Research ArticleABTS-Modified Silica Nanoparticles as Laccase Mediators forDecolorization of Indigo Carmine Dye

Youxun Liu Mingyang Yan Yuanyuan Geng and Juan Huang

School of Basic Medical Sciences Xinxiang Medical University Jinsui Avenue 601 Xinxiang Henan 453003 China

Correspondence should be addressed to Juan Huang huangjuanxxmueducn

Received 1 October 2015 Revised 29 November 2015 Accepted 13 December 2015

Academic Editor Maria P Robalo

Copyright copy 2015 Youxun Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Efficient reuse and regeneration of spent mediators are highly desired for many of the laccasesrsquo biotechnology applicationsThis investigation demonstrates that a redox mediator 221015840-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) covalentlyattached to silica nanoparticles (SNPs) effectively mediated dye decolorization catalyzed by laccase Characteristics of ABTS-modified silica nanoparticles (ABTS-SNPs) were researched by scanning electron microscopy and Fourier-transformed infraredspectroscopy When ABTS and ABTS-SNPs were used as laccase mediators the decolorization yields of 96 and 95 wererespectively obtained for indigo carmine dyeThe results suggest that ABTS immobilized on SNPs can be used as laccase mediatorsas they retain almost the same efficiency as the free ABTS The oxidized ABTS-SNPs were regenerated by their reduction reactionwith ascorbic acid Decolorization efficiency of regenerated ABTS-SNPs and their initial forms were found to be almost equivalentSix reuse cycles for spent ABTS-SNPs were run for the treatment of indigo carmine providing decolorization yields of 96ndash77Compared with free mediator the immobilized mediators have the advantage of being easily recovered regenerated and reusedmaking the whole process environmentally friendly

1 Introduction

It is known that laccase (EC 11032 p-diphenol dioxygenoxidoreductases) belongs to the family of the multicopperoxidases [1 2] It can oxidize various organic substratesincluding phenols and arylamines even some inorganiccompounds In catalytic reactions of laccase oxygen actsas the terminal electron acceptor with water as the onlyby-product [3 4] Due to their broad substrate specificitylaccases play amajor role in a number of biotechnological andindustrial applications [5 6] In general the potential of fugallaccase varies within 430ndash780mV [2]The laccase cannot oxi-dize these substrates whose redox potential exceeds 800mVInterestingly when several chemicals called as mediatorssuch as ABTS are added into catalytic reactions laccase isable to oxidize those substrates with high redox potential likenonphenolics [7 8] Therefore application range of laccaseis further expanded by laccase-mediator systems (LMSs) Ithas been reported that for a number of industrial processesmediated by laccases redox mediators are required in order

to oxide complex substrates [7] LMSs are known to catalyzediverse reactions and in wastewater treatment they are usedto detoxify or remove xenobiotic compounds [9ndash12] ABTSand 1-hydroxybenzotriazole have been found to be efficientlaccase mediators in the decolorization of recalcitrant dyes[13 14] When ABTS is used as a mediator redox interactionwith laccase could produce in situ two reactive intermediatesfrom ABTS namely ABTS+∙ or ABTS2+ [8] However it hasbeen reported that their degradation by-products formed insitu by hydrolysis during the laccase-ABTS reactionsmay alsobe responsible for the observed oxidation of nonphenolics[8]The redox potential of the laccase-ABTS system is around885mvwhichmay be enough to oxidize somedyes [7] but theefficiency of substrate degradation will depend on the redoxpotential of used laccase mediator and substrate

Many mediators used in these applications are soluble inthe reaction medium There are however several disadvan-tages of using dissolved mediators including their interfer-ences during product purification their limited reusabilityand their cost-intensive removal fromwastewater Numerous

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 670194 7 pageshttpdxdoiorg1011552015670194

2 Journal of Chemistry

O

O

S

NH

OHN

O O

SO

Na+

Na+

minusO

minusO

Figure 1 Molecular structure of indigo carmine

ways of laccase immobilization are presented in the liter-ature [15ndash17] whereas the accounts of immobilization ofmediators are scarce Recently the electrode modified withABTS polymer exhibited electrocatalytic activity towardsdioxygen when immersed in laccase solution [18] Thefungal laccase has previously been successfully entrappedin poly-o-phenylenediamine matrix which can mediatethe enzymatic reaction [19] Covalently attached 2266-tetramethylpiperidine1-oxyl to polyethylene glycol has alsobeen successfully utilized as a laccase mediator for dye decol-orization in amembrane reactor [20] On the one hand smallmolecule mediators noncovalently adsorbed to insolublecarriers lead to mediator leakage in use On the other handpolymers or filmsmodifiedwithmoleculemediators ordinar-ily may result in formidable mass-transfer resistance to thelaccase-catalyzed reactions To circumvent these problemsmediators can be covalently attached to nanosized particlesthat significantly reduce the external resistance to masstransfer Further mediator-modified nanoparticles can berecovered easily for repeated use after effectively mediatingthe reactions catalyzed by enzymes Resulting from the abilityof the dication ABTS2+ to undergo reversible oxidation toABTS+∙ radical ABTS is considered as a one of the mostefficient mediator of laccase [21ndash24] It has been reported thatlaccase with ABTS is able to degrade various environmentalcontaminants [8 25] Indigo carmine (Figure 1) is the mostrepresentative indigoid dye and it is widely used as textilecoloring agent [26] It has been proved that the dye iscarcinogenic and can lead to reproductive developmentalneuron and acute toxicity [27]Thus considering the toxicityof this dye numerous attempts have been made to removeindigo carmine from wastewater [26 27] Because indigocarmine is not a typical substrate of laccase the completedecolorization of this dye by laccase needs the help from amediator Therefore it was chosen as the model recalcitrantcompound for carrying out decolorization reaction in thisstudy

In the present study our goal was to utilize ABTS-mod-ified silica nanoparticles (SNPs) as mediators for a laccase-catalyzed decolorization of indigo carmine dye First ABTSwas covalently attached to SNPs to yield ABTS-immobilizedSNPs (ABTS-SNPs) which were utilized as laccase mediatorsto decolorize indigo carmine dye Secondly these immo-bilized ABTS-SNPs were proved to be effective reusablemediators and easily recovered by centrifugation of thereaction medium

2 Materials and Methods

21 Chemicals ABTS and laccase (catalogue number 38429)were purchased from Sigma-Aldrich USA Indigo carmine(purity ge 96 MW 46635) was obtained from AladdinChina The SNPs (catalogue number XF105 purity gt 99)used in this study weremilky white dispersion and purchasedfrom purchased from Nanjing XFNANOMaterials Tech CoLtd ChinaThe diameter of SNPs ranges between 10 nm and50 nm All chemicals were used as received without furtherpurification Deionized water was used throughout the wholeexperiment

22 Preparation and Characteristics of Silica NanoparticlesModified with ABTS The researchers have recently devel-oped a procedure for the modification of the carbon nan-otubeswith residues of ABTS [28 29] By employing the sameprocedure amino-functionalized SNPs were conjugated withABTS In brief first ABTS (ammonium salt) was convertedinto potassium salt by reaction with potassium tert-butoxideThen disulfonyl bromide was obtained by the reaction ofpotassium salt with triphenylphosphineBr

2 Finally ABTS

was covalently bonded to SNPs by the disulfonyl bromidereacted with amino-functionalized SNPs (Scheme 1) Theresulting samples were centrifuged and washed thoroughlyseveral times with dimethyl sulfoxide methanol and distilledwater respectively until unbounded species were completelyremoved ABTS covalently attached to SNPs were obtainedand resuspended in distilled water The functional groupsof SNPs ABTS-SNPs and ABTS were studied using Fouriertransform infrared (FT-IR) spectroscopy (Bruker OpticsTensor 37) in the range 4000ndash450 cmminus1 at a resolution of2 cmminus1These samples were initially dried in drying oven andthen were ground with KBr respectively The mixture wasmolded into a disc which was analyzed by FT-IR Accordingto elemental analysis there were about 009 S in the ABTS-SNPs this meant that the amount of ABTS attached to SNPswas about 7 120583molg The microstructure of ABTS-SNPs wascharacterized by a JEOL JSM-6700F field emission scanningelectron microscope (SEM)

23 Dye Decolorization The effect of pH on decolorizationof indigo carmine (20mgL dye) by 3000UL laccase and10 120583M ABTS at different pH ranging from 25 to 6 (50mMsodium acetate buffer) was studied Results from the pre-liminary experiments indicated that the optimal pH valuewas about 45 so decolorization experiments were performedusing 20mgL dye 3000UL laccase and mediator (10 gLfor ABTS-SNPs containing 70120583M ABTS or 70 120583M ABTS)at pH 45 After the addition of these samples into 5mLcentrifuge tube the reactionmixturewas incubated in 50mMsodium acetate buffer with gentle shaking at 150 rpm at roomtemperature Controls reactionswere also carried out to studythe decolorization efficiency by laccase without a mediatoror with ABTS or ABTS-SNPs alone The data presented arethe average values of measurements from triplicate technicalrepeats The absorption spectra of ABTS (70120583M ABTS) andindigo carmine (20mgL) in 50mM sodium acetate buffer at

Journal of Chemistry 3

S

NN N

S

S

NO

O

NH

NN N

S

S

N

NN N

SS

N

SNPs ABTS

3-Aminopropyltriethoxysilane

+H4NminusO3S

SO3minusNH4

+

tert-BuOK Ph3PBr2 BrO2S

SO2Br

SiO2

SiO2

SiO2NH2

Et3N

SO3H

+

Scheme 1 Structure of dominating SNP modifications obtained by the route for covalent attachment of ABTS

300 400 500 600 70000

05

10

15

20

Wavelength (nm)

Abso

rban

ce

Indigo carmineABTS

340nm 615nm

Figure 2The absorption spectra ofABTS (70 120583MABTS) and indigocarmine (20mgL) in 50mM sodium acetate buffer at pH 45

pH 45 were shown in Figure 2 which indicated that ABTShas a major ultraviolet light absorption peak at 340 nm andindigo carmine has the absorbance peak at around 615 nmDecolorization was determined spectrophotometrically bymonitoring the decrease in absorbance at the maximumwavelength of dye (around 615 nm) using ShimadzuUV-2550spectrophotometer The percentage decolorization of a dyewas calculated based on the formula conversion () = (119860

0minus

1198601)1198600 where 119860

0was the initial absorbance of the dye at

its adsorption maximum and 1198601was its absorbance of the

reaction mixture at the different reaction time

24 Regeneration of the Oxidized ABTS-SNPs After thedecolorization reaction was complete the oxidized ABTS-SNPs were separated and then transferred into a 5mL cen-trifuge tube containing 35mL distilled water With mixing10 120583L of 1M ascorbic acidwas added After shaking at 150 rpmfor 5min the mixture was centrifuged for 2min at 10000timesgThe regenerated ABTS-SNPs were separated and washedthrice with 3mL distilled water again

25 Reusability of ABTS-SNPs The recyclability of ABTS-SNPs was assessed in a discontinuous decolorization reac-tion Decolorization of indigo carmine (20mgL) were per-formed in a 5mL centrifuge tube containing 3000UL oflaccase and 10 gL ABTS-SNPs at pH 45 shaking at 150 rpmfor 30min at room temperature At the end of every cycleABTS-SNPs were separated regenerated and washed thricewith 3mL distilled water Next the ABTS-SNPs was addedinto a fresh reaction solution (20mgL indigo carmine3000UL laccase pH 45) to start a fresh cycle The datapresented are the average values of measurements fromtriplicate technical repeats

3 Results and Discussion

31 Structure Biochemical and Physicochemical Characteris-tics of ABTS-SNPs The chemical structure of ABTS-SNPsis presented in Scheme 1 Morphology of SNPs and ABTS-SNPs as obtained by SEM is shown in Figures 3(a) and 3(b)The particles obtained after covalent attachment of ABTSon SNPs were found to be nanosized (lt50 nm) OriginalSNPs were white but the SNPs covalently attached to ABTSexhibited light yellow color (Figure 3(c)) After ABTS-SNPs

4 Journal of Chemistry

(a) (b)

1 2 3 4 5

(c)

01000200030004000

ABTS

SNPsABTS-SNPs

Wavenumber (cmminus1)

(d)

Figure 3 (a) SEM image of SNPs (b) SEM image of ABTS-SNPs (c) Tube 1 SNPs Tube 2 SNPs covalently attached to ABTS Tube 3 afterreaction of ABTS-SNPs with laccase Tube 4 regenerated ABTS-SNPs Tube 5 after reaction of regenerated ABTS-SNPs with laccase and (d)FT-IR spectra of ABTS ABTS-SNPs and SNPs

weremixedwith laccase for a fewminutes themixture turnedfrom light yellow to green The color change in the reactionwas attributed to the fact that ABTS molecules attached onthe nanoparticle surface were oxidized to green ABTS+∙ bylaccase

The comparison of transmission FT-IR spectral data forABTS and ABTS-SNPs with those for the original SNPs ispresented in Figure 3(d) In FT-IR spectra of ABTS-SNPs theobvious new peak observed at sim3400 cmminus1 was due to the N-H group stretching vibrations [30] Other new peaks at about1550 and 825 cmminus1 in the FT-IR spectra of ABTS-SNPs werecharacteristic bands due to the aryl group [31] Additionallynew strong peaks at 1360 and 1150 cmminus1 were attributed to thecharacteristic absorptions of SO

2-N [30]These results clearly

indicate that ABTS was covalently attached to the SNPs

32 Dye Decolorization The ABTS-SNPs were evaluated aslaccase mediators for dye decolorization The comparisonof decolorization efficiency of laccaseABTS-SNPs systemwith those for ABTS-SNPs laccase and laccaseABTS systemis shown in Figure 4 For ABTS-SNPs only sim4 of dyedecolorization respectively was achieved in 15min Thisdecolorization was caused by the adsorption of ABTS-SNPsFor laccase alone a dismal sim3 decolorization was reachedThe low decolorization rate was due to the fact that indigocarmine is not a typical phenol substrate of laccase Theseresults showed that removal of dye by nanoparticles or laccasewas negligible Almost complete decolorization (96) wasachieved by laccase usingABTS as themediator within 5minwhile about 95 decolorization was achieved within 15minusing ABTS-SNPs as mediators These results suggest that

Journal of Chemistry 5

Laccase

ABTS-SNPs

Dye

Ascorbic acid Laccase

O2

H2O Laccase(ox)

ABTS+∙-SNPs

ABTS+∙-SNPs

Dye(ox)

Scheme 2 The proposed catalytic cycle of decolorizing the dye by ABTS-SNPs mediated laccase oxidation

0

50

5 10 15

100

150

Time (min)

Dec

olor

izat

ion

()

ABTS-SNPsLaccase

LaccaseABTSLaccase ABTS-SNPs

Figure 4 Decolorization of indigo carmine (20mgL) by ABTS-SNPs (10 gL containing 70120583M ABTS) laccase (3000UL) laccaseABTS-SNPs and laccaseABTS (70 120583M ABTS) in 50mM sodiumacetate buffer at pH 45 with shaking at 150 rpm at room temper-ature

ABTS immobilized on SNPs can be used as an efficientmediator of laccase for decolorization in a manner similarto free ABTS Even though the decolorization rate is slowerfor ABTS-SNPs than for the free mediator the immobilizedmediator has the advantage of being easily recovered from thereaction system

33 Regeneration and Catalytic Cycle Mechanism of ABTS-SNPs In the case of laccase the role of mediators in anenzymatic oxidation has been already described by severalauthors [7] First ABTS covalently attached to SNPs isconverted into ABTS+∙ cation radical by the enzymaticreaction Subsequently the ABTS+∙ cation radical takes partin nonenzymatic reactions with dyes which are not oxidizableby laccases alone Simultaneously theABTS+∙ is reduced to itsoriginal form (ABTS) by the dye to be oxidized and thus onecatalytic cycle is completed At the end of all catalytic cyclethe dye molecules are completely decolorized by oxidationandABTSmolecules are oxidized to ABTS+∙ by laccase againMoreover ABTS+∙ can be reduced by some strong reducingregents [23] In our case ABTS+∙-SNPs were reduced with

Cycle

Dec

olor

izat

ion

()

0

50

100

150

1 2 3 4 5 6

Figure 5 Reusability of ABTS-SNPs (10 gL) in six successive cyclesusing 3000UL of laccase and 20mgL of indigo carmine at pH 45with shaking at 150 rpm for 30min at room temperature

ascorbic acid resulting in regeneration of ABTS-SNPs asshown in Scheme 2

As shown in Figure 3(c) after the regeneration pro-cess the oxidized ABTS-SNPs turned from green to lightyellowmdashthe original color of the initial ABTS-SNPs Furtherthe regenerated ABTS-SNPs can be oxidized by laccaseagain Decolorization efficiency of regenerated ABTS-SNPsas mediators was 935 which was almost the same asthat of initial ABTS-SNPs (sim95 decolorization) The resultsshow that ABTS-SNPs after use can be recovered and reusedas efficient mediators for decolorization similar to originalABTS-SNPs

34 Reusability From the economic point of view reusabilityof immobilized mediator plays a key role when LMSs willbe used on a large scale for industrial applications [32]The reusability of ABTS-SNPs was investigated for up to6 cycles (Figure 5) When the reaction time was set to30min which proved to be sufficient for almost completedecolorization comparably high decolorization yields wereobtained in 4 subsequent runs using the same recovered andregenerated ABTS-SNPs Approximately 77 decolorizationcould be maintained after 6 cycles A slight decrease indecolorization efficiency was observed which was attributedto partial destruction of ABTS-SNPs during the reaction

6 Journal of Chemistry

andor the loss in quantity of ABTS-SNPs during supernatantdiscarding in the centrifugal recovery process It has beenreported that the radical cation form of ABTS was themain product observed after reacting with laccase but adegradation product from ABTS was also formed [33] Thedecomposition of ABTS during the laccase-ABTS reactionswill be a drawback for the application of laccaseABTS-SNPssystem

4 Conclusion

This investigation has demonstrated that ABTS covalentlylinked to SNPs can be utilized to mediate dye decoloriza-tion by laccase Even though the decolorization efficiencyof ABTS-SNPs as mediators is slower than that of thefree mediators the ABTS-SNPs can easily be recoveredregenerated and reused therefore improving the efficiencyof mediator utilization in LMSs for biotechnological andindustrial applications This process has the potential toalleviate environmental problems caused by the release of freemediators in the wastewater system Moreover a large scaleapplication of reusable and immobilizedmediators will lowerthe cost of dye treatment using the laccase mediator systems

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was kindly supported by the Doctoral Inno-vation Fund of Xinxiang medical University Nature SciencePlan Program (12B180030) from the Education Departmentof Henan Province and theNational Natural Science Founda-tion of China (no U1304302)

References

[1] P J Hoegger S Kilaru T Y James J R Thacker and UKues ldquoPhylogenetic comparison and classification of laccaseand related multicopper oxidase protein sequencesrdquo The FEBSJournal vol 273 no 10 pp 2308ndash2326 2006

[2] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[3] A I Yaropolov O V Skorobogatrsquoko S S Vartanov and S DVarfolomeyev ldquoLaccase properties catalytic mechanism andapplicabilityrdquo Applied Biochemistry and Biotechnology vol 49no 3 pp 257ndash280 1994

[4] A Leonowicz N S Cho J Luterek et al ldquoFungal laccaseproperties and activity on ligninrdquo Journal of Basic Microbiologyvol 41 no 3-4 pp 185ndash227 2001

[5] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[6] R C Minussi G M Pastore and N Duran ldquoPotential applica-tions of laccase in the food industryrdquo Trends in Food Science andTechnology vol 13 no 6-7 pp 205ndash216 2002

[7] O V Morozova G P Shumakovich S V Shleev and Y IYaropolov ldquoLaccase-mediator systems and their applications areviewrdquo Applied Biochemistry and Microbiology vol 43 no 5pp 523ndash535 2007

[8] B Branchi C Galli and P Gentili ldquoKinetics of oxidation ofbenzyl alcohols by the dication and radical cation of ABTSComparison with laccase-ABTS oxidations an apparent para-doxrdquo Organic and Biomolecular Chemistry vol 3 no 14 pp2604ndash2614 2005

[9] C Johannes and AMajcherczyk ldquoNatural mediators in the oxi-dation of polycyclic aromatic hydrocarbons by laccasemediatorsystemsrdquo Applied and Environmental Microbiology vol 66 no2 pp 524ndash528 2000

[10] M Fabbrini C Galli and P Gentili ldquoComparing the catalyticefficiency of some mediators of laccaserdquo Journal of MolecularCatalysis B Enzymatic vol 16 no 5-6 pp 231ndash240 2002

[11] S Camarero D Ibarra M J Martınez and A T MartınezldquoLignin-derived compounds as efficient laccase mediators fordecolorization of different types of recalcitrant dyesrdquo Appliedand Environmental Microbiology vol 71 no 4 pp 1775ndash17842005

[12] F Xu J J Kulys K Duke et al ldquoRedox chemistry in laccase-catalyzed oxidation of N-hydroxy compoundsrdquo Applied andEnvironmentalMicrobiology vol 66 no 5 pp 2052ndash2056 2000

[13] Y Wong and J Yu ldquoLaccase-catalyzed decolorization of syn-thetic dyesrdquoWater Research vol 33 no 16 pp 3512ndash3520 1999

[14] G S Nyanhongo J Gomes G M Gubitz R Zvauya J Readand W Steiner ldquoDecolorization of textile dyes by laccases froma newly isolated strain of Trametes modestardquo Water Researchvol 36 no 6 pp 1449ndash1456 2002

[15] P Brandi A DrsquoAnnibale C Galli P Gentili and A S N PontesldquoIn search for practical advantages from the immobilisation ofan enzyme the case of laccaserdquo Journal of Molecular CatalysisB Enzymatic vol 41 no 1-2 pp 61ndash69 2006

[16] C Garcia-Galan A Berenguer-Murcia R Fernandez-Lafuenteand R C Rodrigues ldquoPotential of different enzyme immobi-lization strategies to improve enzyme performancerdquo AdvancedSynthesis and Catalysis vol 353 no 16 pp 2885ndash2904 2011

[17] J Huang J-T Wang B Li et al ldquoPreparation of organic-inorganic nanoparticles composite and their laccase immobi-lizationrdquo Chemical Journal of Chinese Universities vol 27 no 11pp 2088ndash2091 2006

[18] Y Tan W Deng Y Li et al ldquoPolymeric bionanocompositecast thin films with in situ laccase-catalyzed polymerizationof dopamine for biosensing and biofuel cell applicationsrdquo TheJournal of Physical Chemistry B vol 114 no 15 pp 5016ndash50242010

[19] B Palys A Bokun and J Rogalski ldquoPoly-o-phenylenediamineas redox mediator for laccaserdquo Electrochimica Acta vol 52 no24 pp 7075ndash7082 2007

[20] L Mendoza M Jonstrup R Hatti-Kaul and B MattiassonldquoAzo dye decolorization by a laccasemediator system in amembrane reactor enzyme and mediator reusabilityrdquo Enzymeand Microbial Technology vol 49 no 5 pp 478ndash484 2011

[21] K Rittstieg A Suurnakki T Suortti K Kruus G M Guebitzand J Buchert ldquoPolymerization of guaiacol and a phenolic 120573-O-4-substructure by Trametes hirsuta Laccase in the presenceof ABTSrdquo Biotechnology Progress vol 19 no 5 pp 1505ndash15092003

[22] A M Osman K K YWong and A Fernyhough ldquoThe laccaseABTS system oxidizes (+)-catechin to oligomeric productsrdquo

Journal of Chemistry 7

Enzyme and Microbial Technology vol 40 no 5 pp 1272ndash12792007

[23] P Maruthamuthu L Venkatasubramanian and P Dharma-lingam ldquoA fast kinetic study of formation and decay of221015840-azinobis(3-ethylbenzothiazole-6-sulfonate) radical cationin aqueous solutionrdquo Bulletin of the Chemical Society of Japanvol 60 no 3 pp 1113ndash1117 1987

[24] M Klis J Rogalski and R Bilewicz ldquoVoltammetric determina-tion of catalytic reaction parameters of laccase based on elec-trooxidation of hydroquinone and ABTSrdquo Bioelectrochemistryvol 71 no 1 pp 2ndash7 2007

[25] M Chhabra S Mishra and T R Sreekrishnan ldquoMediator-assisted decolorization and detoxification of textile dyesdyemixture by cyathus bulleri laccaserdquo Applied Biochemistry andBiotechnology vol 151 no 2-3 pp 587ndash598 2008

[26] N Barka A Assabbane A Nounah and Y A Ichou ldquoPhoto-catalytic degradation of indigo carmine in aqueous solution byTiO2-coated non-woven fibresrdquo Journal ofHazardousMaterials

vol 152 no 3 pp 1054ndash1059 2008[27] E-A Cho J Seo D-W Lee and J-G Pan ldquoDecolorization of

indigo carmine by laccase displayed on Bacillus subtilis sporesrdquoEnzyme and Microbial Technology vol 49 no 1 pp 100ndash1042011

[28] K Sadowska K Stolarczyk J F Biernat K P Roberts JRogalski and R Bilewicz ldquoDerivatization of single-walledcarbon nanotubes with redox mediator for biocatalytic oxygenelectrodesrdquo Bioelectrochemistry vol 80 no 1 pp 73ndash80 2010

[29] R Bilewicz E Nazaruk K Zelechowska et al ldquoCarbon nan-otubes chemically derivatized with redox systems as mediatorsfor biofuel cell applicationsrdquo Biocybernetics and BiomedicalEngineering vol 31 no 4 pp 17ndash30 2011

[30] Z Zhong X Ji R Xing et al ldquoThe preparation and antioxidantactivity of the sulfanilamide derivatives of chitosan and chitosansulfatesrdquo Bioorganic and Medicinal Chemistry vol 15 no 11 pp3775ndash3782 2007

[31] T Kataoka T Iwama T Setta et al ldquoPreparation of sulfon-amides from sodium sulfonates Ph

3PBr2and Ph

3PCl2as a

mild halogenating reagent for sulfonyl bromides and sulfonylchloridesrdquo Synthesis vol 1998 no 4 pp 423ndash426 1998

[32] X Zhang B Pan B Wu W Zhang and L Lv ldquoA new polymer-based laccase for decolorization of AO7 long-term storage andmediator reuserdquo Bioresource Technology vol 164 pp 248ndash2532014

[33] A Marjasvaara J Janis and P Vainiotalo ldquoOxidation of alaccase mediator ABTS as studied by ESI-FTICR mass spec-trometryrdquo Journal of Mass Spectrometry vol 43 no 4 pp 470ndash477 2008

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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CatalystsJournal of

2 Journal of Chemistry

O

O

S

NH

OHN

O O

SO

Na+

Na+

minusO

minusO

Figure 1 Molecular structure of indigo carmine

ways of laccase immobilization are presented in the liter-ature [15ndash17] whereas the accounts of immobilization ofmediators are scarce Recently the electrode modified withABTS polymer exhibited electrocatalytic activity towardsdioxygen when immersed in laccase solution [18] Thefungal laccase has previously been successfully entrappedin poly-o-phenylenediamine matrix which can mediatethe enzymatic reaction [19] Covalently attached 2266-tetramethylpiperidine1-oxyl to polyethylene glycol has alsobeen successfully utilized as a laccase mediator for dye decol-orization in amembrane reactor [20] On the one hand smallmolecule mediators noncovalently adsorbed to insolublecarriers lead to mediator leakage in use On the other handpolymers or filmsmodifiedwithmoleculemediators ordinar-ily may result in formidable mass-transfer resistance to thelaccase-catalyzed reactions To circumvent these problemsmediators can be covalently attached to nanosized particlesthat significantly reduce the external resistance to masstransfer Further mediator-modified nanoparticles can berecovered easily for repeated use after effectively mediatingthe reactions catalyzed by enzymes Resulting from the abilityof the dication ABTS2+ to undergo reversible oxidation toABTS+∙ radical ABTS is considered as a one of the mostefficient mediator of laccase [21ndash24] It has been reported thatlaccase with ABTS is able to degrade various environmentalcontaminants [8 25] Indigo carmine (Figure 1) is the mostrepresentative indigoid dye and it is widely used as textilecoloring agent [26] It has been proved that the dye iscarcinogenic and can lead to reproductive developmentalneuron and acute toxicity [27]Thus considering the toxicityof this dye numerous attempts have been made to removeindigo carmine from wastewater [26 27] Because indigocarmine is not a typical substrate of laccase the completedecolorization of this dye by laccase needs the help from amediator Therefore it was chosen as the model recalcitrantcompound for carrying out decolorization reaction in thisstudy

In the present study our goal was to utilize ABTS-mod-ified silica nanoparticles (SNPs) as mediators for a laccase-catalyzed decolorization of indigo carmine dye First ABTSwas covalently attached to SNPs to yield ABTS-immobilizedSNPs (ABTS-SNPs) which were utilized as laccase mediatorsto decolorize indigo carmine dye Secondly these immo-bilized ABTS-SNPs were proved to be effective reusablemediators and easily recovered by centrifugation of thereaction medium

2 Materials and Methods

21 Chemicals ABTS and laccase (catalogue number 38429)were purchased from Sigma-Aldrich USA Indigo carmine(purity ge 96 MW 46635) was obtained from AladdinChina The SNPs (catalogue number XF105 purity gt 99)used in this study weremilky white dispersion and purchasedfrom purchased from Nanjing XFNANOMaterials Tech CoLtd ChinaThe diameter of SNPs ranges between 10 nm and50 nm All chemicals were used as received without furtherpurification Deionized water was used throughout the wholeexperiment

22 Preparation and Characteristics of Silica NanoparticlesModified with ABTS The researchers have recently devel-oped a procedure for the modification of the carbon nan-otubeswith residues of ABTS [28 29] By employing the sameprocedure amino-functionalized SNPs were conjugated withABTS In brief first ABTS (ammonium salt) was convertedinto potassium salt by reaction with potassium tert-butoxideThen disulfonyl bromide was obtained by the reaction ofpotassium salt with triphenylphosphineBr

2 Finally ABTS

was covalently bonded to SNPs by the disulfonyl bromidereacted with amino-functionalized SNPs (Scheme 1) Theresulting samples were centrifuged and washed thoroughlyseveral times with dimethyl sulfoxide methanol and distilledwater respectively until unbounded species were completelyremoved ABTS covalently attached to SNPs were obtainedand resuspended in distilled water The functional groupsof SNPs ABTS-SNPs and ABTS were studied using Fouriertransform infrared (FT-IR) spectroscopy (Bruker OpticsTensor 37) in the range 4000ndash450 cmminus1 at a resolution of2 cmminus1These samples were initially dried in drying oven andthen were ground with KBr respectively The mixture wasmolded into a disc which was analyzed by FT-IR Accordingto elemental analysis there were about 009 S in the ABTS-SNPs this meant that the amount of ABTS attached to SNPswas about 7 120583molg The microstructure of ABTS-SNPs wascharacterized by a JEOL JSM-6700F field emission scanningelectron microscope (SEM)

23 Dye Decolorization The effect of pH on decolorizationof indigo carmine (20mgL dye) by 3000UL laccase and10 120583M ABTS at different pH ranging from 25 to 6 (50mMsodium acetate buffer) was studied Results from the pre-liminary experiments indicated that the optimal pH valuewas about 45 so decolorization experiments were performedusing 20mgL dye 3000UL laccase and mediator (10 gLfor ABTS-SNPs containing 70120583M ABTS or 70 120583M ABTS)at pH 45 After the addition of these samples into 5mLcentrifuge tube the reactionmixturewas incubated in 50mMsodium acetate buffer with gentle shaking at 150 rpm at roomtemperature Controls reactionswere also carried out to studythe decolorization efficiency by laccase without a mediatoror with ABTS or ABTS-SNPs alone The data presented arethe average values of measurements from triplicate technicalrepeats The absorption spectra of ABTS (70120583M ABTS) andindigo carmine (20mgL) in 50mM sodium acetate buffer at

Journal of Chemistry 3

S

NN N

S

S

NO

O

NH

NN N

S

S

N

NN N

SS

N

SNPs ABTS

3-Aminopropyltriethoxysilane

+H4NminusO3S

SO3minusNH4

+

tert-BuOK Ph3PBr2 BrO2S

SO2Br

SiO2

SiO2

SiO2NH2

Et3N

SO3H

+

Scheme 1 Structure of dominating SNP modifications obtained by the route for covalent attachment of ABTS

300 400 500 600 70000

05

10

15

20

Wavelength (nm)

Abso

rban

ce

Indigo carmineABTS

340nm 615nm

Figure 2The absorption spectra ofABTS (70 120583MABTS) and indigocarmine (20mgL) in 50mM sodium acetate buffer at pH 45

pH 45 were shown in Figure 2 which indicated that ABTShas a major ultraviolet light absorption peak at 340 nm andindigo carmine has the absorbance peak at around 615 nmDecolorization was determined spectrophotometrically bymonitoring the decrease in absorbance at the maximumwavelength of dye (around 615 nm) using ShimadzuUV-2550spectrophotometer The percentage decolorization of a dyewas calculated based on the formula conversion () = (119860

0minus

1198601)1198600 where 119860

0was the initial absorbance of the dye at

its adsorption maximum and 1198601was its absorbance of the

reaction mixture at the different reaction time

24 Regeneration of the Oxidized ABTS-SNPs After thedecolorization reaction was complete the oxidized ABTS-SNPs were separated and then transferred into a 5mL cen-trifuge tube containing 35mL distilled water With mixing10 120583L of 1M ascorbic acidwas added After shaking at 150 rpmfor 5min the mixture was centrifuged for 2min at 10000timesgThe regenerated ABTS-SNPs were separated and washedthrice with 3mL distilled water again

25 Reusability of ABTS-SNPs The recyclability of ABTS-SNPs was assessed in a discontinuous decolorization reac-tion Decolorization of indigo carmine (20mgL) were per-formed in a 5mL centrifuge tube containing 3000UL oflaccase and 10 gL ABTS-SNPs at pH 45 shaking at 150 rpmfor 30min at room temperature At the end of every cycleABTS-SNPs were separated regenerated and washed thricewith 3mL distilled water Next the ABTS-SNPs was addedinto a fresh reaction solution (20mgL indigo carmine3000UL laccase pH 45) to start a fresh cycle The datapresented are the average values of measurements fromtriplicate technical repeats

3 Results and Discussion

31 Structure Biochemical and Physicochemical Characteris-tics of ABTS-SNPs The chemical structure of ABTS-SNPsis presented in Scheme 1 Morphology of SNPs and ABTS-SNPs as obtained by SEM is shown in Figures 3(a) and 3(b)The particles obtained after covalent attachment of ABTSon SNPs were found to be nanosized (lt50 nm) OriginalSNPs were white but the SNPs covalently attached to ABTSexhibited light yellow color (Figure 3(c)) After ABTS-SNPs

4 Journal of Chemistry

(a) (b)

1 2 3 4 5

(c)

01000200030004000

ABTS

SNPsABTS-SNPs

Wavenumber (cmminus1)

(d)

Figure 3 (a) SEM image of SNPs (b) SEM image of ABTS-SNPs (c) Tube 1 SNPs Tube 2 SNPs covalently attached to ABTS Tube 3 afterreaction of ABTS-SNPs with laccase Tube 4 regenerated ABTS-SNPs Tube 5 after reaction of regenerated ABTS-SNPs with laccase and (d)FT-IR spectra of ABTS ABTS-SNPs and SNPs

weremixedwith laccase for a fewminutes themixture turnedfrom light yellow to green The color change in the reactionwas attributed to the fact that ABTS molecules attached onthe nanoparticle surface were oxidized to green ABTS+∙ bylaccase

The comparison of transmission FT-IR spectral data forABTS and ABTS-SNPs with those for the original SNPs ispresented in Figure 3(d) In FT-IR spectra of ABTS-SNPs theobvious new peak observed at sim3400 cmminus1 was due to the N-H group stretching vibrations [30] Other new peaks at about1550 and 825 cmminus1 in the FT-IR spectra of ABTS-SNPs werecharacteristic bands due to the aryl group [31] Additionallynew strong peaks at 1360 and 1150 cmminus1 were attributed to thecharacteristic absorptions of SO

2-N [30]These results clearly

indicate that ABTS was covalently attached to the SNPs

32 Dye Decolorization The ABTS-SNPs were evaluated aslaccase mediators for dye decolorization The comparisonof decolorization efficiency of laccaseABTS-SNPs systemwith those for ABTS-SNPs laccase and laccaseABTS systemis shown in Figure 4 For ABTS-SNPs only sim4 of dyedecolorization respectively was achieved in 15min Thisdecolorization was caused by the adsorption of ABTS-SNPsFor laccase alone a dismal sim3 decolorization was reachedThe low decolorization rate was due to the fact that indigocarmine is not a typical phenol substrate of laccase Theseresults showed that removal of dye by nanoparticles or laccasewas negligible Almost complete decolorization (96) wasachieved by laccase usingABTS as themediator within 5minwhile about 95 decolorization was achieved within 15minusing ABTS-SNPs as mediators These results suggest that

Journal of Chemistry 5

Laccase

ABTS-SNPs

Dye

Ascorbic acid Laccase

O2

H2O Laccase(ox)

ABTS+∙-SNPs

ABTS+∙-SNPs

Dye(ox)

Scheme 2 The proposed catalytic cycle of decolorizing the dye by ABTS-SNPs mediated laccase oxidation

0

50

5 10 15

100

150

Time (min)

Dec

olor

izat

ion

()

ABTS-SNPsLaccase

LaccaseABTSLaccase ABTS-SNPs

Figure 4 Decolorization of indigo carmine (20mgL) by ABTS-SNPs (10 gL containing 70120583M ABTS) laccase (3000UL) laccaseABTS-SNPs and laccaseABTS (70 120583M ABTS) in 50mM sodiumacetate buffer at pH 45 with shaking at 150 rpm at room temper-ature

ABTS immobilized on SNPs can be used as an efficientmediator of laccase for decolorization in a manner similarto free ABTS Even though the decolorization rate is slowerfor ABTS-SNPs than for the free mediator the immobilizedmediator has the advantage of being easily recovered from thereaction system

33 Regeneration and Catalytic Cycle Mechanism of ABTS-SNPs In the case of laccase the role of mediators in anenzymatic oxidation has been already described by severalauthors [7] First ABTS covalently attached to SNPs isconverted into ABTS+∙ cation radical by the enzymaticreaction Subsequently the ABTS+∙ cation radical takes partin nonenzymatic reactions with dyes which are not oxidizableby laccases alone Simultaneously theABTS+∙ is reduced to itsoriginal form (ABTS) by the dye to be oxidized and thus onecatalytic cycle is completed At the end of all catalytic cyclethe dye molecules are completely decolorized by oxidationandABTSmolecules are oxidized to ABTS+∙ by laccase againMoreover ABTS+∙ can be reduced by some strong reducingregents [23] In our case ABTS+∙-SNPs were reduced with

Cycle

Dec

olor

izat

ion

()

0

50

100

150

1 2 3 4 5 6

Figure 5 Reusability of ABTS-SNPs (10 gL) in six successive cyclesusing 3000UL of laccase and 20mgL of indigo carmine at pH 45with shaking at 150 rpm for 30min at room temperature

ascorbic acid resulting in regeneration of ABTS-SNPs asshown in Scheme 2

As shown in Figure 3(c) after the regeneration pro-cess the oxidized ABTS-SNPs turned from green to lightyellowmdashthe original color of the initial ABTS-SNPs Furtherthe regenerated ABTS-SNPs can be oxidized by laccaseagain Decolorization efficiency of regenerated ABTS-SNPsas mediators was 935 which was almost the same asthat of initial ABTS-SNPs (sim95 decolorization) The resultsshow that ABTS-SNPs after use can be recovered and reusedas efficient mediators for decolorization similar to originalABTS-SNPs

34 Reusability From the economic point of view reusabilityof immobilized mediator plays a key role when LMSs willbe used on a large scale for industrial applications [32]The reusability of ABTS-SNPs was investigated for up to6 cycles (Figure 5) When the reaction time was set to30min which proved to be sufficient for almost completedecolorization comparably high decolorization yields wereobtained in 4 subsequent runs using the same recovered andregenerated ABTS-SNPs Approximately 77 decolorizationcould be maintained after 6 cycles A slight decrease indecolorization efficiency was observed which was attributedto partial destruction of ABTS-SNPs during the reaction

6 Journal of Chemistry

andor the loss in quantity of ABTS-SNPs during supernatantdiscarding in the centrifugal recovery process It has beenreported that the radical cation form of ABTS was themain product observed after reacting with laccase but adegradation product from ABTS was also formed [33] Thedecomposition of ABTS during the laccase-ABTS reactionswill be a drawback for the application of laccaseABTS-SNPssystem

4 Conclusion

This investigation has demonstrated that ABTS covalentlylinked to SNPs can be utilized to mediate dye decoloriza-tion by laccase Even though the decolorization efficiencyof ABTS-SNPs as mediators is slower than that of thefree mediators the ABTS-SNPs can easily be recoveredregenerated and reused therefore improving the efficiencyof mediator utilization in LMSs for biotechnological andindustrial applications This process has the potential toalleviate environmental problems caused by the release of freemediators in the wastewater system Moreover a large scaleapplication of reusable and immobilizedmediators will lowerthe cost of dye treatment using the laccase mediator systems

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was kindly supported by the Doctoral Inno-vation Fund of Xinxiang medical University Nature SciencePlan Program (12B180030) from the Education Departmentof Henan Province and theNational Natural Science Founda-tion of China (no U1304302)

References

[1] P J Hoegger S Kilaru T Y James J R Thacker and UKues ldquoPhylogenetic comparison and classification of laccaseand related multicopper oxidase protein sequencesrdquo The FEBSJournal vol 273 no 10 pp 2308ndash2326 2006

[2] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[3] A I Yaropolov O V Skorobogatrsquoko S S Vartanov and S DVarfolomeyev ldquoLaccase properties catalytic mechanism andapplicabilityrdquo Applied Biochemistry and Biotechnology vol 49no 3 pp 257ndash280 1994

[4] A Leonowicz N S Cho J Luterek et al ldquoFungal laccaseproperties and activity on ligninrdquo Journal of Basic Microbiologyvol 41 no 3-4 pp 185ndash227 2001

[5] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[6] R C Minussi G M Pastore and N Duran ldquoPotential applica-tions of laccase in the food industryrdquo Trends in Food Science andTechnology vol 13 no 6-7 pp 205ndash216 2002

[7] O V Morozova G P Shumakovich S V Shleev and Y IYaropolov ldquoLaccase-mediator systems and their applications areviewrdquo Applied Biochemistry and Microbiology vol 43 no 5pp 523ndash535 2007

[8] B Branchi C Galli and P Gentili ldquoKinetics of oxidation ofbenzyl alcohols by the dication and radical cation of ABTSComparison with laccase-ABTS oxidations an apparent para-doxrdquo Organic and Biomolecular Chemistry vol 3 no 14 pp2604ndash2614 2005

[9] C Johannes and AMajcherczyk ldquoNatural mediators in the oxi-dation of polycyclic aromatic hydrocarbons by laccasemediatorsystemsrdquo Applied and Environmental Microbiology vol 66 no2 pp 524ndash528 2000

[10] M Fabbrini C Galli and P Gentili ldquoComparing the catalyticefficiency of some mediators of laccaserdquo Journal of MolecularCatalysis B Enzymatic vol 16 no 5-6 pp 231ndash240 2002

[11] S Camarero D Ibarra M J Martınez and A T MartınezldquoLignin-derived compounds as efficient laccase mediators fordecolorization of different types of recalcitrant dyesrdquo Appliedand Environmental Microbiology vol 71 no 4 pp 1775ndash17842005

[12] F Xu J J Kulys K Duke et al ldquoRedox chemistry in laccase-catalyzed oxidation of N-hydroxy compoundsrdquo Applied andEnvironmentalMicrobiology vol 66 no 5 pp 2052ndash2056 2000

[13] Y Wong and J Yu ldquoLaccase-catalyzed decolorization of syn-thetic dyesrdquoWater Research vol 33 no 16 pp 3512ndash3520 1999

[14] G S Nyanhongo J Gomes G M Gubitz R Zvauya J Readand W Steiner ldquoDecolorization of textile dyes by laccases froma newly isolated strain of Trametes modestardquo Water Researchvol 36 no 6 pp 1449ndash1456 2002

[15] P Brandi A DrsquoAnnibale C Galli P Gentili and A S N PontesldquoIn search for practical advantages from the immobilisation ofan enzyme the case of laccaserdquo Journal of Molecular CatalysisB Enzymatic vol 41 no 1-2 pp 61ndash69 2006

[16] C Garcia-Galan A Berenguer-Murcia R Fernandez-Lafuenteand R C Rodrigues ldquoPotential of different enzyme immobi-lization strategies to improve enzyme performancerdquo AdvancedSynthesis and Catalysis vol 353 no 16 pp 2885ndash2904 2011

[17] J Huang J-T Wang B Li et al ldquoPreparation of organic-inorganic nanoparticles composite and their laccase immobi-lizationrdquo Chemical Journal of Chinese Universities vol 27 no 11pp 2088ndash2091 2006

[18] Y Tan W Deng Y Li et al ldquoPolymeric bionanocompositecast thin films with in situ laccase-catalyzed polymerizationof dopamine for biosensing and biofuel cell applicationsrdquo TheJournal of Physical Chemistry B vol 114 no 15 pp 5016ndash50242010

[19] B Palys A Bokun and J Rogalski ldquoPoly-o-phenylenediamineas redox mediator for laccaserdquo Electrochimica Acta vol 52 no24 pp 7075ndash7082 2007

[20] L Mendoza M Jonstrup R Hatti-Kaul and B MattiassonldquoAzo dye decolorization by a laccasemediator system in amembrane reactor enzyme and mediator reusabilityrdquo Enzymeand Microbial Technology vol 49 no 5 pp 478ndash484 2011

[21] K Rittstieg A Suurnakki T Suortti K Kruus G M Guebitzand J Buchert ldquoPolymerization of guaiacol and a phenolic 120573-O-4-substructure by Trametes hirsuta Laccase in the presenceof ABTSrdquo Biotechnology Progress vol 19 no 5 pp 1505ndash15092003

[22] A M Osman K K YWong and A Fernyhough ldquoThe laccaseABTS system oxidizes (+)-catechin to oligomeric productsrdquo

Journal of Chemistry 7

Enzyme and Microbial Technology vol 40 no 5 pp 1272ndash12792007

[23] P Maruthamuthu L Venkatasubramanian and P Dharma-lingam ldquoA fast kinetic study of formation and decay of221015840-azinobis(3-ethylbenzothiazole-6-sulfonate) radical cationin aqueous solutionrdquo Bulletin of the Chemical Society of Japanvol 60 no 3 pp 1113ndash1117 1987

[24] M Klis J Rogalski and R Bilewicz ldquoVoltammetric determina-tion of catalytic reaction parameters of laccase based on elec-trooxidation of hydroquinone and ABTSrdquo Bioelectrochemistryvol 71 no 1 pp 2ndash7 2007

[25] M Chhabra S Mishra and T R Sreekrishnan ldquoMediator-assisted decolorization and detoxification of textile dyesdyemixture by cyathus bulleri laccaserdquo Applied Biochemistry andBiotechnology vol 151 no 2-3 pp 587ndash598 2008

[26] N Barka A Assabbane A Nounah and Y A Ichou ldquoPhoto-catalytic degradation of indigo carmine in aqueous solution byTiO2-coated non-woven fibresrdquo Journal ofHazardousMaterials

vol 152 no 3 pp 1054ndash1059 2008[27] E-A Cho J Seo D-W Lee and J-G Pan ldquoDecolorization of

indigo carmine by laccase displayed on Bacillus subtilis sporesrdquoEnzyme and Microbial Technology vol 49 no 1 pp 100ndash1042011

[28] K Sadowska K Stolarczyk J F Biernat K P Roberts JRogalski and R Bilewicz ldquoDerivatization of single-walledcarbon nanotubes with redox mediator for biocatalytic oxygenelectrodesrdquo Bioelectrochemistry vol 80 no 1 pp 73ndash80 2010

[29] R Bilewicz E Nazaruk K Zelechowska et al ldquoCarbon nan-otubes chemically derivatized with redox systems as mediatorsfor biofuel cell applicationsrdquo Biocybernetics and BiomedicalEngineering vol 31 no 4 pp 17ndash30 2011

[30] Z Zhong X Ji R Xing et al ldquoThe preparation and antioxidantactivity of the sulfanilamide derivatives of chitosan and chitosansulfatesrdquo Bioorganic and Medicinal Chemistry vol 15 no 11 pp3775ndash3782 2007

[31] T Kataoka T Iwama T Setta et al ldquoPreparation of sulfon-amides from sodium sulfonates Ph

3PBr2and Ph

3PCl2as a

mild halogenating reagent for sulfonyl bromides and sulfonylchloridesrdquo Synthesis vol 1998 no 4 pp 423ndash426 1998

[32] X Zhang B Pan B Wu W Zhang and L Lv ldquoA new polymer-based laccase for decolorization of AO7 long-term storage andmediator reuserdquo Bioresource Technology vol 164 pp 248ndash2532014

[33] A Marjasvaara J Janis and P Vainiotalo ldquoOxidation of alaccase mediator ABTS as studied by ESI-FTICR mass spec-trometryrdquo Journal of Mass Spectrometry vol 43 no 4 pp 470ndash477 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 3

S

NN N

S

S

NO

O

NH

NN N

S

S

N

NN N

SS

N

SNPs ABTS

3-Aminopropyltriethoxysilane

+H4NminusO3S

SO3minusNH4

+

tert-BuOK Ph3PBr2 BrO2S

SO2Br

SiO2

SiO2

SiO2NH2

Et3N

SO3H

+

Scheme 1 Structure of dominating SNP modifications obtained by the route for covalent attachment of ABTS

300 400 500 600 70000

05

10

15

20

Wavelength (nm)

Abso

rban

ce

Indigo carmineABTS

340nm 615nm

Figure 2The absorption spectra ofABTS (70 120583MABTS) and indigocarmine (20mgL) in 50mM sodium acetate buffer at pH 45

pH 45 were shown in Figure 2 which indicated that ABTShas a major ultraviolet light absorption peak at 340 nm andindigo carmine has the absorbance peak at around 615 nmDecolorization was determined spectrophotometrically bymonitoring the decrease in absorbance at the maximumwavelength of dye (around 615 nm) using ShimadzuUV-2550spectrophotometer The percentage decolorization of a dyewas calculated based on the formula conversion () = (119860

0minus

1198601)1198600 where 119860

0was the initial absorbance of the dye at

its adsorption maximum and 1198601was its absorbance of the

reaction mixture at the different reaction time

24 Regeneration of the Oxidized ABTS-SNPs After thedecolorization reaction was complete the oxidized ABTS-SNPs were separated and then transferred into a 5mL cen-trifuge tube containing 35mL distilled water With mixing10 120583L of 1M ascorbic acidwas added After shaking at 150 rpmfor 5min the mixture was centrifuged for 2min at 10000timesgThe regenerated ABTS-SNPs were separated and washedthrice with 3mL distilled water again

25 Reusability of ABTS-SNPs The recyclability of ABTS-SNPs was assessed in a discontinuous decolorization reac-tion Decolorization of indigo carmine (20mgL) were per-formed in a 5mL centrifuge tube containing 3000UL oflaccase and 10 gL ABTS-SNPs at pH 45 shaking at 150 rpmfor 30min at room temperature At the end of every cycleABTS-SNPs were separated regenerated and washed thricewith 3mL distilled water Next the ABTS-SNPs was addedinto a fresh reaction solution (20mgL indigo carmine3000UL laccase pH 45) to start a fresh cycle The datapresented are the average values of measurements fromtriplicate technical repeats

3 Results and Discussion

31 Structure Biochemical and Physicochemical Characteris-tics of ABTS-SNPs The chemical structure of ABTS-SNPsis presented in Scheme 1 Morphology of SNPs and ABTS-SNPs as obtained by SEM is shown in Figures 3(a) and 3(b)The particles obtained after covalent attachment of ABTSon SNPs were found to be nanosized (lt50 nm) OriginalSNPs were white but the SNPs covalently attached to ABTSexhibited light yellow color (Figure 3(c)) After ABTS-SNPs

4 Journal of Chemistry

(a) (b)

1 2 3 4 5

(c)

01000200030004000

ABTS

SNPsABTS-SNPs

Wavenumber (cmminus1)

(d)

Figure 3 (a) SEM image of SNPs (b) SEM image of ABTS-SNPs (c) Tube 1 SNPs Tube 2 SNPs covalently attached to ABTS Tube 3 afterreaction of ABTS-SNPs with laccase Tube 4 regenerated ABTS-SNPs Tube 5 after reaction of regenerated ABTS-SNPs with laccase and (d)FT-IR spectra of ABTS ABTS-SNPs and SNPs

weremixedwith laccase for a fewminutes themixture turnedfrom light yellow to green The color change in the reactionwas attributed to the fact that ABTS molecules attached onthe nanoparticle surface were oxidized to green ABTS+∙ bylaccase

The comparison of transmission FT-IR spectral data forABTS and ABTS-SNPs with those for the original SNPs ispresented in Figure 3(d) In FT-IR spectra of ABTS-SNPs theobvious new peak observed at sim3400 cmminus1 was due to the N-H group stretching vibrations [30] Other new peaks at about1550 and 825 cmminus1 in the FT-IR spectra of ABTS-SNPs werecharacteristic bands due to the aryl group [31] Additionallynew strong peaks at 1360 and 1150 cmminus1 were attributed to thecharacteristic absorptions of SO

2-N [30]These results clearly

indicate that ABTS was covalently attached to the SNPs

32 Dye Decolorization The ABTS-SNPs were evaluated aslaccase mediators for dye decolorization The comparisonof decolorization efficiency of laccaseABTS-SNPs systemwith those for ABTS-SNPs laccase and laccaseABTS systemis shown in Figure 4 For ABTS-SNPs only sim4 of dyedecolorization respectively was achieved in 15min Thisdecolorization was caused by the adsorption of ABTS-SNPsFor laccase alone a dismal sim3 decolorization was reachedThe low decolorization rate was due to the fact that indigocarmine is not a typical phenol substrate of laccase Theseresults showed that removal of dye by nanoparticles or laccasewas negligible Almost complete decolorization (96) wasachieved by laccase usingABTS as themediator within 5minwhile about 95 decolorization was achieved within 15minusing ABTS-SNPs as mediators These results suggest that

Journal of Chemistry 5

Laccase

ABTS-SNPs

Dye

Ascorbic acid Laccase

O2

H2O Laccase(ox)

ABTS+∙-SNPs

ABTS+∙-SNPs

Dye(ox)

Scheme 2 The proposed catalytic cycle of decolorizing the dye by ABTS-SNPs mediated laccase oxidation

0

50

5 10 15

100

150

Time (min)

Dec

olor

izat

ion

()

ABTS-SNPsLaccase

LaccaseABTSLaccase ABTS-SNPs

Figure 4 Decolorization of indigo carmine (20mgL) by ABTS-SNPs (10 gL containing 70120583M ABTS) laccase (3000UL) laccaseABTS-SNPs and laccaseABTS (70 120583M ABTS) in 50mM sodiumacetate buffer at pH 45 with shaking at 150 rpm at room temper-ature

ABTS immobilized on SNPs can be used as an efficientmediator of laccase for decolorization in a manner similarto free ABTS Even though the decolorization rate is slowerfor ABTS-SNPs than for the free mediator the immobilizedmediator has the advantage of being easily recovered from thereaction system

33 Regeneration and Catalytic Cycle Mechanism of ABTS-SNPs In the case of laccase the role of mediators in anenzymatic oxidation has been already described by severalauthors [7] First ABTS covalently attached to SNPs isconverted into ABTS+∙ cation radical by the enzymaticreaction Subsequently the ABTS+∙ cation radical takes partin nonenzymatic reactions with dyes which are not oxidizableby laccases alone Simultaneously theABTS+∙ is reduced to itsoriginal form (ABTS) by the dye to be oxidized and thus onecatalytic cycle is completed At the end of all catalytic cyclethe dye molecules are completely decolorized by oxidationandABTSmolecules are oxidized to ABTS+∙ by laccase againMoreover ABTS+∙ can be reduced by some strong reducingregents [23] In our case ABTS+∙-SNPs were reduced with

Cycle

Dec

olor

izat

ion

()

0

50

100

150

1 2 3 4 5 6

Figure 5 Reusability of ABTS-SNPs (10 gL) in six successive cyclesusing 3000UL of laccase and 20mgL of indigo carmine at pH 45with shaking at 150 rpm for 30min at room temperature

ascorbic acid resulting in regeneration of ABTS-SNPs asshown in Scheme 2

As shown in Figure 3(c) after the regeneration pro-cess the oxidized ABTS-SNPs turned from green to lightyellowmdashthe original color of the initial ABTS-SNPs Furtherthe regenerated ABTS-SNPs can be oxidized by laccaseagain Decolorization efficiency of regenerated ABTS-SNPsas mediators was 935 which was almost the same asthat of initial ABTS-SNPs (sim95 decolorization) The resultsshow that ABTS-SNPs after use can be recovered and reusedas efficient mediators for decolorization similar to originalABTS-SNPs

34 Reusability From the economic point of view reusabilityof immobilized mediator plays a key role when LMSs willbe used on a large scale for industrial applications [32]The reusability of ABTS-SNPs was investigated for up to6 cycles (Figure 5) When the reaction time was set to30min which proved to be sufficient for almost completedecolorization comparably high decolorization yields wereobtained in 4 subsequent runs using the same recovered andregenerated ABTS-SNPs Approximately 77 decolorizationcould be maintained after 6 cycles A slight decrease indecolorization efficiency was observed which was attributedto partial destruction of ABTS-SNPs during the reaction

6 Journal of Chemistry

andor the loss in quantity of ABTS-SNPs during supernatantdiscarding in the centrifugal recovery process It has beenreported that the radical cation form of ABTS was themain product observed after reacting with laccase but adegradation product from ABTS was also formed [33] Thedecomposition of ABTS during the laccase-ABTS reactionswill be a drawback for the application of laccaseABTS-SNPssystem

4 Conclusion

This investigation has demonstrated that ABTS covalentlylinked to SNPs can be utilized to mediate dye decoloriza-tion by laccase Even though the decolorization efficiencyof ABTS-SNPs as mediators is slower than that of thefree mediators the ABTS-SNPs can easily be recoveredregenerated and reused therefore improving the efficiencyof mediator utilization in LMSs for biotechnological andindustrial applications This process has the potential toalleviate environmental problems caused by the release of freemediators in the wastewater system Moreover a large scaleapplication of reusable and immobilizedmediators will lowerthe cost of dye treatment using the laccase mediator systems

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was kindly supported by the Doctoral Inno-vation Fund of Xinxiang medical University Nature SciencePlan Program (12B180030) from the Education Departmentof Henan Province and theNational Natural Science Founda-tion of China (no U1304302)

References

[1] P J Hoegger S Kilaru T Y James J R Thacker and UKues ldquoPhylogenetic comparison and classification of laccaseand related multicopper oxidase protein sequencesrdquo The FEBSJournal vol 273 no 10 pp 2308ndash2326 2006

[2] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[3] A I Yaropolov O V Skorobogatrsquoko S S Vartanov and S DVarfolomeyev ldquoLaccase properties catalytic mechanism andapplicabilityrdquo Applied Biochemistry and Biotechnology vol 49no 3 pp 257ndash280 1994

[4] A Leonowicz N S Cho J Luterek et al ldquoFungal laccaseproperties and activity on ligninrdquo Journal of Basic Microbiologyvol 41 no 3-4 pp 185ndash227 2001

[5] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[6] R C Minussi G M Pastore and N Duran ldquoPotential applica-tions of laccase in the food industryrdquo Trends in Food Science andTechnology vol 13 no 6-7 pp 205ndash216 2002

[7] O V Morozova G P Shumakovich S V Shleev and Y IYaropolov ldquoLaccase-mediator systems and their applications areviewrdquo Applied Biochemistry and Microbiology vol 43 no 5pp 523ndash535 2007

[8] B Branchi C Galli and P Gentili ldquoKinetics of oxidation ofbenzyl alcohols by the dication and radical cation of ABTSComparison with laccase-ABTS oxidations an apparent para-doxrdquo Organic and Biomolecular Chemistry vol 3 no 14 pp2604ndash2614 2005

[9] C Johannes and AMajcherczyk ldquoNatural mediators in the oxi-dation of polycyclic aromatic hydrocarbons by laccasemediatorsystemsrdquo Applied and Environmental Microbiology vol 66 no2 pp 524ndash528 2000

[10] M Fabbrini C Galli and P Gentili ldquoComparing the catalyticefficiency of some mediators of laccaserdquo Journal of MolecularCatalysis B Enzymatic vol 16 no 5-6 pp 231ndash240 2002

[11] S Camarero D Ibarra M J Martınez and A T MartınezldquoLignin-derived compounds as efficient laccase mediators fordecolorization of different types of recalcitrant dyesrdquo Appliedand Environmental Microbiology vol 71 no 4 pp 1775ndash17842005

[12] F Xu J J Kulys K Duke et al ldquoRedox chemistry in laccase-catalyzed oxidation of N-hydroxy compoundsrdquo Applied andEnvironmentalMicrobiology vol 66 no 5 pp 2052ndash2056 2000

[13] Y Wong and J Yu ldquoLaccase-catalyzed decolorization of syn-thetic dyesrdquoWater Research vol 33 no 16 pp 3512ndash3520 1999

[14] G S Nyanhongo J Gomes G M Gubitz R Zvauya J Readand W Steiner ldquoDecolorization of textile dyes by laccases froma newly isolated strain of Trametes modestardquo Water Researchvol 36 no 6 pp 1449ndash1456 2002

[15] P Brandi A DrsquoAnnibale C Galli P Gentili and A S N PontesldquoIn search for practical advantages from the immobilisation ofan enzyme the case of laccaserdquo Journal of Molecular CatalysisB Enzymatic vol 41 no 1-2 pp 61ndash69 2006

[16] C Garcia-Galan A Berenguer-Murcia R Fernandez-Lafuenteand R C Rodrigues ldquoPotential of different enzyme immobi-lization strategies to improve enzyme performancerdquo AdvancedSynthesis and Catalysis vol 353 no 16 pp 2885ndash2904 2011

[17] J Huang J-T Wang B Li et al ldquoPreparation of organic-inorganic nanoparticles composite and their laccase immobi-lizationrdquo Chemical Journal of Chinese Universities vol 27 no 11pp 2088ndash2091 2006

[18] Y Tan W Deng Y Li et al ldquoPolymeric bionanocompositecast thin films with in situ laccase-catalyzed polymerizationof dopamine for biosensing and biofuel cell applicationsrdquo TheJournal of Physical Chemistry B vol 114 no 15 pp 5016ndash50242010

[19] B Palys A Bokun and J Rogalski ldquoPoly-o-phenylenediamineas redox mediator for laccaserdquo Electrochimica Acta vol 52 no24 pp 7075ndash7082 2007

[20] L Mendoza M Jonstrup R Hatti-Kaul and B MattiassonldquoAzo dye decolorization by a laccasemediator system in amembrane reactor enzyme and mediator reusabilityrdquo Enzymeand Microbial Technology vol 49 no 5 pp 478ndash484 2011

[21] K Rittstieg A Suurnakki T Suortti K Kruus G M Guebitzand J Buchert ldquoPolymerization of guaiacol and a phenolic 120573-O-4-substructure by Trametes hirsuta Laccase in the presenceof ABTSrdquo Biotechnology Progress vol 19 no 5 pp 1505ndash15092003

[22] A M Osman K K YWong and A Fernyhough ldquoThe laccaseABTS system oxidizes (+)-catechin to oligomeric productsrdquo

Journal of Chemistry 7

Enzyme and Microbial Technology vol 40 no 5 pp 1272ndash12792007

[23] P Maruthamuthu L Venkatasubramanian and P Dharma-lingam ldquoA fast kinetic study of formation and decay of221015840-azinobis(3-ethylbenzothiazole-6-sulfonate) radical cationin aqueous solutionrdquo Bulletin of the Chemical Society of Japanvol 60 no 3 pp 1113ndash1117 1987

[24] M Klis J Rogalski and R Bilewicz ldquoVoltammetric determina-tion of catalytic reaction parameters of laccase based on elec-trooxidation of hydroquinone and ABTSrdquo Bioelectrochemistryvol 71 no 1 pp 2ndash7 2007

[25] M Chhabra S Mishra and T R Sreekrishnan ldquoMediator-assisted decolorization and detoxification of textile dyesdyemixture by cyathus bulleri laccaserdquo Applied Biochemistry andBiotechnology vol 151 no 2-3 pp 587ndash598 2008

[26] N Barka A Assabbane A Nounah and Y A Ichou ldquoPhoto-catalytic degradation of indigo carmine in aqueous solution byTiO2-coated non-woven fibresrdquo Journal ofHazardousMaterials

vol 152 no 3 pp 1054ndash1059 2008[27] E-A Cho J Seo D-W Lee and J-G Pan ldquoDecolorization of

indigo carmine by laccase displayed on Bacillus subtilis sporesrdquoEnzyme and Microbial Technology vol 49 no 1 pp 100ndash1042011

[28] K Sadowska K Stolarczyk J F Biernat K P Roberts JRogalski and R Bilewicz ldquoDerivatization of single-walledcarbon nanotubes with redox mediator for biocatalytic oxygenelectrodesrdquo Bioelectrochemistry vol 80 no 1 pp 73ndash80 2010

[29] R Bilewicz E Nazaruk K Zelechowska et al ldquoCarbon nan-otubes chemically derivatized with redox systems as mediatorsfor biofuel cell applicationsrdquo Biocybernetics and BiomedicalEngineering vol 31 no 4 pp 17ndash30 2011

[30] Z Zhong X Ji R Xing et al ldquoThe preparation and antioxidantactivity of the sulfanilamide derivatives of chitosan and chitosansulfatesrdquo Bioorganic and Medicinal Chemistry vol 15 no 11 pp3775ndash3782 2007

[31] T Kataoka T Iwama T Setta et al ldquoPreparation of sulfon-amides from sodium sulfonates Ph

3PBr2and Ph

3PCl2as a

mild halogenating reagent for sulfonyl bromides and sulfonylchloridesrdquo Synthesis vol 1998 no 4 pp 423ndash426 1998

[32] X Zhang B Pan B Wu W Zhang and L Lv ldquoA new polymer-based laccase for decolorization of AO7 long-term storage andmediator reuserdquo Bioresource Technology vol 164 pp 248ndash2532014

[33] A Marjasvaara J Janis and P Vainiotalo ldquoOxidation of alaccase mediator ABTS as studied by ESI-FTICR mass spec-trometryrdquo Journal of Mass Spectrometry vol 43 no 4 pp 470ndash477 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 Journal of Chemistry

(a) (b)

1 2 3 4 5

(c)

01000200030004000

ABTS

SNPsABTS-SNPs

Wavenumber (cmminus1)

(d)

Figure 3 (a) SEM image of SNPs (b) SEM image of ABTS-SNPs (c) Tube 1 SNPs Tube 2 SNPs covalently attached to ABTS Tube 3 afterreaction of ABTS-SNPs with laccase Tube 4 regenerated ABTS-SNPs Tube 5 after reaction of regenerated ABTS-SNPs with laccase and (d)FT-IR spectra of ABTS ABTS-SNPs and SNPs

weremixedwith laccase for a fewminutes themixture turnedfrom light yellow to green The color change in the reactionwas attributed to the fact that ABTS molecules attached onthe nanoparticle surface were oxidized to green ABTS+∙ bylaccase

The comparison of transmission FT-IR spectral data forABTS and ABTS-SNPs with those for the original SNPs ispresented in Figure 3(d) In FT-IR spectra of ABTS-SNPs theobvious new peak observed at sim3400 cmminus1 was due to the N-H group stretching vibrations [30] Other new peaks at about1550 and 825 cmminus1 in the FT-IR spectra of ABTS-SNPs werecharacteristic bands due to the aryl group [31] Additionallynew strong peaks at 1360 and 1150 cmminus1 were attributed to thecharacteristic absorptions of SO

2-N [30]These results clearly

indicate that ABTS was covalently attached to the SNPs

32 Dye Decolorization The ABTS-SNPs were evaluated aslaccase mediators for dye decolorization The comparisonof decolorization efficiency of laccaseABTS-SNPs systemwith those for ABTS-SNPs laccase and laccaseABTS systemis shown in Figure 4 For ABTS-SNPs only sim4 of dyedecolorization respectively was achieved in 15min Thisdecolorization was caused by the adsorption of ABTS-SNPsFor laccase alone a dismal sim3 decolorization was reachedThe low decolorization rate was due to the fact that indigocarmine is not a typical phenol substrate of laccase Theseresults showed that removal of dye by nanoparticles or laccasewas negligible Almost complete decolorization (96) wasachieved by laccase usingABTS as themediator within 5minwhile about 95 decolorization was achieved within 15minusing ABTS-SNPs as mediators These results suggest that

Journal of Chemistry 5

Laccase

ABTS-SNPs

Dye

Ascorbic acid Laccase

O2

H2O Laccase(ox)

ABTS+∙-SNPs

ABTS+∙-SNPs

Dye(ox)

Scheme 2 The proposed catalytic cycle of decolorizing the dye by ABTS-SNPs mediated laccase oxidation

0

50

5 10 15

100

150

Time (min)

Dec

olor

izat

ion

()

ABTS-SNPsLaccase

LaccaseABTSLaccase ABTS-SNPs

Figure 4 Decolorization of indigo carmine (20mgL) by ABTS-SNPs (10 gL containing 70120583M ABTS) laccase (3000UL) laccaseABTS-SNPs and laccaseABTS (70 120583M ABTS) in 50mM sodiumacetate buffer at pH 45 with shaking at 150 rpm at room temper-ature

ABTS immobilized on SNPs can be used as an efficientmediator of laccase for decolorization in a manner similarto free ABTS Even though the decolorization rate is slowerfor ABTS-SNPs than for the free mediator the immobilizedmediator has the advantage of being easily recovered from thereaction system

33 Regeneration and Catalytic Cycle Mechanism of ABTS-SNPs In the case of laccase the role of mediators in anenzymatic oxidation has been already described by severalauthors [7] First ABTS covalently attached to SNPs isconverted into ABTS+∙ cation radical by the enzymaticreaction Subsequently the ABTS+∙ cation radical takes partin nonenzymatic reactions with dyes which are not oxidizableby laccases alone Simultaneously theABTS+∙ is reduced to itsoriginal form (ABTS) by the dye to be oxidized and thus onecatalytic cycle is completed At the end of all catalytic cyclethe dye molecules are completely decolorized by oxidationandABTSmolecules are oxidized to ABTS+∙ by laccase againMoreover ABTS+∙ can be reduced by some strong reducingregents [23] In our case ABTS+∙-SNPs were reduced with

Cycle

Dec

olor

izat

ion

()

0

50

100

150

1 2 3 4 5 6

Figure 5 Reusability of ABTS-SNPs (10 gL) in six successive cyclesusing 3000UL of laccase and 20mgL of indigo carmine at pH 45with shaking at 150 rpm for 30min at room temperature

ascorbic acid resulting in regeneration of ABTS-SNPs asshown in Scheme 2

As shown in Figure 3(c) after the regeneration pro-cess the oxidized ABTS-SNPs turned from green to lightyellowmdashthe original color of the initial ABTS-SNPs Furtherthe regenerated ABTS-SNPs can be oxidized by laccaseagain Decolorization efficiency of regenerated ABTS-SNPsas mediators was 935 which was almost the same asthat of initial ABTS-SNPs (sim95 decolorization) The resultsshow that ABTS-SNPs after use can be recovered and reusedas efficient mediators for decolorization similar to originalABTS-SNPs

34 Reusability From the economic point of view reusabilityof immobilized mediator plays a key role when LMSs willbe used on a large scale for industrial applications [32]The reusability of ABTS-SNPs was investigated for up to6 cycles (Figure 5) When the reaction time was set to30min which proved to be sufficient for almost completedecolorization comparably high decolorization yields wereobtained in 4 subsequent runs using the same recovered andregenerated ABTS-SNPs Approximately 77 decolorizationcould be maintained after 6 cycles A slight decrease indecolorization efficiency was observed which was attributedto partial destruction of ABTS-SNPs during the reaction

6 Journal of Chemistry

andor the loss in quantity of ABTS-SNPs during supernatantdiscarding in the centrifugal recovery process It has beenreported that the radical cation form of ABTS was themain product observed after reacting with laccase but adegradation product from ABTS was also formed [33] Thedecomposition of ABTS during the laccase-ABTS reactionswill be a drawback for the application of laccaseABTS-SNPssystem

4 Conclusion

This investigation has demonstrated that ABTS covalentlylinked to SNPs can be utilized to mediate dye decoloriza-tion by laccase Even though the decolorization efficiencyof ABTS-SNPs as mediators is slower than that of thefree mediators the ABTS-SNPs can easily be recoveredregenerated and reused therefore improving the efficiencyof mediator utilization in LMSs for biotechnological andindustrial applications This process has the potential toalleviate environmental problems caused by the release of freemediators in the wastewater system Moreover a large scaleapplication of reusable and immobilizedmediators will lowerthe cost of dye treatment using the laccase mediator systems

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was kindly supported by the Doctoral Inno-vation Fund of Xinxiang medical University Nature SciencePlan Program (12B180030) from the Education Departmentof Henan Province and theNational Natural Science Founda-tion of China (no U1304302)

References

[1] P J Hoegger S Kilaru T Y James J R Thacker and UKues ldquoPhylogenetic comparison and classification of laccaseand related multicopper oxidase protein sequencesrdquo The FEBSJournal vol 273 no 10 pp 2308ndash2326 2006

[2] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[3] A I Yaropolov O V Skorobogatrsquoko S S Vartanov and S DVarfolomeyev ldquoLaccase properties catalytic mechanism andapplicabilityrdquo Applied Biochemistry and Biotechnology vol 49no 3 pp 257ndash280 1994

[4] A Leonowicz N S Cho J Luterek et al ldquoFungal laccaseproperties and activity on ligninrdquo Journal of Basic Microbiologyvol 41 no 3-4 pp 185ndash227 2001

[5] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[6] R C Minussi G M Pastore and N Duran ldquoPotential applica-tions of laccase in the food industryrdquo Trends in Food Science andTechnology vol 13 no 6-7 pp 205ndash216 2002

[7] O V Morozova G P Shumakovich S V Shleev and Y IYaropolov ldquoLaccase-mediator systems and their applications areviewrdquo Applied Biochemistry and Microbiology vol 43 no 5pp 523ndash535 2007

[8] B Branchi C Galli and P Gentili ldquoKinetics of oxidation ofbenzyl alcohols by the dication and radical cation of ABTSComparison with laccase-ABTS oxidations an apparent para-doxrdquo Organic and Biomolecular Chemistry vol 3 no 14 pp2604ndash2614 2005

[9] C Johannes and AMajcherczyk ldquoNatural mediators in the oxi-dation of polycyclic aromatic hydrocarbons by laccasemediatorsystemsrdquo Applied and Environmental Microbiology vol 66 no2 pp 524ndash528 2000

[10] M Fabbrini C Galli and P Gentili ldquoComparing the catalyticefficiency of some mediators of laccaserdquo Journal of MolecularCatalysis B Enzymatic vol 16 no 5-6 pp 231ndash240 2002

[11] S Camarero D Ibarra M J Martınez and A T MartınezldquoLignin-derived compounds as efficient laccase mediators fordecolorization of different types of recalcitrant dyesrdquo Appliedand Environmental Microbiology vol 71 no 4 pp 1775ndash17842005

[12] F Xu J J Kulys K Duke et al ldquoRedox chemistry in laccase-catalyzed oxidation of N-hydroxy compoundsrdquo Applied andEnvironmentalMicrobiology vol 66 no 5 pp 2052ndash2056 2000

[13] Y Wong and J Yu ldquoLaccase-catalyzed decolorization of syn-thetic dyesrdquoWater Research vol 33 no 16 pp 3512ndash3520 1999

[14] G S Nyanhongo J Gomes G M Gubitz R Zvauya J Readand W Steiner ldquoDecolorization of textile dyes by laccases froma newly isolated strain of Trametes modestardquo Water Researchvol 36 no 6 pp 1449ndash1456 2002

[15] P Brandi A DrsquoAnnibale C Galli P Gentili and A S N PontesldquoIn search for practical advantages from the immobilisation ofan enzyme the case of laccaserdquo Journal of Molecular CatalysisB Enzymatic vol 41 no 1-2 pp 61ndash69 2006

[16] C Garcia-Galan A Berenguer-Murcia R Fernandez-Lafuenteand R C Rodrigues ldquoPotential of different enzyme immobi-lization strategies to improve enzyme performancerdquo AdvancedSynthesis and Catalysis vol 353 no 16 pp 2885ndash2904 2011

[17] J Huang J-T Wang B Li et al ldquoPreparation of organic-inorganic nanoparticles composite and their laccase immobi-lizationrdquo Chemical Journal of Chinese Universities vol 27 no 11pp 2088ndash2091 2006

[18] Y Tan W Deng Y Li et al ldquoPolymeric bionanocompositecast thin films with in situ laccase-catalyzed polymerizationof dopamine for biosensing and biofuel cell applicationsrdquo TheJournal of Physical Chemistry B vol 114 no 15 pp 5016ndash50242010

[19] B Palys A Bokun and J Rogalski ldquoPoly-o-phenylenediamineas redox mediator for laccaserdquo Electrochimica Acta vol 52 no24 pp 7075ndash7082 2007

[20] L Mendoza M Jonstrup R Hatti-Kaul and B MattiassonldquoAzo dye decolorization by a laccasemediator system in amembrane reactor enzyme and mediator reusabilityrdquo Enzymeand Microbial Technology vol 49 no 5 pp 478ndash484 2011

[21] K Rittstieg A Suurnakki T Suortti K Kruus G M Guebitzand J Buchert ldquoPolymerization of guaiacol and a phenolic 120573-O-4-substructure by Trametes hirsuta Laccase in the presenceof ABTSrdquo Biotechnology Progress vol 19 no 5 pp 1505ndash15092003

[22] A M Osman K K YWong and A Fernyhough ldquoThe laccaseABTS system oxidizes (+)-catechin to oligomeric productsrdquo

Journal of Chemistry 7

Enzyme and Microbial Technology vol 40 no 5 pp 1272ndash12792007

[23] P Maruthamuthu L Venkatasubramanian and P Dharma-lingam ldquoA fast kinetic study of formation and decay of221015840-azinobis(3-ethylbenzothiazole-6-sulfonate) radical cationin aqueous solutionrdquo Bulletin of the Chemical Society of Japanvol 60 no 3 pp 1113ndash1117 1987

[24] M Klis J Rogalski and R Bilewicz ldquoVoltammetric determina-tion of catalytic reaction parameters of laccase based on elec-trooxidation of hydroquinone and ABTSrdquo Bioelectrochemistryvol 71 no 1 pp 2ndash7 2007

[25] M Chhabra S Mishra and T R Sreekrishnan ldquoMediator-assisted decolorization and detoxification of textile dyesdyemixture by cyathus bulleri laccaserdquo Applied Biochemistry andBiotechnology vol 151 no 2-3 pp 587ndash598 2008

[26] N Barka A Assabbane A Nounah and Y A Ichou ldquoPhoto-catalytic degradation of indigo carmine in aqueous solution byTiO2-coated non-woven fibresrdquo Journal ofHazardousMaterials

vol 152 no 3 pp 1054ndash1059 2008[27] E-A Cho J Seo D-W Lee and J-G Pan ldquoDecolorization of

indigo carmine by laccase displayed on Bacillus subtilis sporesrdquoEnzyme and Microbial Technology vol 49 no 1 pp 100ndash1042011

[28] K Sadowska K Stolarczyk J F Biernat K P Roberts JRogalski and R Bilewicz ldquoDerivatization of single-walledcarbon nanotubes with redox mediator for biocatalytic oxygenelectrodesrdquo Bioelectrochemistry vol 80 no 1 pp 73ndash80 2010

[29] R Bilewicz E Nazaruk K Zelechowska et al ldquoCarbon nan-otubes chemically derivatized with redox systems as mediatorsfor biofuel cell applicationsrdquo Biocybernetics and BiomedicalEngineering vol 31 no 4 pp 17ndash30 2011

[30] Z Zhong X Ji R Xing et al ldquoThe preparation and antioxidantactivity of the sulfanilamide derivatives of chitosan and chitosansulfatesrdquo Bioorganic and Medicinal Chemistry vol 15 no 11 pp3775ndash3782 2007

[31] T Kataoka T Iwama T Setta et al ldquoPreparation of sulfon-amides from sodium sulfonates Ph

3PBr2and Ph

3PCl2as a

mild halogenating reagent for sulfonyl bromides and sulfonylchloridesrdquo Synthesis vol 1998 no 4 pp 423ndash426 1998

[32] X Zhang B Pan B Wu W Zhang and L Lv ldquoA new polymer-based laccase for decolorization of AO7 long-term storage andmediator reuserdquo Bioresource Technology vol 164 pp 248ndash2532014

[33] A Marjasvaara J Janis and P Vainiotalo ldquoOxidation of alaccase mediator ABTS as studied by ESI-FTICR mass spec-trometryrdquo Journal of Mass Spectrometry vol 43 no 4 pp 470ndash477 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 5

Laccase

ABTS-SNPs

Dye

Ascorbic acid Laccase

O2

H2O Laccase(ox)

ABTS+∙-SNPs

ABTS+∙-SNPs

Dye(ox)

Scheme 2 The proposed catalytic cycle of decolorizing the dye by ABTS-SNPs mediated laccase oxidation

0

50

5 10 15

100

150

Time (min)

Dec

olor

izat

ion

()

ABTS-SNPsLaccase

LaccaseABTSLaccase ABTS-SNPs

Figure 4 Decolorization of indigo carmine (20mgL) by ABTS-SNPs (10 gL containing 70120583M ABTS) laccase (3000UL) laccaseABTS-SNPs and laccaseABTS (70 120583M ABTS) in 50mM sodiumacetate buffer at pH 45 with shaking at 150 rpm at room temper-ature

ABTS immobilized on SNPs can be used as an efficientmediator of laccase for decolorization in a manner similarto free ABTS Even though the decolorization rate is slowerfor ABTS-SNPs than for the free mediator the immobilizedmediator has the advantage of being easily recovered from thereaction system

33 Regeneration and Catalytic Cycle Mechanism of ABTS-SNPs In the case of laccase the role of mediators in anenzymatic oxidation has been already described by severalauthors [7] First ABTS covalently attached to SNPs isconverted into ABTS+∙ cation radical by the enzymaticreaction Subsequently the ABTS+∙ cation radical takes partin nonenzymatic reactions with dyes which are not oxidizableby laccases alone Simultaneously theABTS+∙ is reduced to itsoriginal form (ABTS) by the dye to be oxidized and thus onecatalytic cycle is completed At the end of all catalytic cyclethe dye molecules are completely decolorized by oxidationandABTSmolecules are oxidized to ABTS+∙ by laccase againMoreover ABTS+∙ can be reduced by some strong reducingregents [23] In our case ABTS+∙-SNPs were reduced with

Cycle

Dec

olor

izat

ion

()

0

50

100

150

1 2 3 4 5 6

Figure 5 Reusability of ABTS-SNPs (10 gL) in six successive cyclesusing 3000UL of laccase and 20mgL of indigo carmine at pH 45with shaking at 150 rpm for 30min at room temperature

ascorbic acid resulting in regeneration of ABTS-SNPs asshown in Scheme 2

As shown in Figure 3(c) after the regeneration pro-cess the oxidized ABTS-SNPs turned from green to lightyellowmdashthe original color of the initial ABTS-SNPs Furtherthe regenerated ABTS-SNPs can be oxidized by laccaseagain Decolorization efficiency of regenerated ABTS-SNPsas mediators was 935 which was almost the same asthat of initial ABTS-SNPs (sim95 decolorization) The resultsshow that ABTS-SNPs after use can be recovered and reusedas efficient mediators for decolorization similar to originalABTS-SNPs

34 Reusability From the economic point of view reusabilityof immobilized mediator plays a key role when LMSs willbe used on a large scale for industrial applications [32]The reusability of ABTS-SNPs was investigated for up to6 cycles (Figure 5) When the reaction time was set to30min which proved to be sufficient for almost completedecolorization comparably high decolorization yields wereobtained in 4 subsequent runs using the same recovered andregenerated ABTS-SNPs Approximately 77 decolorizationcould be maintained after 6 cycles A slight decrease indecolorization efficiency was observed which was attributedto partial destruction of ABTS-SNPs during the reaction

6 Journal of Chemistry

andor the loss in quantity of ABTS-SNPs during supernatantdiscarding in the centrifugal recovery process It has beenreported that the radical cation form of ABTS was themain product observed after reacting with laccase but adegradation product from ABTS was also formed [33] Thedecomposition of ABTS during the laccase-ABTS reactionswill be a drawback for the application of laccaseABTS-SNPssystem

4 Conclusion

This investigation has demonstrated that ABTS covalentlylinked to SNPs can be utilized to mediate dye decoloriza-tion by laccase Even though the decolorization efficiencyof ABTS-SNPs as mediators is slower than that of thefree mediators the ABTS-SNPs can easily be recoveredregenerated and reused therefore improving the efficiencyof mediator utilization in LMSs for biotechnological andindustrial applications This process has the potential toalleviate environmental problems caused by the release of freemediators in the wastewater system Moreover a large scaleapplication of reusable and immobilizedmediators will lowerthe cost of dye treatment using the laccase mediator systems

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was kindly supported by the Doctoral Inno-vation Fund of Xinxiang medical University Nature SciencePlan Program (12B180030) from the Education Departmentof Henan Province and theNational Natural Science Founda-tion of China (no U1304302)

References

[1] P J Hoegger S Kilaru T Y James J R Thacker and UKues ldquoPhylogenetic comparison and classification of laccaseand related multicopper oxidase protein sequencesrdquo The FEBSJournal vol 273 no 10 pp 2308ndash2326 2006

[2] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[3] A I Yaropolov O V Skorobogatrsquoko S S Vartanov and S DVarfolomeyev ldquoLaccase properties catalytic mechanism andapplicabilityrdquo Applied Biochemistry and Biotechnology vol 49no 3 pp 257ndash280 1994

[4] A Leonowicz N S Cho J Luterek et al ldquoFungal laccaseproperties and activity on ligninrdquo Journal of Basic Microbiologyvol 41 no 3-4 pp 185ndash227 2001

[5] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[6] R C Minussi G M Pastore and N Duran ldquoPotential applica-tions of laccase in the food industryrdquo Trends in Food Science andTechnology vol 13 no 6-7 pp 205ndash216 2002

[7] O V Morozova G P Shumakovich S V Shleev and Y IYaropolov ldquoLaccase-mediator systems and their applications areviewrdquo Applied Biochemistry and Microbiology vol 43 no 5pp 523ndash535 2007

[8] B Branchi C Galli and P Gentili ldquoKinetics of oxidation ofbenzyl alcohols by the dication and radical cation of ABTSComparison with laccase-ABTS oxidations an apparent para-doxrdquo Organic and Biomolecular Chemistry vol 3 no 14 pp2604ndash2614 2005

[9] C Johannes and AMajcherczyk ldquoNatural mediators in the oxi-dation of polycyclic aromatic hydrocarbons by laccasemediatorsystemsrdquo Applied and Environmental Microbiology vol 66 no2 pp 524ndash528 2000

[10] M Fabbrini C Galli and P Gentili ldquoComparing the catalyticefficiency of some mediators of laccaserdquo Journal of MolecularCatalysis B Enzymatic vol 16 no 5-6 pp 231ndash240 2002

[11] S Camarero D Ibarra M J Martınez and A T MartınezldquoLignin-derived compounds as efficient laccase mediators fordecolorization of different types of recalcitrant dyesrdquo Appliedand Environmental Microbiology vol 71 no 4 pp 1775ndash17842005

[12] F Xu J J Kulys K Duke et al ldquoRedox chemistry in laccase-catalyzed oxidation of N-hydroxy compoundsrdquo Applied andEnvironmentalMicrobiology vol 66 no 5 pp 2052ndash2056 2000

[13] Y Wong and J Yu ldquoLaccase-catalyzed decolorization of syn-thetic dyesrdquoWater Research vol 33 no 16 pp 3512ndash3520 1999

[14] G S Nyanhongo J Gomes G M Gubitz R Zvauya J Readand W Steiner ldquoDecolorization of textile dyes by laccases froma newly isolated strain of Trametes modestardquo Water Researchvol 36 no 6 pp 1449ndash1456 2002

[15] P Brandi A DrsquoAnnibale C Galli P Gentili and A S N PontesldquoIn search for practical advantages from the immobilisation ofan enzyme the case of laccaserdquo Journal of Molecular CatalysisB Enzymatic vol 41 no 1-2 pp 61ndash69 2006

[16] C Garcia-Galan A Berenguer-Murcia R Fernandez-Lafuenteand R C Rodrigues ldquoPotential of different enzyme immobi-lization strategies to improve enzyme performancerdquo AdvancedSynthesis and Catalysis vol 353 no 16 pp 2885ndash2904 2011

[17] J Huang J-T Wang B Li et al ldquoPreparation of organic-inorganic nanoparticles composite and their laccase immobi-lizationrdquo Chemical Journal of Chinese Universities vol 27 no 11pp 2088ndash2091 2006

[18] Y Tan W Deng Y Li et al ldquoPolymeric bionanocompositecast thin films with in situ laccase-catalyzed polymerizationof dopamine for biosensing and biofuel cell applicationsrdquo TheJournal of Physical Chemistry B vol 114 no 15 pp 5016ndash50242010

[19] B Palys A Bokun and J Rogalski ldquoPoly-o-phenylenediamineas redox mediator for laccaserdquo Electrochimica Acta vol 52 no24 pp 7075ndash7082 2007

[20] L Mendoza M Jonstrup R Hatti-Kaul and B MattiassonldquoAzo dye decolorization by a laccasemediator system in amembrane reactor enzyme and mediator reusabilityrdquo Enzymeand Microbial Technology vol 49 no 5 pp 478ndash484 2011

[21] K Rittstieg A Suurnakki T Suortti K Kruus G M Guebitzand J Buchert ldquoPolymerization of guaiacol and a phenolic 120573-O-4-substructure by Trametes hirsuta Laccase in the presenceof ABTSrdquo Biotechnology Progress vol 19 no 5 pp 1505ndash15092003

[22] A M Osman K K YWong and A Fernyhough ldquoThe laccaseABTS system oxidizes (+)-catechin to oligomeric productsrdquo

Journal of Chemistry 7

Enzyme and Microbial Technology vol 40 no 5 pp 1272ndash12792007

[23] P Maruthamuthu L Venkatasubramanian and P Dharma-lingam ldquoA fast kinetic study of formation and decay of221015840-azinobis(3-ethylbenzothiazole-6-sulfonate) radical cationin aqueous solutionrdquo Bulletin of the Chemical Society of Japanvol 60 no 3 pp 1113ndash1117 1987

[24] M Klis J Rogalski and R Bilewicz ldquoVoltammetric determina-tion of catalytic reaction parameters of laccase based on elec-trooxidation of hydroquinone and ABTSrdquo Bioelectrochemistryvol 71 no 1 pp 2ndash7 2007

[25] M Chhabra S Mishra and T R Sreekrishnan ldquoMediator-assisted decolorization and detoxification of textile dyesdyemixture by cyathus bulleri laccaserdquo Applied Biochemistry andBiotechnology vol 151 no 2-3 pp 587ndash598 2008

[26] N Barka A Assabbane A Nounah and Y A Ichou ldquoPhoto-catalytic degradation of indigo carmine in aqueous solution byTiO2-coated non-woven fibresrdquo Journal ofHazardousMaterials

vol 152 no 3 pp 1054ndash1059 2008[27] E-A Cho J Seo D-W Lee and J-G Pan ldquoDecolorization of

indigo carmine by laccase displayed on Bacillus subtilis sporesrdquoEnzyme and Microbial Technology vol 49 no 1 pp 100ndash1042011

[28] K Sadowska K Stolarczyk J F Biernat K P Roberts JRogalski and R Bilewicz ldquoDerivatization of single-walledcarbon nanotubes with redox mediator for biocatalytic oxygenelectrodesrdquo Bioelectrochemistry vol 80 no 1 pp 73ndash80 2010

[29] R Bilewicz E Nazaruk K Zelechowska et al ldquoCarbon nan-otubes chemically derivatized with redox systems as mediatorsfor biofuel cell applicationsrdquo Biocybernetics and BiomedicalEngineering vol 31 no 4 pp 17ndash30 2011

[30] Z Zhong X Ji R Xing et al ldquoThe preparation and antioxidantactivity of the sulfanilamide derivatives of chitosan and chitosansulfatesrdquo Bioorganic and Medicinal Chemistry vol 15 no 11 pp3775ndash3782 2007

[31] T Kataoka T Iwama T Setta et al ldquoPreparation of sulfon-amides from sodium sulfonates Ph

3PBr2and Ph

3PCl2as a

mild halogenating reagent for sulfonyl bromides and sulfonylchloridesrdquo Synthesis vol 1998 no 4 pp 423ndash426 1998

[32] X Zhang B Pan B Wu W Zhang and L Lv ldquoA new polymer-based laccase for decolorization of AO7 long-term storage andmediator reuserdquo Bioresource Technology vol 164 pp 248ndash2532014

[33] A Marjasvaara J Janis and P Vainiotalo ldquoOxidation of alaccase mediator ABTS as studied by ESI-FTICR mass spec-trometryrdquo Journal of Mass Spectrometry vol 43 no 4 pp 470ndash477 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 Journal of Chemistry

andor the loss in quantity of ABTS-SNPs during supernatantdiscarding in the centrifugal recovery process It has beenreported that the radical cation form of ABTS was themain product observed after reacting with laccase but adegradation product from ABTS was also formed [33] Thedecomposition of ABTS during the laccase-ABTS reactionswill be a drawback for the application of laccaseABTS-SNPssystem

4 Conclusion

This investigation has demonstrated that ABTS covalentlylinked to SNPs can be utilized to mediate dye decoloriza-tion by laccase Even though the decolorization efficiencyof ABTS-SNPs as mediators is slower than that of thefree mediators the ABTS-SNPs can easily be recoveredregenerated and reused therefore improving the efficiencyof mediator utilization in LMSs for biotechnological andindustrial applications This process has the potential toalleviate environmental problems caused by the release of freemediators in the wastewater system Moreover a large scaleapplication of reusable and immobilizedmediators will lowerthe cost of dye treatment using the laccase mediator systems

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was kindly supported by the Doctoral Inno-vation Fund of Xinxiang medical University Nature SciencePlan Program (12B180030) from the Education Departmentof Henan Province and theNational Natural Science Founda-tion of China (no U1304302)

References

[1] P J Hoegger S Kilaru T Y James J R Thacker and UKues ldquoPhylogenetic comparison and classification of laccaseand related multicopper oxidase protein sequencesrdquo The FEBSJournal vol 273 no 10 pp 2308ndash2326 2006

[2] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[3] A I Yaropolov O V Skorobogatrsquoko S S Vartanov and S DVarfolomeyev ldquoLaccase properties catalytic mechanism andapplicabilityrdquo Applied Biochemistry and Biotechnology vol 49no 3 pp 257ndash280 1994

[4] A Leonowicz N S Cho J Luterek et al ldquoFungal laccaseproperties and activity on ligninrdquo Journal of Basic Microbiologyvol 41 no 3-4 pp 185ndash227 2001

[5] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[6] R C Minussi G M Pastore and N Duran ldquoPotential applica-tions of laccase in the food industryrdquo Trends in Food Science andTechnology vol 13 no 6-7 pp 205ndash216 2002

[7] O V Morozova G P Shumakovich S V Shleev and Y IYaropolov ldquoLaccase-mediator systems and their applications areviewrdquo Applied Biochemistry and Microbiology vol 43 no 5pp 523ndash535 2007

[8] B Branchi C Galli and P Gentili ldquoKinetics of oxidation ofbenzyl alcohols by the dication and radical cation of ABTSComparison with laccase-ABTS oxidations an apparent para-doxrdquo Organic and Biomolecular Chemistry vol 3 no 14 pp2604ndash2614 2005

[9] C Johannes and AMajcherczyk ldquoNatural mediators in the oxi-dation of polycyclic aromatic hydrocarbons by laccasemediatorsystemsrdquo Applied and Environmental Microbiology vol 66 no2 pp 524ndash528 2000

[10] M Fabbrini C Galli and P Gentili ldquoComparing the catalyticefficiency of some mediators of laccaserdquo Journal of MolecularCatalysis B Enzymatic vol 16 no 5-6 pp 231ndash240 2002

[11] S Camarero D Ibarra M J Martınez and A T MartınezldquoLignin-derived compounds as efficient laccase mediators fordecolorization of different types of recalcitrant dyesrdquo Appliedand Environmental Microbiology vol 71 no 4 pp 1775ndash17842005

[12] F Xu J J Kulys K Duke et al ldquoRedox chemistry in laccase-catalyzed oxidation of N-hydroxy compoundsrdquo Applied andEnvironmentalMicrobiology vol 66 no 5 pp 2052ndash2056 2000

[13] Y Wong and J Yu ldquoLaccase-catalyzed decolorization of syn-thetic dyesrdquoWater Research vol 33 no 16 pp 3512ndash3520 1999

[14] G S Nyanhongo J Gomes G M Gubitz R Zvauya J Readand W Steiner ldquoDecolorization of textile dyes by laccases froma newly isolated strain of Trametes modestardquo Water Researchvol 36 no 6 pp 1449ndash1456 2002

[15] P Brandi A DrsquoAnnibale C Galli P Gentili and A S N PontesldquoIn search for practical advantages from the immobilisation ofan enzyme the case of laccaserdquo Journal of Molecular CatalysisB Enzymatic vol 41 no 1-2 pp 61ndash69 2006

[16] C Garcia-Galan A Berenguer-Murcia R Fernandez-Lafuenteand R C Rodrigues ldquoPotential of different enzyme immobi-lization strategies to improve enzyme performancerdquo AdvancedSynthesis and Catalysis vol 353 no 16 pp 2885ndash2904 2011

[17] J Huang J-T Wang B Li et al ldquoPreparation of organic-inorganic nanoparticles composite and their laccase immobi-lizationrdquo Chemical Journal of Chinese Universities vol 27 no 11pp 2088ndash2091 2006

[18] Y Tan W Deng Y Li et al ldquoPolymeric bionanocompositecast thin films with in situ laccase-catalyzed polymerizationof dopamine for biosensing and biofuel cell applicationsrdquo TheJournal of Physical Chemistry B vol 114 no 15 pp 5016ndash50242010

[19] B Palys A Bokun and J Rogalski ldquoPoly-o-phenylenediamineas redox mediator for laccaserdquo Electrochimica Acta vol 52 no24 pp 7075ndash7082 2007

[20] L Mendoza M Jonstrup R Hatti-Kaul and B MattiassonldquoAzo dye decolorization by a laccasemediator system in amembrane reactor enzyme and mediator reusabilityrdquo Enzymeand Microbial Technology vol 49 no 5 pp 478ndash484 2011

[21] K Rittstieg A Suurnakki T Suortti K Kruus G M Guebitzand J Buchert ldquoPolymerization of guaiacol and a phenolic 120573-O-4-substructure by Trametes hirsuta Laccase in the presenceof ABTSrdquo Biotechnology Progress vol 19 no 5 pp 1505ndash15092003

[22] A M Osman K K YWong and A Fernyhough ldquoThe laccaseABTS system oxidizes (+)-catechin to oligomeric productsrdquo

Journal of Chemistry 7

Enzyme and Microbial Technology vol 40 no 5 pp 1272ndash12792007

[23] P Maruthamuthu L Venkatasubramanian and P Dharma-lingam ldquoA fast kinetic study of formation and decay of221015840-azinobis(3-ethylbenzothiazole-6-sulfonate) radical cationin aqueous solutionrdquo Bulletin of the Chemical Society of Japanvol 60 no 3 pp 1113ndash1117 1987

[24] M Klis J Rogalski and R Bilewicz ldquoVoltammetric determina-tion of catalytic reaction parameters of laccase based on elec-trooxidation of hydroquinone and ABTSrdquo Bioelectrochemistryvol 71 no 1 pp 2ndash7 2007

[25] M Chhabra S Mishra and T R Sreekrishnan ldquoMediator-assisted decolorization and detoxification of textile dyesdyemixture by cyathus bulleri laccaserdquo Applied Biochemistry andBiotechnology vol 151 no 2-3 pp 587ndash598 2008

[26] N Barka A Assabbane A Nounah and Y A Ichou ldquoPhoto-catalytic degradation of indigo carmine in aqueous solution byTiO2-coated non-woven fibresrdquo Journal ofHazardousMaterials

vol 152 no 3 pp 1054ndash1059 2008[27] E-A Cho J Seo D-W Lee and J-G Pan ldquoDecolorization of

indigo carmine by laccase displayed on Bacillus subtilis sporesrdquoEnzyme and Microbial Technology vol 49 no 1 pp 100ndash1042011

[28] K Sadowska K Stolarczyk J F Biernat K P Roberts JRogalski and R Bilewicz ldquoDerivatization of single-walledcarbon nanotubes with redox mediator for biocatalytic oxygenelectrodesrdquo Bioelectrochemistry vol 80 no 1 pp 73ndash80 2010

[29] R Bilewicz E Nazaruk K Zelechowska et al ldquoCarbon nan-otubes chemically derivatized with redox systems as mediatorsfor biofuel cell applicationsrdquo Biocybernetics and BiomedicalEngineering vol 31 no 4 pp 17ndash30 2011

[30] Z Zhong X Ji R Xing et al ldquoThe preparation and antioxidantactivity of the sulfanilamide derivatives of chitosan and chitosansulfatesrdquo Bioorganic and Medicinal Chemistry vol 15 no 11 pp3775ndash3782 2007

[31] T Kataoka T Iwama T Setta et al ldquoPreparation of sulfon-amides from sodium sulfonates Ph

3PBr2and Ph

3PCl2as a

mild halogenating reagent for sulfonyl bromides and sulfonylchloridesrdquo Synthesis vol 1998 no 4 pp 423ndash426 1998

[32] X Zhang B Pan B Wu W Zhang and L Lv ldquoA new polymer-based laccase for decolorization of AO7 long-term storage andmediator reuserdquo Bioresource Technology vol 164 pp 248ndash2532014

[33] A Marjasvaara J Janis and P Vainiotalo ldquoOxidation of alaccase mediator ABTS as studied by ESI-FTICR mass spec-trometryrdquo Journal of Mass Spectrometry vol 43 no 4 pp 470ndash477 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 7

Enzyme and Microbial Technology vol 40 no 5 pp 1272ndash12792007

[23] P Maruthamuthu L Venkatasubramanian and P Dharma-lingam ldquoA fast kinetic study of formation and decay of221015840-azinobis(3-ethylbenzothiazole-6-sulfonate) radical cationin aqueous solutionrdquo Bulletin of the Chemical Society of Japanvol 60 no 3 pp 1113ndash1117 1987

[24] M Klis J Rogalski and R Bilewicz ldquoVoltammetric determina-tion of catalytic reaction parameters of laccase based on elec-trooxidation of hydroquinone and ABTSrdquo Bioelectrochemistryvol 71 no 1 pp 2ndash7 2007

[25] M Chhabra S Mishra and T R Sreekrishnan ldquoMediator-assisted decolorization and detoxification of textile dyesdyemixture by cyathus bulleri laccaserdquo Applied Biochemistry andBiotechnology vol 151 no 2-3 pp 587ndash598 2008

[26] N Barka A Assabbane A Nounah and Y A Ichou ldquoPhoto-catalytic degradation of indigo carmine in aqueous solution byTiO2-coated non-woven fibresrdquo Journal ofHazardousMaterials

vol 152 no 3 pp 1054ndash1059 2008[27] E-A Cho J Seo D-W Lee and J-G Pan ldquoDecolorization of

indigo carmine by laccase displayed on Bacillus subtilis sporesrdquoEnzyme and Microbial Technology vol 49 no 1 pp 100ndash1042011

[28] K Sadowska K Stolarczyk J F Biernat K P Roberts JRogalski and R Bilewicz ldquoDerivatization of single-walledcarbon nanotubes with redox mediator for biocatalytic oxygenelectrodesrdquo Bioelectrochemistry vol 80 no 1 pp 73ndash80 2010

[29] R Bilewicz E Nazaruk K Zelechowska et al ldquoCarbon nan-otubes chemically derivatized with redox systems as mediatorsfor biofuel cell applicationsrdquo Biocybernetics and BiomedicalEngineering vol 31 no 4 pp 17ndash30 2011

[30] Z Zhong X Ji R Xing et al ldquoThe preparation and antioxidantactivity of the sulfanilamide derivatives of chitosan and chitosansulfatesrdquo Bioorganic and Medicinal Chemistry vol 15 no 11 pp3775ndash3782 2007

[31] T Kataoka T Iwama T Setta et al ldquoPreparation of sulfon-amides from sodium sulfonates Ph

3PBr2and Ph

3PCl2as a

mild halogenating reagent for sulfonyl bromides and sulfonylchloridesrdquo Synthesis vol 1998 no 4 pp 423ndash426 1998

[32] X Zhang B Pan B Wu W Zhang and L Lv ldquoA new polymer-based laccase for decolorization of AO7 long-term storage andmediator reuserdquo Bioresource Technology vol 164 pp 248ndash2532014

[33] A Marjasvaara J Janis and P Vainiotalo ldquoOxidation of alaccase mediator ABTS as studied by ESI-FTICR mass spec-trometryrdquo Journal of Mass Spectrometry vol 43 no 4 pp 470ndash477 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of