concerning toxicity degradation and bioremediation of PAHs have been reported, but very little information is available on the biodegradation and toxicity change of ANT (Bonnet et al., 2005; Sverdrup et al., 2002). Moreover, because of low molecular weight compared to most of the other PAHs, ANT has a higher solubility and can be found at more significant levels in water. Therefore, it is important to determine the impact of this molecule on living organisms and ANT was selected as the target compound in this study for bioremediation using immobilized laccase. A variety of bacterial species have been isolated that have the ability to utilize ANT or phenanthrene as the sole source of carbon and energy (Moody et al., 2001; Cerniglia, 1992; Sutherland et al., 1995). Laccase (EC.1.10.3.2) belongs to the group of polyphenol oxidases and is produced by selected fungi, plants and bacteria. The most extensively studied are the extracellular laccases from lignin degrading basidiomycetes (Pozdnyakova et al., 2004). It is commonly accepted that typical substrates of laccases are substituted monophenols, polyphenolic compounds and phenolic groups * Corresponding author. Fax: +1 601 9792778. E-mail address: [email protected] (H.-m. Hwang). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.03.089
of lignin polymer. However, it was shown recently that laccases could oxidize non-phenolic aromatic compounds in the presence of aromatic electron-transfer or radical-forming mediators, such as 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and 1-hydroxybenzotriazole (HBT) (Pozdnyakova et al., 2004; Hu et al., 2007a). It was reported that the Trametes versicolor laccase catalyzed the oxidation of ANT in the presence of ABTS and HBT (Johannes and Majcherczyk, 2000; Johannes et al., 1996). The versatile catalytic properties of laccases promise improvements in many applications, but the short lifetimes of enzymes presently limit their usefulness. Improvement in enzyme stability can enable its further applications. It can reduce the required amount of the enzyme, prolong the lifetime of the enzyme, increase the potential for enzyme reuse, or maintain the good signal of biosensor (Kim et al., 2006). Enzyme immobilization is the most straightforward way to implement continuous enzyme-catalyzed processes. Russo et al. (2008) reported the conversion of an anthraquinone-dye by means of both free or immobilized laccase mixtures on the acrylic resin with epoxy functionalities. We have developed the immobilization of laccase on functionalized nanoparticles such as kaolinite and nanoparticles SBA-15 to improve the stability and reusability of the free laccase (Dodor et al., 2004; Hu et al., 2007a). Many studies have been conducted on studying toxicity change of PAHs after environmental transformations. Many of the transformation products generated through environmental photomodification exhibit greater toxicity than the parent PAHs. Mallakin et al. (1999) found that several hydroxylated ANQs inhibited growth in Lemna gibba. El-Alawi et al. (2001) and Brack et al. (2003) observed toxicity of ANT photoproducts to Vibrio fischeri.
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In this work, we aimed to identify the biodegradation products of ANT by immobilized laccase on SBA-15 mesoporous silica nanoparticles and to determine the cytotoxicity and genotoxicity of ANT and its degradation products to keratinocyte HaCat cells and lymphocyte A3 cells.
2. Methods 2.1. Chemicals ANT, anthroquinone (ANQ), benzo[k]fluoranthene, HBT, ABTS, acetone and acetonitrile were obtained from Sigma (St. Louis, MO). Laccase from T. versicolor was also purchased from Sigma (St. Louis, MO) with an activity of 34 U mg1 protein and used without further purification. 2.2. Biodegradation of ANT by immobilized laccase Laccase (structure weight 53368) from T. versicolor was immobilized on nanoparticles SBA-15 according to procedures reported in our previous study (Hu et al., 2007a). All oxidation treatments were performed in 20 ml reactants in 40 ml amber bottles containing suitable amount of immobilized laccase dissolved in phosphate buffer (pH 7). ANT dissolved in dimethyl sulfoxide (DMSO) was added to all treatments to make the final concentration 20 lM. Tween 80 was added to reach 1% (v/v) to increase ANT bioavailability. The concentration of the mediator ABTS or HBT reached 1 mM in the reaction mixture. The reaction bottles were tightly closed with Teflon-lined screw caps and incubated in a horizontal shaker at the speed of 150 rpm at 37 °C. After the incubation period, the reaction mixture was centrifuged at 18,000g for 10 min, the supernatant was decanted, and then the mixture was added to the same volume of acetonitrile to inactivate the enzyme and shaken for 0.5 h to extract the ANT. For the immobilized enzyme system, the controls were either boiled immobilized laccase or nanoparticles without the laccase for evaluating the sorption of ANT by the support. The percentage of ANT oxidized was calculated from the difference between the ANT levels in the experimental assay and the corresponding control. All treatments, including controls, were replicated three times. 2.3. High performance liquid chromatography (HPLC) analysis The separation and quantification of ANT and its major oxidation products were conducted with a Waters high performance liquid chromatography (HPLC) system, which consists of Waters 515 quaternary pumps, a Waters 717 plus autosampler and a diode array detector (Waters 996). The Supelco Discovery stainless steel octadecyl silica column with 5 lm packing (250 4.6 mm I.D., Supelco, Bellefonte, PA) and a RP-18 guard column (Supelco, Bellefonte, PA) were used in the analysis. The HPLC system was programmed in isocratic mode, with 40% water and 60% acetonitrile at a flow rate of 1 ml min1 as the eluent. UV-light adsorption was monitored at 2-nm intervals from 200 to 600 nm; the wavelength was set at 254 nm to integrate the peaks. The injection volume was 10 ll with an autosampler at a fixed temperature of 10 °C. Calibration models were constructed in the range of 1–10 lM. Linearity was achieved with a correlation coefficient (R2) of 0.999. For identification of the oxidation products, samples were concentrated using solid phase extraction (SPE) with LC–NH2 tube (SupelcleanÒ 3 ml, Supelco, Bellefonte, PA). The extracted solution dissolved in acetonitrile was subjected to HPLC analysis using the same protocol aforementioned. Identification of the products was confirmed by comparison of their retention time and spectrum with those of authentic standards.
2.4. Gas chromatograph/mass spectrometry (GC/MS) analysis The samples were extracted and concentrated with CH2Cl2. The analysis was performed with a Hewlett–Packard (Palo Alto, CA, USA) HP 5890 series II gas chromatography coupled to a HP 5972 series mass selective detector via a heated transfer capillary line (300 °C). Samples were separated on a 30 m 0.25 mm i.d. DB-5 MS fused-silica capillary column with film thickness of 0.25 lm. The oven temperature program was as follows: 50 °C for 2 min, from 50 °C to 100 °C at a rate of 25 °C min1, from 100 °C to 290 °C at a rate of 10 °C min1, and kept at 290 °C for 5 min. The injector temperature was 250 °C. The mass spectrometer was operated in the full-scan mode, scanning from 50 to 550 m/z at a rate of 1.53 scans s1. 2.5. Cytotoxicity of the main oxidation products The human keratinocyte HaCaT cells at passage number 30–35 were grown in T-25 Falcon flasks (Becton Dickinson Labware, Bedford, MA) at 37 °C in a 95% air/5% CO2 humidified incubator (Isotemp; Fisher Scientific, Houston, TX) using the recommended DMEM medium (Pfeifer et al., 2005). The lymphocyte A3 cells were cultured in RPMI 1640 medium recommended by the ATCC. The A3 cells used for the experiments were between passages 14 and 16 (Hu et al., 2007b). 2.6. Treatment of cells with ANT and its degradation intermediates HaCaT cells were harvested by spreading 1 ml of 0.25% trypsin over the stationary cultures and incubating at 37 °C for 5 min. Then, 1 ml of medium was added to stop the reaction. The A3 cells were collected directly. Both the A3 cells and the dislodged HaCaT cells were collected by centrifugation at 129 g for 5 min at 4 °C. The pellets were washed twice with 1 PBS (phosphate buffer solution, pH 7.2). The viability of cells processed in this way was over 95% using the Trypan blue dye method (Jolanda et al., 2003). The cell density was adjusted to 1 106 ml1 with 1 PBS. One hundred microlitre aliquots of HaCaT cells and A3 cells (1 105 cells) were seeded into the wells of 96-well plates. Then, 100 ll of different concentrations of ANT and ANQ in DMSO (final DMSO concentration 4%) were added to each well to achieve the final test chemical concentrations of 0, 5, and 10 lM. The concentration range of the test chemicals was chosen according to their solubility in PBS (containing 8% DMSO). A solvent control containing PBS with 4% DMSO was included in all the experiments. Positive control, cultures treated with 100 lM H2O2, was also included to assure the sensitivity of the assays. Each treatment concentration was assayed in eight replicate wells. The plates were covered with aluminum foil to avoid ambient irradiation. 2.7. Fluorescein diacetate uptake (FDA) test After 30 min of treatment at room temperature, 100 ll of an aliquot containing 10 ng ml1 of FDA were added to each well of the 96-well treatment plates. The plates were incubated at 37 °C for 35 min in a CO2 incubator and the fluorescence read using a microplate spectrofluorimeter (Triad series; Dynex Technologies, Chantilly, VA) at an emission wavelength of 538 nm and excitation of 485 nm. 2.8. Statistical analysis All results are expressed as the mean ± standard deviation (SD). For the FDA test, FDA uptake was measured by the fluorescence intensity of FDA, and the fluorescence intensity of treated cells was compared to the corresponding PBS control group (containing
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AU
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4% DMSO), whose fluorescence intensity was set as 100%. The experiment was conducted at least 3 times, and each treatment had eight replicates. For the purpose of statistical analysis, the experiment was used as the experimental unit; thus, for most data points, then =3. The SAS System for Windows, V8 (SAS Institute, Gary, NC) was used for statistical evaluations. Means ± SD were calculated for normalized FDA uptake. Differences among treatment and control groups were tested by one-way analysis of variance (ANOVA), followed by pair-wise comparisons between groups using Tukey’s test. Differences at p < 0.05 were considered significant.
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0.00 5.00
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RT min Fig. 2. HPLC chromatogram of the oxidation products of ANT.
3. Results 3.1. Efficiency of ANT oxidation by immobilized laccase The free laccase from T. versicolor was immobilized on nanoparticles according to the method reported in our previous study (Hu et al., 2007a). The nanoparticles were dispersed in 100% ethanol and subsequently fixed on the pre-cleaned silica. The diameter of the nanoparticles was determined with atomic force microscope (AFM) (Veeco, Somerset, NJ). Based on the microscopic measurement and calculation, the average diameter of the nanoparticles SBA-15 is <10 nm. Reactions of ANT and immobilized laccases were investigated with separate addition of two mediators, ABTS and HBT. To improve the bioavailability of ANT, 1% Tween 80 was added to the reaction solutions as well. Fig. 1 indicates that the oxidation of ANT was enhanced by adding 1 mM of HBT than that by 1 mM ABTS. Specifically, after 48 h oxidation HBT group showed significant oxidation with ANT residue of 58% relative to 88% of the ABTS group. Overall, the immobilized laccase showed higher oxidation levels in the presence of 1 mM HBT than the 1 mM ABTS group. Laccase alone is also capable of oxidizing xenobiotics, but the non-mediated laccase activity is too low for practical applications (Alcalde et al., 2002). Our results showed that HBT was a more effective mediator for the laccase oxidation system.
were administered to the HPLC system with the separation elution conditions described in Section 2. The HPLC chromatogram of ANT standard showed its retention time was 22.49 min. The absorption spectrum of ANT consists of several bands, with the major bands maxima at 190.6, 252.2, 358.2 and 377.7 nm (data not shown). After 24 h of oxidation, the ANT was converted to its quinone derivatives. The HPLC chromatogram (Fig. 2) showed several product peaks besides the ANT peak. The retention times of these products were all shorter than that of ANT, indicating that the polarity of the products was increased after the oxidation. The oxidation product with a retention time of 9.74 min was identified as ANQ based on comparison to the authentic standard (Fig. 2). The UV– vis spectrum of ANQ presented three main maxima at 206.0, 253.4 and 327.4 nm (data not shown). The trivial amount of oxidation product eluted closely adjacent to the ANQ at a retention time of 12.52 min has not been identified with an absorption spectrum of three main bands peaking at 190.6 and 254.6 nm (Fig. 2). The oxidation products solution was extracted with CH2Cl2 (Rasmussen et al., 1986) after 48 h reaction. According to the GC/ MS results, the ANT was found at 15.39 min with MW = 178 and the fragments at m/z 152, 126, 89, 76 and 63. The ANQ was found at 17.22 min with MW = 208 and the fragments at m/z 180, 152, 126, 76 and 63. The result is consistent with the HPLC result. Therefore, the main oxidation intermediate product for ANT is determined as ANQ (Fig. 3).
3.2. Oxidation products identification 3.3. Cytotoxicity of ANT and its oxidation products The oxidation products of ANT by immobilized laccase were analyzed with HPLC with a photodiode array detector and GC/ MS. The oxidation products of ANT were concentrated with the SPE method using LC–NH2 tubes. After the extraction, the samples
To understand the toxicity changes after enzymatic treatment of ANT, the viability of the keratinocyte cells treated with ANT and its degradation intermediates was measured using the FDA
Fig. 1. Oxidation of ANT by immobilized laccase on nanoparticles SBA-15.
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test. The FDA test assesses viability by measuring the fluorescence intensity of the treated cells relative to control (Zheng et al., 2004). HaCaT and A3 cells were treated with 0, 5, and 10 lM of ANT and ANQ. The concentrations of the test compounds were limited by their solubility in the cell culture medium; thus, the LD50 (lethal dose causing the death of 50% of the cells) could not be obtained in this study. For both cell lines, ANT and ANQ caused significant decreases in cell viability. In the HaCaT cells (Fig. 4), all the treatments of ANT and ANQ at the concentrations of 5 and 10 lM exhibited cytotoxicity compared to the PBS control group containing 4% DMSO (P < 0.05). The percent decrease in viability of the HaCaT cells ranged from 31% to 44% after treatment with ANT and ANQ. In the experiment with A3 lymphocytes (Fig. 5), significant decreases in cell viability (the percent decreases in viability ranged from 13% to 24%) were detected for all the treatments of ANT and ANQ at 5 and 10 lM. Although all the test compounds reduced cell viability, none of these treatments resulted in decreases >30%. These two results illustrated that after oxidation by immobilized laccase, the oxidation product ANQ is slightly less toxic than the parent compound ANT. Moreover, the ANT and ANQ showed higher cytotoxicity in HaCaT cells than that in the A3 cells, which indicated that HaCaT cells are more sensitive to ANT and ANQ. The alkaline Comet Assay was used to determine the genotoxicity of ANT and ANQ at the concentrations of 5 and 10 lM. The re-
sults showed there was no significant genotoxicity found on ANT and ANQ compared to the PBS buffer group (Table 1). 4. Discussion Many approaches, e.g., enzyme immobilization, enzyme modification, protein engineering, and medium engineering were used to improve enzyme’s stability for various scientific and industrial applications. Enzyme immobilization represents the attachment or incorporation of enzyme molecules onto or into large structures, via simple physical adsorption, covalent attachment, or encapsulation (Tischer and Kasche, 1999; Livage et al., 2001). The multipoint attachment between enzyme molecules and host materials reduces protein unfolding, and hence improves its stability (Mozhaev et al., 1990). Ordered mesoporous materials, such as SBA-15 are receiving great attention because of their unique properties of high controlled and uniform pore size and high values of surface area and pore volume (Salis et al., 2009). SBA-15 can provide large surface for enzyme immobilization compared to kaolinite. It just reported that the laccase from Pleurotus sajor-caju can be successfully immobilized through chemical bonding on an ordered mesoporous support (Salis et al., 2009). Therefore, the nanoparticles were adopted as the matrix in the enzyme immobilization in this research.
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Fig. 3. GC/MS chromatography for the oxidation products of ANT.
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Fig. 4. Normalized fluorescein diacetate uptake by HaCaT cells following 30 min treatment with 0, 5 and 10 lM of ANT or ANQ. *Significantly different from the corresponding PBS control (P < 0.05).
Fig. 5. Normalized fluorescein diacetate uptake by A3 lymphocytes following 30 min treatment with 0, 5 and 10 lM of ANT or ANQ. *Significantly different from the corresponding PBS control (P < 0.05).
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could also be removed from the system by being mineralized to CO2 (Cerniglia, 1992; Zhao et al., 2007). Since the surface active substances are commonly produced by bacteria, yeast and fungi, the 1% Tween 80 was added to the reaction solution to simulate the biosurfactants to improve solubility of ANT. The principle of bioremediation is to remove or detoxify a contaminant from a given environment using microorganisms or enzymes. The toxicity of many PAHs is known to be enhanced by light irradiation via photosensitized reactions (production of reactive oxygen species ROS) and photomodification of the chemicals (e.g., oxidation) to more toxic compounds. ANT toxicity in particular has been found to increase dramatically following photomodification (Mallakin et al., 1999; Lampi et al., 2005). Brack et al. (2003) also showed that ANT genotoxicity was manifested only after ultraviolet irradiation. In our study, we found that after oxidation by immobilized laccase, ANT was converted to ANQ which showed a lower cytotoxicity than its parent compound ANT to both HaCaT and A3 cells (Figs. 4 and 5). Both ANT and ANQ showed no genotoxicity according to the alkaline Comet assay (Table 1). This is contrary to our previous study that BaP’s quinone products are more toxic than BaP itself. Therefore, we speculate that on one hand, the number of benzo-ring and oxy-positions on the ring play more important roles in the cytotoxicity. On the other hand, compared to the photodegradation, enzymatic oxidation of ANT might produce less ROS which is known to cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA (Bolton et al., 2000). In conclusion, our finding indicates that the enzymatic oxidation of ANT can be adopted as a green chemistry approach for industrial applications and in the field of waste water treatment. Acknowledgements
Table 1 Genotoxicity of ANT and ANQ. HaCaT cells
PBS 5 lM ANT 5 lM ANQ 10 lM ANT 10 lM ANQ
A3 cells
Moment
% DNA
Moment
% DNA
0.500 ± 0.692 0.370 ± 0.523 0.015 ± 0.007 1.810 ± 0.778 1.140 ± 0.382
2.330 ± 2.263 1.585 ± 1.421 0.680 ± 0.028 2.555 ± 0.969 2.660 ± 0.891
1.150 ± 0.730 0.089 ± 0.070 0.675 ± 0.305 1.060 ± 0.580 1.655 ± 0.835
2.655 ± 0.195 2.665 ± 0.335 1.785 ± 0.265 3.125 ± 0.385 3.300 ± 1.630
DNA damage measured by Comet tail moment and tail DNA content following 30 min treatment of HaCaT cells and A3 cells with 0, 5 and 10 lM of ANT and ANQ. * None of the treatment data was significantly different from the PBS control (P < 0.05).
The mechanism of oxidation by laccase-mediator system is still under study. It has been suggested that the mediator acts as a diffusible electron carrier or a cooxidant that enables the laccase to accept electrons directly from the nonnatural substrates (Alcalde et al., 2002; Bourbonnais et al., 1995; Potthast et al., 1995). Our results showed that the ANT could be reduced to 58% of the original amount after 48 h transformation by immobilized laccase in the presence of 1 mM HBT (Fig. 1). The main intermediate oxidation product was identified as ANQ (Figs. 2 and 3). These results are consistent with the report that the oxidation of the ANT by laccase of T. versicolor under in vitro conditions led to the formation of ANQ (Johannes et al., 1996). The reaction was enhanced significantly by adding of ABTS or HBT (Collins et al., 1996). Our finding that HBT displayed a higher increase in the reaction rate is in agreement with a previous study by Majcherczyk et al. (1998). Quinones and hydroxyl derivatives have been reported to be common end products in the biodegradation of PAH by fungi. Furthermore, in the laccase-mediator system, the quinone intermediate products
This research was supported by the U.S. Department of the Army Research and Development Grant # W912HZ-04-2-0002 to Jackson State University. We appreciate Dr. Xi Liu and Dr. Quinton L. Williams of Jackson State University for the AFM results and kind help. References Alcalde, M., Bulter, T., Arnold, F.H., 2002. Colorimetric assays for biodegradation of polycyclic aromatic hydrocarbons by fungal laccases. J. Biomol. Screen. 7, 537– 543. Bolton, J.L., Trush, M.A., Penning, T.M., Dryhurst, G., Monks, T.J., 2000. Role of quinones in toxicology. Chem. Res. Toxicol. 13, 135–160. Bonnet, J.L., Guiraud, P., Dusser, M., Kadri, M., Laffosse, J., Steinman, R., Bohatier, J., 2005. Assessment of anthracene toxicity toward environmental eukaryotic microorganisms: Tetrahymena pyriformis and selected micromycetes. Ecotoxicol. Environ. Safe. 60, 87–100. Bourbonnais, R., Paice, M.G., Reid, I.D., Lanthier, P., Yaguchi, M., 1995. Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator in kraft lignin 2,20 -azinobis(3-ethylbenzthiazoline-6-sulfonate) depolymerization. Appl. Environ. Microbiol. 61, 1876–1880. Brack, W., Altenburger, R., Kuster, E., Meissner, B., Wenzel, K-D., Schüürmann, G., 2003. Identification of toxic products of anthracene photomodification in simulated sunlight. Environ. Toxicol. Chem. 22, 2228–2237. Cerniglia, C.E., 1992. Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3, 351–368. Collins, P.J., Kotterman, M.J.J., Field, J.A., Dobsoon, A.D.W., 1996. Oxidation of anthracene and benzo[a]pyrene by laccases from Trametes versicolor. Appl. Environ. Microbiol. 62, 4563–4567. Dodor, D.E., Hwang, H.-M., Ekunwe, S.IN., 2004. Oxidation of anthracene and benzo[a]pyrene by immobilized laccase from Trametes versicolor. Enzyme Microbiol. Technol. 35, 210–217. El-Alawi, Y.S., Dixon, D.G., Greenberg, B.M., 2001. Effects of a preincubation period on the photoinduced toxicity of polycyclic aromatic hydrocarbons to the luminescent bacterium Vibrio fischeri. Environ. Toxicol. 16, 277–286. Hu, X., Zhang, Y., Zhao, X., Hwang, H.-M., 2007a. Biodegradation of benzo[a]pyrene with immobilized laccase: Genotoxicity of the products in HaCaT and A3 cells. Environ. Mol. Mutagen. 48, 106–113. Hu, X., Zhao, X., Hwang, H-M., 2007b. Comparative study of immobilized Trametes versicolor laccase on nanoparticles and kaolinite. Chemosphere 66, 1618–1626.
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Johannes, C., Majcherczyk, A., 2000. Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems. Appl. Environ. Microbiol. 66, 524–528. Johannes, C., Majcherczyk, A., Hüttermann, A., 1996. Degradation of anthracene by laccase of Tremetes versicolor in the presence of different mediator compounds. Appl. Microbiol. Biotechnol. 46, 313–317. Jolanda, M.R., Ralf, M.W.M., Elena, P., Gerard, M.J.B.Van H., 2003. Photoprotection by antioxidants against UVB-radiation-induced damage in pig skin organ culture. Radiat. Res. 159, 210–217. Kim, J., Grate, J.W., Wang, P., 2006. Nanostructures for enzyme stabilization. Chem. Eng. Sci. 61, 1017–1026. Lampi, M.A., Gurska, J., McDonald, K., Xie, F., Huang, X.-D., Dixon, D.G., Greenberg, B.M., 2005. Photoinduced toxicity of polycyclic aromatic hydrocarbons to Daphnia magna: ultraviolet-mediated effects and the toxicity of polycyclic aromatic hydrocarbon photoproducts. Environ. Toxicol. Chem. 25, 1979–1987. Livage, J., Coradin, T., Roux, C., 2001. Encapsulation of biomolecules in silica gels. J. Phys.: Condens. Matter. 13, R673–R691. Majcherczyk, A., Johannes, C., Hüttermann, A., 1998. Oxidation of polycyclic aromatic hydrocarbons (PAH) by laccase of Trametes versicolor. Enzyme Microbial. Technol. 22, 335–341. Mallakin, A., McConkey, B.J., Miao, G., McKibben, B., Snieckus, V., Dixon, D.G., Greenberg, B.M., 1999. Impacts of structural photo-modification on the toxicity of environmental contaminants: anthracene photooxidation products. Ecotoxicol. Environ. Safe. 43, 204–212. Moody, J.D., Freeman, J.P., Doerge, D.R., Cerniglia, C.E., 2001. Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. Strain PYR-1. Appl. Environ. Microbiol. 67, 1476–1483. Mozhaev, V.V., Melik-Nubarov, N.S., Sergeeva, M.V., Siksnis, V., Martinek, K., 1990. Strategy for stabilizing enzymes. Part one: increasing stability of enzymes via their multi-point interaction with a support. Biocatalysis 3, 179–187. Pfeifer, G.P., You, Y.H., Besaratinia, A., 2005. Mutations induced by ultraviolet light. Mutat. Res. 571, 19–31.
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Oxidation of anthracene by immobilized laccase from Trametes versicolor Xiaoke Hu, Peng Wang, Huey-min Hwang * Department of Biology, Jackson State University, P.O. Box 18540, Jackson, MS 39217, USA
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Article history: Received 7 January 2008 Received in revised form 9 March 2009 Accepted 9 March 2009 Available online 28 June 2009 Keywords: Transformation Anthracene Trametes versicolor Laccase
a b s t r a c t The laccase of Trametes versicolor was immobilized on the functionalized nanoparticles SBA-15 with the average diameter less than 10 nm. Laccase mediated oxidations of anthracene (ANT) were investigated in the presence of two mediators, 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and 1-hydroxybenzotriazole (HBT). Oxidation of ANT was more efficiently enhanced by adding 1 mM of HBT than that by adding ABTS. After 48 h oxidation HBT group significantly oxidized ANT with residue 58% relative to 88% in the ABTS group. HPLC and GC/MS analyses indicated the main product of ANT oxidation was anthraquinone (ANQ). The fluorescein diacetate (FDA) uptake of two human cell lines was used to assess the cytotoxicity and genotoxicity of ANT and ANQ. Treatments with ANT and ANQ at 5 and 10 lM exhibited significant cytotoxicity to the HaCaT cells and the A3 lymphocytes and no significant genotoxicity was observed. The results illustrated that ANQ is less toxic than ANT as well. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Anthracene (ANT) is a tricyclic aromatic hydrocarbon compound that is found in high concentrations in polycyclic aromatic hydrocarbons (PAHs) – contaminated sediments, surface soils, and waste sites (Moody et al., 2001). There is evidence that it is absorbed following oral and dermal exposure. Targets for ANT toxicity could be the skin, hematopoietic system, lymphoid system, and gastrointestinal tract (Risk Assessment Information System, 2003). Concerning environmental hazards, some data concerning toxicity degradation and bioremediation of PAHs have been reported, but very little information is available on the biodegradation and toxicity change of ANT (Bonnet et al., 2005; Sverdrup et al., 2002). Moreover, because of low molecular weight compared to most of the other PAHs, ANT has a higher solubility and can be found at more significant levels in water. Therefore, it is important to determine the impact of this molecule on living organisms and ANT was selected as the target compound in this study for bioremediation using immobilized laccase. A variety of bacterial species have been isolated that have the ability to utilize ANT or phenanthrene as the sole source of carbon and energy (Moody et al., 2001; Cerniglia, 1992; Sutherland et al., 1995). Laccase (EC.1.10.3.2) belongs to the group of polyphenol oxidases and is produced by selected fungi, plants and bacteria. The most extensively studied are the extracellular laccases from lignin degrading basidiomycetes (Pozdnyakova et al., 2004). It is commonly accepted that typical substrates of laccases are substituted monophenols, polyphenolic compounds and phenolic groups * Corresponding author. Fax: +1 601 9792778. E-mail address: [email protected] (H.-m. Hwang). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.03.089
of lignin polymer. However, it was shown recently that laccases could oxidize non-phenolic aromatic compounds in the presence of aromatic electron-transfer or radical-forming mediators, such as 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and 1-hydroxybenzotriazole (HBT) (Pozdnyakova et al., 2004; Hu et al., 2007a). It was reported that the Trametes versicolor laccase catalyzed the oxidation of ANT in the presence of ABTS and HBT (Johannes and Majcherczyk, 2000; Johannes et al., 1996). The versatile catalytic properties of laccases promise improvements in many applications, but the short lifetimes of enzymes presently limit their usefulness. Improvement in enzyme stability can enable its further applications. It can reduce the required amount of the enzyme, prolong the lifetime of the enzyme, increase the potential for enzyme reuse, or maintain the good signal of biosensor (Kim et al., 2006). Enzyme immobilization is the most straightforward way to implement continuous enzyme-catalyzed processes. Russo et al. (2008) reported the conversion of an anthraquinone-dye by means of both free or immobilized laccase mixtures on the acrylic resin with epoxy functionalities. We have developed the immobilization of laccase on functionalized nanoparticles such as kaolinite and nanoparticles SBA-15 to improve the stability and reusability of the free laccase (Dodor et al., 2004; Hu et al., 2007a). Many studies have been conducted on studying toxicity change of PAHs after environmental transformations. Many of the transformation products generated through environmental photomodification exhibit greater toxicity than the parent PAHs. Mallakin et al. (1999) found that several hydroxylated ANQs inhibited growth in Lemna gibba. El-Alawi et al. (2001) and Brack et al. (2003) observed toxicity of ANT photoproducts to Vibrio fischeri.
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In this work, we aimed to identify the biodegradation products of ANT by immobilized laccase on SBA-15 mesoporous silica nanoparticles and to determine the cytotoxicity and genotoxicity of ANT and its degradation products to keratinocyte HaCat cells and lymphocyte A3 cells.
2. Methods 2.1. Chemicals ANT, anthroquinone (ANQ), benzo[k]fluoranthene, HBT, ABTS, acetone and acetonitrile were obtained from Sigma (St. Louis, MO). Laccase from T. versicolor was also purchased from Sigma (St. Louis, MO) with an activity of 34 U mg1 protein and used without further purification. 2.2. Biodegradation of ANT by immobilized laccase Laccase (structure weight 53368) from T. versicolor was immobilized on nanoparticles SBA-15 according to procedures reported in our previous study (Hu et al., 2007a). All oxidation treatments were performed in 20 ml reactants in 40 ml amber bottles containing suitable amount of immobilized laccase dissolved in phosphate buffer (pH 7). ANT dissolved in dimethyl sulfoxide (DMSO) was added to all treatments to make the final concentration 20 lM. Tween 80 was added to reach 1% (v/v) to increase ANT bioavailability. The concentration of the mediator ABTS or HBT reached 1 mM in the reaction mixture. The reaction bottles were tightly closed with Teflon-lined screw caps and incubated in a horizontal shaker at the speed of 150 rpm at 37 °C. After the incubation period, the reaction mixture was centrifuged at 18,000g for 10 min, the supernatant was decanted, and then the mixture was added to the same volume of acetonitrile to inactivate the enzyme and shaken for 0.5 h to extract the ANT. For the immobilized enzyme system, the controls were either boiled immobilized laccase or nanoparticles without the laccase for evaluating the sorption of ANT by the support. The percentage of ANT oxidized was calculated from the difference between the ANT levels in the experimental assay and the corresponding control. All treatments, including controls, were replicated three times. 2.3. High performance liquid chromatography (HPLC) analysis The separation and quantification of ANT and its major oxidation products were conducted with a Waters high performance liquid chromatography (HPLC) system, which consists of Waters 515 quaternary pumps, a Waters 717 plus autosampler and a diode array detector (Waters 996). The Supelco Discovery stainless steel octadecyl silica column with 5 lm packing (250 4.6 mm I.D., Supelco, Bellefonte, PA) and a RP-18 guard column (Supelco, Bellefonte, PA) were used in the analysis. The HPLC system was programmed in isocratic mode, with 40% water and 60% acetonitrile at a flow rate of 1 ml min1 as the eluent. UV-light adsorption was monitored at 2-nm intervals from 200 to 600 nm; the wavelength was set at 254 nm to integrate the peaks. The injection volume was 10 ll with an autosampler at a fixed temperature of 10 °C. Calibration models were constructed in the range of 1–10 lM. Linearity was achieved with a correlation coefficient (R2) of 0.999. For identification of the oxidation products, samples were concentrated using solid phase extraction (SPE) with LC–NH2 tube (SupelcleanÒ 3 ml, Supelco, Bellefonte, PA). The extracted solution dissolved in acetonitrile was subjected to HPLC analysis using the same protocol aforementioned. Identification of the products was confirmed by comparison of their retention time and spectrum with those of authentic standards.
2.4. Gas chromatograph/mass spectrometry (GC/MS) analysis The samples were extracted and concentrated with CH2Cl2. The analysis was performed with a Hewlett–Packard (Palo Alto, CA, USA) HP 5890 series II gas chromatography coupled to a HP 5972 series mass selective detector via a heated transfer capillary line (300 °C). Samples were separated on a 30 m 0.25 mm i.d. DB-5 MS fused-silica capillary column with film thickness of 0.25 lm. The oven temperature program was as follows: 50 °C for 2 min, from 50 °C to 100 °C at a rate of 25 °C min1, from 100 °C to 290 °C at a rate of 10 °C min1, and kept at 290 °C for 5 min. The injector temperature was 250 °C. The mass spectrometer was operated in the full-scan mode, scanning from 50 to 550 m/z at a rate of 1.53 scans s1. 2.5. Cytotoxicity of the main oxidation products The human keratinocyte HaCaT cells at passage number 30–35 were grown in T-25 Falcon flasks (Becton Dickinson Labware, Bedford, MA) at 37 °C in a 95% air/5% CO2 humidified incubator (Isotemp; Fisher Scientific, Houston, TX) using the recommended DMEM medium (Pfeifer et al., 2005). The lymphocyte A3 cells were cultured in RPMI 1640 medium recommended by the ATCC. The A3 cells used for the experiments were between passages 14 and 16 (Hu et al., 2007b). 2.6. Treatment of cells with ANT and its degradation intermediates HaCaT cells were harvested by spreading 1 ml of 0.25% trypsin over the stationary cultures and incubating at 37 °C for 5 min. Then, 1 ml of medium was added to stop the reaction. The A3 cells were collected directly. Both the A3 cells and the dislodged HaCaT cells were collected by centrifugation at 129 g for 5 min at 4 °C. The pellets were washed twice with 1 PBS (phosphate buffer solution, pH 7.2). The viability of cells processed in this way was over 95% using the Trypan blue dye method (Jolanda et al., 2003). The cell density was adjusted to 1 106 ml1 with 1 PBS. One hundred microlitre aliquots of HaCaT cells and A3 cells (1 105 cells) were seeded into the wells of 96-well plates. Then, 100 ll of different concentrations of ANT and ANQ in DMSO (final DMSO concentration 4%) were added to each well to achieve the final test chemical concentrations of 0, 5, and 10 lM. The concentration range of the test chemicals was chosen according to their solubility in PBS (containing 8% DMSO). A solvent control containing PBS with 4% DMSO was included in all the experiments. Positive control, cultures treated with 100 lM H2O2, was also included to assure the sensitivity of the assays. Each treatment concentration was assayed in eight replicate wells. The plates were covered with aluminum foil to avoid ambient irradiation. 2.7. Fluorescein diacetate uptake (FDA) test After 30 min of treatment at room temperature, 100 ll of an aliquot containing 10 ng ml1 of FDA were added to each well of the 96-well treatment plates. The plates were incubated at 37 °C for 35 min in a CO2 incubator and the fluorescence read using a microplate spectrofluorimeter (Triad series; Dynex Technologies, Chantilly, VA) at an emission wavelength of 538 nm and excitation of 485 nm. 2.8. Statistical analysis All results are expressed as the mean ± standard deviation (SD). For the FDA test, FDA uptake was measured by the fluorescence intensity of FDA, and the fluorescence intensity of treated cells was compared to the corresponding PBS control group (containing
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0.20 0.10
12.525
AU
9.740 ANQ
4% DMSO), whose fluorescence intensity was set as 100%. The experiment was conducted at least 3 times, and each treatment had eight replicates. For the purpose of statistical analysis, the experiment was used as the experimental unit; thus, for most data points, then =3. The SAS System for Windows, V8 (SAS Institute, Gary, NC) was used for statistical evaluations. Means ± SD were calculated for normalized FDA uptake. Differences among treatment and control groups were tested by one-way analysis of variance (ANOVA), followed by pair-wise comparisons between groups using Tukey’s test. Differences at p < 0.05 were considered significant.
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0.00 5.00
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RT min Fig. 2. HPLC chromatogram of the oxidation products of ANT.
3. Results 3.1. Efficiency of ANT oxidation by immobilized laccase The free laccase from T. versicolor was immobilized on nanoparticles according to the method reported in our previous study (Hu et al., 2007a). The nanoparticles were dispersed in 100% ethanol and subsequently fixed on the pre-cleaned silica. The diameter of the nanoparticles was determined with atomic force microscope (AFM) (Veeco, Somerset, NJ). Based on the microscopic measurement and calculation, the average diameter of the nanoparticles SBA-15 is <10 nm. Reactions of ANT and immobilized laccases were investigated with separate addition of two mediators, ABTS and HBT. To improve the bioavailability of ANT, 1% Tween 80 was added to the reaction solutions as well. Fig. 1 indicates that the oxidation of ANT was enhanced by adding 1 mM of HBT than that by 1 mM ABTS. Specifically, after 48 h oxidation HBT group showed significant oxidation with ANT residue of 58% relative to 88% of the ABTS group. Overall, the immobilized laccase showed higher oxidation levels in the presence of 1 mM HBT than the 1 mM ABTS group. Laccase alone is also capable of oxidizing xenobiotics, but the non-mediated laccase activity is too low for practical applications (Alcalde et al., 2002). Our results showed that HBT was a more effective mediator for the laccase oxidation system.
were administered to the HPLC system with the separation elution conditions described in Section 2. The HPLC chromatogram of ANT standard showed its retention time was 22.49 min. The absorption spectrum of ANT consists of several bands, with the major bands maxima at 190.6, 252.2, 358.2 and 377.7 nm (data not shown). After 24 h of oxidation, the ANT was converted to its quinone derivatives. The HPLC chromatogram (Fig. 2) showed several product peaks besides the ANT peak. The retention times of these products were all shorter than that of ANT, indicating that the polarity of the products was increased after the oxidation. The oxidation product with a retention time of 9.74 min was identified as ANQ based on comparison to the authentic standard (Fig. 2). The UV– vis spectrum of ANQ presented three main maxima at 206.0, 253.4 and 327.4 nm (data not shown). The trivial amount of oxidation product eluted closely adjacent to the ANQ at a retention time of 12.52 min has not been identified with an absorption spectrum of three main bands peaking at 190.6 and 254.6 nm (Fig. 2). The oxidation products solution was extracted with CH2Cl2 (Rasmussen et al., 1986) after 48 h reaction. According to the GC/ MS results, the ANT was found at 15.39 min with MW = 178 and the fragments at m/z 152, 126, 89, 76 and 63. The ANQ was found at 17.22 min with MW = 208 and the fragments at m/z 180, 152, 126, 76 and 63. The result is consistent with the HPLC result. Therefore, the main oxidation intermediate product for ANT is determined as ANQ (Fig. 3).
3.2. Oxidation products identification 3.3. Cytotoxicity of ANT and its oxidation products The oxidation products of ANT by immobilized laccase were analyzed with HPLC with a photodiode array detector and GC/ MS. The oxidation products of ANT were concentrated with the SPE method using LC–NH2 tubes. After the extraction, the samples
To understand the toxicity changes after enzymatic treatment of ANT, the viability of the keratinocyte cells treated with ANT and its degradation intermediates was measured using the FDA
Fig. 1. Oxidation of ANT by immobilized laccase on nanoparticles SBA-15.
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test. The FDA test assesses viability by measuring the fluorescence intensity of the treated cells relative to control (Zheng et al., 2004). HaCaT and A3 cells were treated with 0, 5, and 10 lM of ANT and ANQ. The concentrations of the test compounds were limited by their solubility in the cell culture medium; thus, the LD50 (lethal dose causing the death of 50% of the cells) could not be obtained in this study. For both cell lines, ANT and ANQ caused significant decreases in cell viability. In the HaCaT cells (Fig. 4), all the treatments of ANT and ANQ at the concentrations of 5 and 10 lM exhibited cytotoxicity compared to the PBS control group containing 4% DMSO (P < 0.05). The percent decrease in viability of the HaCaT cells ranged from 31% to 44% after treatment with ANT and ANQ. In the experiment with A3 lymphocytes (Fig. 5), significant decreases in cell viability (the percent decreases in viability ranged from 13% to 24%) were detected for all the treatments of ANT and ANQ at 5 and 10 lM. Although all the test compounds reduced cell viability, none of these treatments resulted in decreases >30%. These two results illustrated that after oxidation by immobilized laccase, the oxidation product ANQ is slightly less toxic than the parent compound ANT. Moreover, the ANT and ANQ showed higher cytotoxicity in HaCaT cells than that in the A3 cells, which indicated that HaCaT cells are more sensitive to ANT and ANQ. The alkaline Comet Assay was used to determine the genotoxicity of ANT and ANQ at the concentrations of 5 and 10 lM. The re-
sults showed there was no significant genotoxicity found on ANT and ANQ compared to the PBS buffer group (Table 1). 4. Discussion Many approaches, e.g., enzyme immobilization, enzyme modification, protein engineering, and medium engineering were used to improve enzyme’s stability for various scientific and industrial applications. Enzyme immobilization represents the attachment or incorporation of enzyme molecules onto or into large structures, via simple physical adsorption, covalent attachment, or encapsulation (Tischer and Kasche, 1999; Livage et al., 2001). The multipoint attachment between enzyme molecules and host materials reduces protein unfolding, and hence improves its stability (Mozhaev et al., 1990). Ordered mesoporous materials, such as SBA-15 are receiving great attention because of their unique properties of high controlled and uniform pore size and high values of surface area and pore volume (Salis et al., 2009). SBA-15 can provide large surface for enzyme immobilization compared to kaolinite. It just reported that the laccase from Pleurotus sajor-caju can be successfully immobilized through chemical bonding on an ordered mesoporous support (Salis et al., 2009). Therefore, the nanoparticles were adopted as the matrix in the enzyme immobilization in this research.
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Fig. 3. GC/MS chromatography for the oxidation products of ANT.
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Fig. 4. Normalized fluorescein diacetate uptake by HaCaT cells following 30 min treatment with 0, 5 and 10 lM of ANT or ANQ. *Significantly different from the corresponding PBS control (P < 0.05).
Fig. 5. Normalized fluorescein diacetate uptake by A3 lymphocytes following 30 min treatment with 0, 5 and 10 lM of ANT or ANQ. *Significantly different from the corresponding PBS control (P < 0.05).
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could also be removed from the system by being mineralized to CO2 (Cerniglia, 1992; Zhao et al., 2007). Since the surface active substances are commonly produced by bacteria, yeast and fungi, the 1% Tween 80 was added to the reaction solution to simulate the biosurfactants to improve solubility of ANT. The principle of bioremediation is to remove or detoxify a contaminant from a given environment using microorganisms or enzymes. The toxicity of many PAHs is known to be enhanced by light irradiation via photosensitized reactions (production of reactive oxygen species ROS) and photomodification of the chemicals (e.g., oxidation) to more toxic compounds. ANT toxicity in particular has been found to increase dramatically following photomodification (Mallakin et al., 1999; Lampi et al., 2005). Brack et al. (2003) also showed that ANT genotoxicity was manifested only after ultraviolet irradiation. In our study, we found that after oxidation by immobilized laccase, ANT was converted to ANQ which showed a lower cytotoxicity than its parent compound ANT to both HaCaT and A3 cells (Figs. 4 and 5). Both ANT and ANQ showed no genotoxicity according to the alkaline Comet assay (Table 1). This is contrary to our previous study that BaP’s quinone products are more toxic than BaP itself. Therefore, we speculate that on one hand, the number of benzo-ring and oxy-positions on the ring play more important roles in the cytotoxicity. On the other hand, compared to the photodegradation, enzymatic oxidation of ANT might produce less ROS which is known to cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA (Bolton et al., 2000). In conclusion, our finding indicates that the enzymatic oxidation of ANT can be adopted as a green chemistry approach for industrial applications and in the field of waste water treatment. Acknowledgements
Table 1 Genotoxicity of ANT and ANQ. HaCaT cells
PBS 5 lM ANT 5 lM ANQ 10 lM ANT 10 lM ANQ
A3 cells
Moment
% DNA
Moment
% DNA
0.500 ± 0.692 0.370 ± 0.523 0.015 ± 0.007 1.810 ± 0.778 1.140 ± 0.382
2.330 ± 2.263 1.585 ± 1.421 0.680 ± 0.028 2.555 ± 0.969 2.660 ± 0.891
1.150 ± 0.730 0.089 ± 0.070 0.675 ± 0.305 1.060 ± 0.580 1.655 ± 0.835
2.655 ± 0.195 2.665 ± 0.335 1.785 ± 0.265 3.125 ± 0.385 3.300 ± 1.630
DNA damage measured by Comet tail moment and tail DNA content following 30 min treatment of HaCaT cells and A3 cells with 0, 5 and 10 lM of ANT and ANQ. * None of the treatment data was significantly different from the PBS control (P < 0.05).
The mechanism of oxidation by laccase-mediator system is still under study. It has been suggested that the mediator acts as a diffusible electron carrier or a cooxidant that enables the laccase to accept electrons directly from the nonnatural substrates (Alcalde et al., 2002; Bourbonnais et al., 1995; Potthast et al., 1995). Our results showed that the ANT could be reduced to 58% of the original amount after 48 h transformation by immobilized laccase in the presence of 1 mM HBT (Fig. 1). The main intermediate oxidation product was identified as ANQ (Figs. 2 and 3). These results are consistent with the report that the oxidation of the ANT by laccase of T. versicolor under in vitro conditions led to the formation of ANQ (Johannes et al., 1996). The reaction was enhanced significantly by adding of ABTS or HBT (Collins et al., 1996). Our finding that HBT displayed a higher increase in the reaction rate is in agreement with a previous study by Majcherczyk et al. (1998). Quinones and hydroxyl derivatives have been reported to be common end products in the biodegradation of PAH by fungi. Furthermore, in the laccase-mediator system, the quinone intermediate products
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