oxidative biodegradation of single-walled carbon nanotubes by partially purified lignin peroxidase...

Journal of Industrial and Engineering Chemistry xxx (2013) xxx–xxx

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JIEC-1752; No. of Pages 8

Oxidative biodegradation of single-walled carbon nanotubes bypartially purified lignin peroxidase from Sparassis latifolia mushroom

Gayathri Chandrasekaran a, Soo-Kyung Choi a, Young-Chul Lee b, Geun-Joong Kim c,Hyun-Jae Shin a,*a Department of Chemical and Biochemical Engineering, Chosun University, Gwangju 501-759, Republic of Koreab Department of Biological Engineering, College of Engineering, Inha University, Incheon 402-751, Republic of Koreac Department of Biological Science, College of Natural Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea

A R T I C L E I N F O

Article history:

Received 10 September 2013

Accepted 8 December 2013

Available online xxx

Keywords:

Single-walled carbon nanotubes

Lignin peroxidase

Sparassis latifolia

Biodegradation

Bioremediation

A B S T R A C T

Two types of carbon nanotubes (usually single-walled carbon nanotubes (SWCNTs) and multi-walled

carbon nanotubes (MWCNTs)) have been intensively focused on academic researches and mass-

produced for wide applications such as composite materials, biosensors, and drug delivery systems.

However, due to oxidative stress-dependent and physically-induced cellular toxicity of CNTs, many

efforts to render biocompatible and biodegradable properties in CNTs have been highlighted. Thus,

taking into the consideration of exposure in human health and the environment, biodegradation of CNTs

as a potential disposal is highly addressed. In this study, lignin peroxidase (LiP) was isolated and partially

purified from the fruiting bodies of the edible mushroom Sparassis latifolia (S. latifolia). The

biodegradation of raw grade and thermally-treated carboxylated SWCNTs (denoted as ASA and AST)

with this enzyme was investigated, prior to more biodegradation-resistant MWCNTs. The interactions

between the SWCNTs and LiP were investigated using various techniques, and the intermediate by-

products of the LiP degradation were identified. Our findings demonstrated that both ASA and AST were

efficiently degraded by LiP where the producing radicals by the LiP played a critical role in the

biodegradation of SWCNTs. The final degraded products were confirmed with the generation of CO2 gas.

Conclusively, the low extraction cost of partially purified enzyme from mushrooms can make this

approach a promising alternative in environmental bioremediation as a practical application.

� 2013 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering

Chemistry.

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Journal of Industrial and Engineering Chemistry

jou r n al h o mep ag e: w ww .e lsev ier . co m / loc ate / j iec

1. Introduction

Carbon-based nanotechnology has become one of the mostimportant and exciting aspects of research in various fields andespecially in engineering and biology [1–6]. Presently, theenvironmental and human health concerns over engineerednanomaterials (ENMs), especially on single-walled carbon nano-tubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs),are increasing [7–10], an important issue to be taken intoconsideration. Moreover, it was stated that carbon nanotubes(CNTs) affect the environment as much as they affect humans [11–13], and the levels of risk are proportional to the amount of ENMsproduced globally.

Various reports have stated that the CNTs cause skin cancer,oxidative stress, granuloma formation, fibrosis, lung cancer,genotoxicity, and mutagenicity [12]. Moreover, the dermal toxicity

* Corresponding author. Tel.: +82 62 2307518; fax: +82 62 2307226.

E-mail address: [email protected] (H.-J. Shin).

Please cite this article in press as: G. Chandrasekaran, et al., J. Ind. E

1226-086X/$ – see front matter � 2013 Published by Elsevier B.V. on behalf of The Ko

http://dx.doi.org/10.1016/j.jiec.2013.12.022

of the SWCNTs causes oxidative stress to the skin. Various types ofCNT products, including raw grade and thermally-treated grades,are available; they contain iron, nickel, and yttrium, whichexhibited a proven pulmonary toxicity [9,12]. Although theycontain lower amounts of catalysts, the overall research hasrevealed that CNTs tend to cause epithelioid granulomas andinterstitial inflammation in animals [14]. Recently, it has beendemonstrated that CNTs can trigger some biological responsessimilar to those of asbestos. For example, MWCNTs (less than20 mm) are short and soft, which could cause serious healthproblems with the identical mechanism [15]. As a result, suchfunctionalized CNTs to reduce toxicity and grant biocompatibleand biodegradable characteristics have been developed [16].However, it is still stressed that the disposal of CNTs is criticalissues. Concerning the environmental hazards of CNTs, those trialsare reported that SWCNTs and MWCNTs could be degraded byenzyme-catalyzed oxidations within a few weeks or months [17].

Among several peroxidases which are activated by H2O2 togenerate unstable radicals for degradation of carbonaceousnanomaterials, white-rot fungi (WRF) can degrade a wide range

ng. Chem. (2013), http://dx.doi.org/10.1016/j.jiec.2013.12.022

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G. Chandrasekaran et al. / Journal of Industrial and Engineering Chemistry xxx (2013) xxx–xxx2

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of organopollutants whose degradable ability is closely related tothe lignin-degrading systems and catalyze the oxidation of non-phenolic and various organic compounds. Specifically, this fungusproduces mainly two types of extracellular peroxidases, ligninperoxidases (LiP) (EC.1.11.1.14) and manganese dependent perox-idase (MnP) (EC.1.11.1.13), while others secrete laccase andversatile peroxidise [18–20]. LiP consists of a single polypeptidechain, with an iron protoporphyrin prosthetic group. It has aunique ability to degrade lignin polymer through an oxidativeelectron transfer mechanism. Based on properties of WRF, theywere applied in the paper and pulp industries as well as in thetextile industry for the decolorization of dyes and transformationof polyaromatic hydrocarbons [21].

Recently, a proteomic analysis of the fruiting bodies of thismushroom and the economic importance of lignin structures wasreported [20]. As a result, the microbial degradation by lignolyticfungi has been intensively studied over the past few years due tothe irregular structure of lignin; the lignolytic fungi produceextracellular enzymes with low substrate specificity that allowsdegradation of substrate compounds.

Peroxidase enzymes can be an alternative that provides a newstrategy to detoxicify organic pollutants from wastewater and soils.However, the bioremediation of carbonaceous materials by LiPextracted from this mushroom is not reported yet despite the lowextraction cost of partially purified enzyme. Based on the first reporton the enzymatic biodegradation of CNTs by horseradish peroxidase(HRP) [17,22], in this study, the biodegradation of two different typesof carboxylated SWCNTs including both a raw grade and thermally-treated using LiP, which is partially purified from the fruiting bodiesof the mushroom Sparassis latifolia (formerly S. crispa) was carriedout. The findings showed that LiP could play an important role in thebiodegradation of carboxylated SWCNTs. We demonstrate feasibilitythat LiP, partially extracted from mushroom S. latifolia, could be apromising option in large-scale settings as an economic and eco-friendly bioremediation for carbonaceous nanomaterials.

2. Experimental

2.1. Materials

SWCNTs purchased from Hanwha Nanotech Corporation(Incheon, Korea) were used. The product name of a raw grade ofASA-100 F is manufactured by the arc-discharge process (denotedas ASA) while that of AST-100 F is prepared by thermal treatment(denoted as AST). The S. latifolia mushroom fruiting bodies werecollected from Forest Resources Research Institute (Naju, Jeonnam,Korea). Lyophilized HRP type VI (analytical grade) and hydrogenperoxide (30 wt%, analytical grade), and all the substratesincluding pyrogallol, 2,6-dimethoxy phenol, veratryl alcohol,2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS),and guaiacol and methanol, formic acid, and acetonitrile ofanalytical grade were purchased from Sigma–Aldrich (St. Louis,MO, USA). DEAE–Sepharose Fast Flow and PD-10 desaltingcolumns were acquired from Amersham Biosciences (GE Health-care, Sweden). Unless otherwise stated, all of the chemicals in thisstudy were used as received from the supplier. Distilled deionizedwater was utilized through the experiments (resistance > 18 mV,DI water).

2.2. Media compositions of S. latifolia mushroom culture

For the media compositions and their effects on enzymeproduction, five different sawdust media compositions withcontrolled percentages of media moisture were used such as:(a) Larix leptolepis (100%) + DI water, (b) Larix leptolepis

(100%) + 10% corn syrup solution, (c) Larix leptolepis (90%) + 10%


wheat flour + 10% corn syrup solution, (d) Larix leptolepis

(80%) + wheat flour (20%) + 10% corn syrup solution, and (e) Larix

leptolepis (80%) + wheat flour (10%) + corn pellet (10%) + 10% cornsyrup solution. S. latifolia fruiting bodies samples were preparedusing these media, and their LiP activities also were tested.

2.3. Enzyme extraction

The fresh fruiting body of the mushroom was isolated andfreeze-dried. Each frozen fresh fruiting body (50 g) of S. latifolia

mushroom was ground to a fine powder in liquid nitrogen using apre-chilled ceramic mortar pestle. Next, the mushroom powderswere extracted in acetate buffer (pH 5.0) separately, phosphatebuffer (pH 7.0), and Tris–HCl buffer (pH 9.0) containing 2 mMEDTA, 1 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride(PMSF) at 4 8C. The concentrations of all the buffers were 10 mM.The mixture was centrifuged at 10,000 rpm for 10 min at 4 8C. Theresultant supernatants were used as crude enzymes [23].

2.4. Protein purification

The enzyme was purified from the crude supernatants by thefollowing steps: 1) ammonium sulfate precipitation and 2) DEAE–Sepharose anion exchange chromatography. The crude extractswere precipitated by addition of ammonium sulfate to 65%saturation. The solution was then centrifuged at 8000 rpm for15 min at 4 8C. The precipitate was dissolved in a Tris–HCl buffer(10 mM, pH 9.0) and desalted over a PD-10 desalting column in theidentical buffer. The filtered enzyme solution was applied to aDEAE–anion exchange column (5 ml) that had been equilibratedwith a Tris–HCl buffer (100 mM, pH 9.0) containing 0.15 M NaCland then eluted with a linear salt gradient at a flow rate of 0.5 ml/min. The fractions with a high LiP specific activity were collected,concentrated, and used with following studies.

2.5. Enzyme assay

The mixture consists of 50 mM of pyrogallol (2.5 ml),100 mM of sodium acetate buffer pH 5.4 (16 ml), and 50 mMof H2O2 (1.5 ml). Approximately 250 ml of the mixture wasaliquotted to each well in the 96-well microtiter plate; 10 ml ofthe enzyme sample (0.05 U/ml) was added and incubated at37 8C for a few minutes. The appearance of a dark-colorindicated the presence of peroxidase activity [24,25]. Theactivity of LiP was quantitatively assayed by the reportedmethod [26] using veratryl alcohol as a substrate and monitor-ing the formation of veratraldehyde at 310 nm spectrophoto-metrically. The reaction solution (1 ml) consisted of 2 mM ofveratryl alcohol and 0.4 mM of H2O2 in 50 nM of sodiumtartarate buffer, pH 3, at 25 8C. The reaction was begun byadding 50 ml of the enzyme solution. The MnP and laccase werethen assayed by the reported methods [27,28].

2.6. Electrophoretic and zymographic analysis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS–PAGE) was performed using a 10% polyacrylamide gel [29]. Ahigh-range molecular weight marker (EBM-1031, Elpis BiotechInc.) was used as a standard. The proteins were quantified by thebicinchoninic acid (BCA) assay using bovine serum albumin (BSA)as a standard [30]. For the assay of peroxidase activity, a zymogramanalysis was performed in the polyacrylamide gels; the proteinsamples were dissolved in loading buffer without SDS or thiol-reducing agents and separated on a 7.5% Native-PAGE gel. After theprotein separation, the gels were equilibrated for 30 min in 50 mMof sodium acetate buffer (pH 5.4) prior to their incubation with



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80 mM of pyrogallol and 80 mM of H2O2 in fresh sodium acetatebuffer.

2.7. Carboxylation and its examination of SWCNTs by scanning

electron microscopy (SEM) observation

SWCNTs were carboxylated as described previously [31].Briefly, approximately 25 mg of each ASA and AST was sonicatedin H2SO4/H2O2 (30%) at a ratio of 3:1 for 24 h at 0 8C. After 10-15 h,2.0 ml H2O2 was added to the reaction to replace the spent H2O2.The final dispersion was then heated at 70 8C for 10 min, andsubsequently diluted 10-fold and filtered through a 0.22 mmTeflon membrane filter; the sample was washed with copiousamounts of water to become a neutral pH. The carboxylated ASAand AST were subjected to an SEM (Hitachi S-2400N, Japan)analysis. This reaction is known as carboxylation, and it wasconfirmed through the SEM studies.

2.8. Transmission electron microscopy (TEM) measurement

The samples in PBS solution were centrifuged at 3400 rpm for3 h to remove salts from the buffer [17,22]. The supernatant wasremoved and the pellet was re-suspended in DMF and sonicated for1 min. The sample was dropped on a lacey carbon grid (pacific-GridTech) and allowed to dry for 1 hr and subsequently take TEMimages (FEI Morgagni, 80 keV or JEOL 2100F, 200 keV).

2.9. Treatment of SWCNTs with LiP and H2O2

Approximately 1 mg of each carboxylated ASA and AST wasadded to 4.0 ml of phosphate buffered saline (PBS) and sonicated for1 min. As a positive control, 0.385 mg/ml of lyophilized HRP type VIwas solubilized in PBS. The negative control was performed withbuffer alone. The experimental samples consisted of the LiP purifiedfrom S. latifolia and solubilized in PBS at the identical concentration.Four ml of each enzyme solution was added to the vials containingthe carboxylated SWCNTs and brought to a total volume of 8 ml. Allthe vials were then sealed with a septum and wrapped withparafilms. From the first day of incubation, 8 ml of 40 mM H2O2 wasadded through 20 daily additions of 250 ml by syringes to the allvials; during the 20 days, the vials were kept at 25 8C [17,22]. Alsocrude LiP was incubated under the identical condition except thatthe incubation temperature was lowered to 4 8C.

2.10. Visible near infrared (vis-NIR) and Raman spectroscopy

150 ml of the aqueous samples of both carboxylated SWCNTswere analyzed in 2 ml-glass cuvettes using a Lambda 900spectrophotometer (Perkin-Elmer, Norwalk, CT). The SWCNTswere scanned from 600 to 1300 nm. Six samples as mentionedabove were subjected to the vis-NIR spectroscopy. All the sampleswere centrifuged at 3400 rpm for 3 h to remove salts from thebuffer. The precipitated samples were then treated with MeOHfollowing a 2 min sonication. Approximately 20 ml of the sampleswere placed on the microscope slide and dried. All the spectra werecollected on a Renishaw inVia Raman microscope using anexcitation wavelength of 633 nm. The samples were scannedfrom 1000 to 1800 cm�1 to visualize the changes in the D and Gband intensities resulting from the degradation process. All thespectra were collected using a 15 s exposure period and averaged 5scans per sample [17,22].

2.11. Electron spin resonance (ESR) spectroscopy

ESR spectra were recorded on a JES-FA ESR spectrometer (JEOL,Tokyo, Japan). The measurements were performed at room


temperature in gas permeable Teflon tubing. The tubing was filledwith 10 ml of sample, folded over, and placed in the ESR quartztube with an open 3.0 mm internal diameter. The ESR solutionswere prepared by incubating LiP with 0.02 mg/ml SWCNTs in PBSfor 1 min at room temperature; pyrogallol was then added and theperoxidase reaction was initiated by adding H2O2 (80 mM). As acontrol, HRP (0.35 mM) was incubated with SWCNTs (0.02 mg/ml)in PBS for 1 min at room temperature, and ascorbate (100 mM) wasthen added. The ESR spectra of the radicals were recorded 1 minafter addition of H2O2. The spectra of these radicals were recordedusing the following conditions: 3270 G, center field; 10 G, sweepwidth; 10 mW, microwave power; 0.4, field modulation; 103,receiver gain; 0.1 s, time constant; 1 min, scan time [22].

2.12. Liquid chromatography–mass spectrometry (LC–MS)

Approximately 3 ml of the aqueous samples of both ASA andAST were acidified by the addition of 500 ml of 0.1 M of HCl andextracted with dichloromethane (3 ml). The dichloromethane wasthen removed and the products were then re-dispersed in pureMeOH (500 ml). Approximately 5 ml of the concentrated samplewas injected onto a C18 column (100 � 2.1 mm, 1.7 mm) at 40 8C ina mobile phase of 20:80 (v/v, formic acid and water:formic acid andacetonitrile). The samples were then analyzed for positive ionsusing electrospray mass spectrometry. An accurate mass mea-surement was performed with a Synapt high-definition massspectrometry system (HDMS; Waters Co.). Leucine enkephalin wasused as an independent reference lock-mass via the LockSpray toensure mass accuracy and reproducibility. The LC–MS profilingwas performed on an Acquity UPLC system (Waters Co., Milford,MA) equipped with a binary solvent delivery system and anautosampler. The chromatographic separation was performed onan Acquity UPLC BEH [17,22].

2.13. Gas chromatography (GC)

Approximately 2 ml headspace of each biodegradation for ASAand AST sample (total headspace volume: 5 ml) was taken throughthe septum of the vials and injected into a Shimadzu QP5050A GC-MS unit with an XFI-F capillary column. The temperature programwas set to hold at 100 8C for 1 min, followed by temperatureramping at a rate of 10 8C/min until a maximum temperature of325 8C was achieved and held for an additional 10 min.

3. Results and discussion

3.1. Identification and partial purification of LiP

The crude S. latifolia mushroom fruiting bodies exhibited aperoxidase activity toward the substrate pyrogallol in the 96-well-plate. The appearance of a dark reddish-brown color in the 96-well-plate indicated the presence of the peroxidase activity.Generally, there are three types of oxidoreductase enzymes in thefungal sources; therefore, we sought to screen for the enzymestypes including LiP, MnP, and laccase using the spectrophotometricmethod. The screening results indicated that veratrylaldehyde wasformed by LiP activity. Other enzymes such as MnP and laccasewere not found. The relative specificity of LiP toward the substrateswas ranked in the order of pyrogallol > veratryl alco-hol > ABTS > guaiacol (Supplementary data, Table S1).

S. latifolia LiP was partially purified by ammonium sulfateprecipitation followed by DEAE column chromatography (Fig. 1a).Furthermore, the peroxidase protein of the 65% fraction waspurified by anion exchange chromatography from the otherproteins present. The unbound fraction (Fig. 1b lane 1) demon-strates the absence of the LiP band, but two major peaks were



Fig. 1. SDS-PAGE (a) M, standard protein marker; lane 1, 40% ammonium sulfate precipitate; lane 2, 65% ammonium sulfate precipitate. SDS-PAGE (b) M, standard protein

marker; lane 1, DEAE unbound fraction; lane 2, DEAE 1st bound fraction; lane 3, DEAE 2nd bound fraction. Native PAGE (7.5%) zymography (c) of peroxidase from S. latifolia.


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observed within the bound fractions. The first peak did not exhibitLiP activity (Fig. 1b lane 2), while the second peak showed thepresence of LiP that was confirmed by the enzyme assay (Fig. 1blane 3). Therefore, the DEAE fractions were used in thecharacterization and application studies without further purifica-tion. The zymographic analysis strongly suggested that the proteinretained its peroxidase activity (Fig. 1c). Furthermore, the lowermolecular weight protein at 26 kDa was purified and subjected itto the enzyme activity assay; there was no LiP enzyme activitypresent (see Supplementary data, Fig. S1).

The LiP of S. latifolia was purified from its fruiting bodies. SDS–PAGE revealed that the molecular mass of the LiP was approxi-mately 45 kDa (Fig. 1b). Some fungal LiP, including LiP isoenzyme41 with a 44 kDa enzyme from Irpex lacteus and a 45 kDa fromPhanerochaete sordida YK-624, have been reported with amolecular mass similar to that of the S. latifolia LiP [32,33].

3.2. Enzymatic degradation of SWCNTs

SWCNTs treatment imparts carboxylic acid groups andimproves the CNTs dispersion in aqueous solution [31] andremoves the residual metal catalyst. The SEM analysis (Supple-mentary data, Fig. S2) clearly shows both ASA and AST werecarboxylated, and observed that the metal impurities aresignificantly reduced after the acid treatment, compared to thepristine SWCNTs. After the acid treatment, the SWCNTs are notonly cut into short pipes but also purified because the acid mixtureis known to intercalate and exfoliate graphite. As-prepared shortenand carboxylated SWCNTs were used throughout the study.

The photograph demonstrating the enzyme degradation isshown (Fig. 2a). The vial (1) contained the carboxylated ASTwithout degradation at day 1, while vial (2) is a positive controlcontaining HRP, vial (3) contains the AST with S. latifolia LiP. Thevials (2) and (3) exhibited the noticeable color change thatindicates degradation after 20 days incubation. This resultconfirmed that both enzymes degrade the AST. In regard tothe degradation of the pristine AST, the visual observationindicated that complete oxidation did not occur, correspondingto TEM image (Supplementary data, Fig. S3). Because thedegradation rate was considerably lower than that of thecarboxylated SWCNTs. Thus the degradation studies of thatmaterial have not further pursued. In addition, pristine andcarboxylated ASA showed a similar trend, compared to AST cases(data not shown).


Furthermore, TEM analyses were performed to compare thedegradation of the sample during a 20 day incubation with LiP andH2O2 (Fig. 2b–d): the non-degraded AST on the initial day, after 10days, the degradation of the carboxylated AST by LiP with H2O2;this degradation is indicated by the reduced lengths of theSWCNTs. When compared with the initial stage, the 10 day and 20day samples showed the presence of non-tubular structures,indicating the degradation of the SWCNTs. However, it was unableto discern complete oxidation at day 20, which would be indicatedby the presence of the intermediates of carbonaceous products.

To observe the longevity of the enzyme, a microtiter plate assaywas performed using pyrogallol as a substrate for the LiP. Thisassay was performed for each sample after the 20 day incubation,and the results showed that enzyme activity was nearly retained(Supplementary data, Table S1).

3.3. Visible near infrared and Raman spectra

The visible near infrared (vis-NIR) and Raman spectroscopicmethods can be used to monitor the degradation of CNTs [17]. Thedegradation of ASA and AST was monitored using a vis-NIRspectroscopy after a 5 days incubation with the partially purifiedLiP and 40 ml H2O2 (aq). Non-degraded carboxylated SWCNTs aremixtures of various diameters and helicities, exhibiting metallicand semiconducting electronic properties (Fig. 3). It shows thespectral range of the M1 metallic band between 650 and 750 nm, aswell as the broad S2 semiconducting band of the CNTs absorbingbetween 1000 and 1100 nm [34]. By monitoring these bands overthe 5 days incubation period with partially purified LiP and H2O2 at25 8C, the M1 and S2 band of the CNTs decreased. The graphiticstructure of the CNTs diminished and completely disappeared as aresult of the enzymatic degradation during the 10 days incubationperiod (Fig. 3a and b). Also, observed results on the 20th dayconfirmed this phenomenon, indicating similar results for bothSWCNTs. Furthermore, when the incubation of the SWCNTs withcrude LiP and H2O2 took place at 4 8C, the degradation processslowed and required 60 days (Supplementary data, Fig. S4). Thisresult indicated that at 25 8C, the degradation was faster than at4 8C. The results are similar to those of the HRP reaction that serveas a control. Temperature has a considerable effect on the ability ofthe HRP to degrade CNTs [17]. Because approximately 60 days at alower temperature are required to achieve the same level ofdegradation, this demonstrates that CNTs degradation was slowercompared with the degradation at 25 8C.



Fig. 2. Photograph (a) of the enzymatic degradation of the carboxylated AST: vial 1, carboxylated AST; vial 2, after 10 days of incubation of carboxylated AST with HRP; vial 3,

after 10 days of incubation of carboxylated AST with S. latifolia LiP. TEM micrographs (b–d) confirming the degradation of the carboxylated AST according to incubation time

(1, 10, and 20 days), scale bars = 200 nm.


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The degradation of SWCNTs was also confirmed by Ramanspectroscopy, and the results showed that at 4 8C, the carboxylatedAST display D and G bands. After 60 days incubation with crude LiP,these bands were decreased, indicating the degradation of the

Fig. 3. Vis-NIR spectra of carboxylated ASA (a) degraded by HRP and S. latifolia LiP after 5,

after 5, 10, and 20 days at 25 8C.


SWCNTs (Fig. 4). The D and G bands disappeared after theincubation with HRP. When the samples were incubated with LiP,however, the bands did not completely disappear; due tovariations in the samples, some fluctuations in the D:G ratios

10, and 20 days at 25 8C and carboxylated AST (b) degraded by HRP and S. latifolia LiP



Fig. 4. Raman spectra of carboxylated AST after 60 days incubation at 25 8C with S.

latifolia crude LiP (black), HRP (red), and buffer alone (green). (For interpretation of

the references to colour in this figure legend, the reader is referred to the web

version of this article.)

Fig. 5. The ESR spectra to characterize the peroxidase activity of HRP (a) and S.

latifolia LiP (b) in the presence and absence of SWCNTs. In (a), the radicals produced

by the HRP with ascorbate as a substrate. (1) HRP and H2O2; (2) SWCNTs, HRP, and

H2O2; (3) SWCNTs and H2O2; (4) SWCNTs and HRP. In (b), the radicals produced by

the S. latifolia LiP with pyrogallol as a substrate. (1) LiP and H2O2; (2) SWCNTs, LiP,

and H2O2; (3) SWCNTs and H2O2; (4) SWCNTs and LiP.


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were observed that were consistent with another study [35].However, this result indicated the LiP reduced the D and G bands. Itis indicated that the degradation does occur but rate is low,compared to the partially purified samples (data not shown).Clearly, the crude LiP was sufficient to initiate and promote thedegradation process. The results are consistent with other reportedstudies for the enzymatic degradation by crude MnP of polyaro-matic hydrocarbons [36].

3.4. ESR spectra

ESR measurement of the LiP and HRP activity in the presence ofthe SWCNTs was used to detect the radicals formed during the one-electron oxidation of pyrogallol and ascorbate compounds by LiPand HRP, respectively. The addition of H2O2 to LiP in the presenceof pyrogallol produced the characteristic ESR signals of a pyrogalloloxygen radical (pyrogallol-O*). The free radical spectrum ischaracterized by a signal at g = 2.00 (Fig. 5a, 1 and 2). The signalof the ascorbate radical was detected upon the incubation of HRPwith ascorbate in the presence of H2O2 (Fig. 5b, 1 and 2). Theaddition of the SWCNTs to the incubation mixture did not changethe ESR signals of the ascorbate and pyrogallol radical (#2 in Fig. 5aand b). In the absence of LiP and H2O2 or HRP and H2O2, the freeradical signal was several-fold lower, thus confirming that theoxidation of pyrogallol and ascorbate occurred mainly via theperoxidase reaction (Fig. 5a and b, 3 and 4). The spectra wereidentical in the presence and in the absence of SWCNTs. LiPproduced free radical signals from the substrate in the presence ofH2O2 similarly to that produced by HRP. Therefore, both enzymesproduced strong signals from the respective peroxidase substrates,thus demonstrating that CNTs did not inactivate the enzymes.Other studies reported that ESR signals showed that cation radicalswere produced during the degradation of organopollutants, whichproves the cation radical oxidizes these compounds [37]. Thecatalytic cycle of LiP is similar to that of other peroxidase whereinferric enzyme is first oxidized by H2O2 to generate the two-electron oxidized intermediate, Compound I [38]. The degradationmechanism occurs through a classical compound I spectrum,similar to that described for other peroxidases and indicative of theformation of a porphyrin p cation radical [39]. Generally,Compound I contains two oxidizing equivalents; one is stored inthe enzyme as an oxoferryl moiety [Fe (IV) = 0]2+ and the other isOH radicals. It is interesting that in all the peroxidases and otherheme-containing proteins that react with H2O2, the radical is a p-orbital delocalized porphyrin radical. Moreover, there is a report


that porphyrins physisorb onto SWCNTs providing close proximalcontact with the iron site, which further promotes degradation[17]. The enzymatic breakdown of the raw grade and thermallytreated SWCNTs is proposed to occur through a mechanism ofaction that includes the generation of an aryl cation radical byreaction with H2O2. It has been proven that the biodegradation ofSWCNTs is induced by free radicals which aid in the oxidation ofSWCNTs [40]. Conclusively, the interaction of LiP with H2O2

molecules produces free radicals. Those unstable radicals coulddegrade SWCNTs non-selectively.

Alternatively, there is a report on the induction of extracellularOH radicals production in WRF through quinone redox cycling.This mechanism can be explained by the production of OH radicalsfrom the generation of Fe2+ and H2O2 (i.e., Fenton reaction). Likethe WRF, S. latifolia as a brown rot fungi (BRF) [41] can also able toproduce OH radicals during the depolymerization of cellulose.However, the production of reactive oxygen species (ROS) in theextracellular environment in both WRF and BRF is evidence ofquinine redox cycling. This was explained as a simple strategy forthe WRF to induce extracellular OH radicals production [42,43].

3.5. LS–MS analysis

To identify the final products of the degradation of the ASA andAST by LiP and HRP, LC–MS was performed, with monitoring fornegative ions with a positive detector (Supplementary data, Fig.S5). The results revealed that the major degraded products of bothASA and AST are acidic by-products and aldehydes. For example, inthis study we found salicylic acid as the degradation product; thisidentification was also confirmed later with an authentic standard.Thus, these compounds are within a class of compounds similar to




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the identified biodegradation products for the SWCNTs by HRP.However, the entire spectra of the products might be likelyimpossible to identify or interpret.

As shown in Supplementary data, Fig. S5a, the mass to charge(m/z) value, 136.93, was observed for the LiP-degraded SWCNTs,indicative of salicylic acid. The product identified was similar tothose observed in the studies of the bioremediation of polyaro-matic hydrocarbons (PAHs) [21]. The molecular masses of thedegraded products were very similar in the studies of the HRP- andLiP-treated CNTs. This indicated that the enzymes perform asimilar cleavage of the CNTs. However, it is important to note thatmass spectrometry cannot distinguish between molecular ions andfragments, and the preparative scale experiments involvingproduct separation still must be performed. Furthermore, theLiP purified from Gloeophyllum sepiarium MTCC-1170, whichdegrades coal humic acid, and the mechanism involves thegeneration of an aryl cation radical during the reaction withH2O2 [44]. It has been proven that P. chrysosporium oxidizes PAHcompounds through a radical mechanism. HRP also degrades thePAHs through cation radical intermediates. The first enzyme toattack lignin-type compounds was the LiP isolated from P.

chrysosporium. This lignolytic capacity makes most taxa of fungifor use in bioremediation. Pollutants such as chlorophenols,nitrophenols, and polyaromatic hydrocarbons can be transformedby the lignolytic enzymes due to the free radical reactions [8].

3.6. GC analysis

GC was used to analyze the final degradation products of theSWCNTs. Furthermore, the evolution of CO2 gas in the sampleheadspace on the10th day of incubation was monitored and alsocompared with the degradation process of the carboxylated ASAand AST incubated with LiP and H2O2 or with HRP and H2O2 (datanot shown). Both ASA and AST in the presence of H2O2 did notproduce any significant concentration of CO2 in the headspace overthe course of 10 days. In contrast, when carboxylated ASA and ASTwere incubated with LiP and H2O2 and HRP and H2O2, CO2 wasmeasured in the headspace, which was evidence of the degradationof the SWCNTs. In particular, it was observed that the ASTproduced more CO2 gas than the ASA. The ASA incubated with HRPand H2O2 exhibited a higher CO2 gas concentration, compared tothose incubated with the LiP and H2O2.

The analysis of products indicates that complete degradationproduces CO2 gas. A previous study proposed that the lignolyticfungus Pleurotus ostreatus degrades phenanthrene, producingintermediate products such as 9,10-phenanthrenequinone and2,2-diphenic acid before forming CO2 gas [39]. Therefore, theresults were agreed with the other findings and proposed thatmost of the fungal degradation products for the organopollutantsare oxidized to carboxylic acids and finally to form CO2 gas [45].

4. Conclusions

The crude fruiting bodies of S. latifolia can potentially be used inbioremediations for carbon-based nanomaterials. S. latifolia crudeextracts could be a highly efficient and low cost source of thematerials for environmental remediation. Furthermore, the resultsare consistent with other studies that show crude purification ofthe enzyme is sufficient to degrade organic pollutants. Forexample, crude MnP was sufficient to initiate and promote thedegradation of anthracene and pyrene [46]. Similarly, anotherstudy reports that crude LiP from WRF P. chrysosporium candegrade pharmaceutically active compounds such as carbamaze-pine and diclofenac [47].

In summary, LiP isolated from the S. latifolia mushroomcatalyzed the oxidation of a raw grade and thermally-treated


SWCNTs. This study proposes that LiP employs a mechanism basedon the formation of cation radicals for its enzymatic action. Thus,the partially purified enzyme from the S. latifolia mushroom caneconomically perform the bioremediation and especially onbiodegradation of CNTs [48] in which the partially purifiedenzymes with a low concentration of H2O2 facilitate the oxidation.

Acknowledgements

This study was carried out with the support of ‘Forest Science &Technology Projects (Project No. 2009-project-02)’ provided byKorea Forest Service.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.jiec.2013.12.022.

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