Laccase-thermodynamics.pdf

RESEARCH ARTICLE

Laccase-catalyzed decoloriof Acid Blue 92: statistical

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effluents, rich in complex aromatic structures into theenvironment [1,2]. Almost all synthetic colorants espe-

still remains as a major challenge [1]. Among variousphysicochemical and biotechnological techniques, the

JOURNAL OF ENVIRONMENTAL HEALTHSCIENCE & ENGINEERING

Rezaei et al. Journal of Environmental Health Science & Engineering (2015) 13:31 DOI 10.1186/s40201-015-0183-1compounds such as benzenethiols, substituted phenols,Sciences, Tehran 1417614411, IranFull list of author information is available at the end of the articlecially the azo dyes the most common dye group intextile dyeing processes and/or their degradation prod-ucts have been reported to be toxic, mutagenic, andcarcinogenic [3]. Resistance of the environmentally haz-ardous dyes to light, biological treatment procedures,

enzymatic removal of synthetic dyes is the most pre-ferred method due to its simplicity, efficiency at highand low pollutant concentration over a wide range ofpH and temperature, low energy required, minimal im-pact on ecosystem, and less sludge production in thedecolorization process [6-9].Laccases (benzenediol:oxygen oxidoreductase, EC

1.10.3.2) are multi-copper containing oxidase mainlyfound in fungi, plants, and some bacterial strains [5,10].The ability of laccases to oxidize a broad range of aromatic

* Correspondence: [email protected] of Pharmaceutical Biotechnology, Faculty of Pharmacy andBiotechnology Research Center, Tehran University of Medical Sciences, P.O.Box 141556451, Tehran 1417614411, Iran2Pharmaceutical Sciences Research Center, Tehran University of Medicalorder to obtain the optimal condition for laccase-mediated (purified from the ascomycete Paraconiothyrium variabile)decolorization of Acid Blue 92; a monoazo dye, using response surface methodology (RSM). So, a D-optimal designwith three variables, including pH, enzyme activity, and dye concentration, was applied to optimize the decolorizationprocess. In addition, the kinetic and energetic parameters of the above mentioned enzymatic removal of Acid Blue 92was investigated.

Results: Decolorization of Acid Blue 92 was maximally (94.1% 2.61) occurred at pH 8.0, laccase activity of 2.5 U/mL,and dye concentration of 75 mg/mL. The obtained results of kinetic and energetic studies introduced thelaccase-catalyzed decolorization of Acid Blue 92 as an endothermic reaction (Ea, 39 kJ/mol; S, 131 J/mol K; and H,40 kJ/mol) with Km and Vmax values of 0.48 mM and 227 mM/min mg, respectively. Furthermore, the results of microtoxicitystudy revealed that the toxicity of laccase-treated dye was significantly reduced compared to the untreated dye.

Conclusions: To sum up, the present investigation introduced the Paraconiothyrium variabile laccase as an efficientbiocatalyst for decolorization of synthetic dye Acid Blue 92.

Keywords: Enzyme Biocatalysis, Optimization, Waste Treatment, Bioremediation, Laccase, Decolorization

IntroductionThe wide usage of synthetic dyes in industries suchas textile, paper, plastics, printing, leather, cosmetics,and pharmaceuticals has led to releasing dye containing

ozone, or other degradative environmental procedures isthe main problem in the elimination of dyes dischargedfrom wastewaters [4,5]. So, development of efficient andeconomical processes for treatment of the synthetic dyesmicrotoxicity, kinetics, anShahla Rezaei1, Hamed Tahmasbi1, Mehdi Mogharabi1,2, AMohammad Reza Khoshayand5 and Mohammad Ali Fara

Abstract

Background: In recent years, enzymatic-assisted removaland eco-friendly method compared to those of physicoch 2015 Rezaei et al.; licensee BioMed Central.Commons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.Open Access

zation and detoxificationoptimization,energetics

eh Ameri3, Hamid Forootanfar4,arzi1,2*

hazardous dyes has been considered as an alternativemical techniques. The present study was designed inThis is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,

part of full factorial design matrix with two-level factorvariations containing 2kp runs (1/2p fraction of the 2k

design); where k and p are the number of independentvariables and size of the fraction, respectively. Differentfactors including pH (A), temperature (B), enzyme activ-ity (C), dye concentration (D), and incubation time (E)were implemented using 251 fractional factorial designwith resolution V. The design matrix was built using thestatistical software package, Design-Expert (version 7.0.0;Stat-Ease, Inc., Minneapolis, Minnesota, USA). Factorsand corresponding response presented in Table 1. All ofthe experiments were accomplished in triplicate and theaverages considered as responses.

Optimization study

Rezaei et al. Journal of Environmental Health Science & Engineering (2015) 13:31 Page 2 of 9and polyaromatic hydrocarbons (PAHs) in the presence ofmolecular oxygen as a co-substrate introduces this bio-technologically important enzyme as the first choice forxenobiotic removal experiments [11,12]. Decolorizationand detoxification of synthetic dyes assisted by laccase andlaccase-mediated system (LMS) have received great atten-tion during two last decades [13,14]. However, high cost ofenzymatic removal (due to low production yield) limitsextensive application of laccases in xenobiotic elimin-ation [15,16]. This constrain could be overcome viaoptimization of removal reaction conditions using stat-istical approaches [17,18].The response surface methodology (RSM) which is

utilized broadly in biotechnological processes, involves acollection of useful statistical and mathematical tech-niques for analyzing the causal relationship between in-dependent variables, responses, and their interactionsthrough the construction of polynomial mathematicalmodels which leads to time and cost saving [19]. Severalstudies have been recently published on the potential ap-plications of RSM in the enzymatic decolorization ofreactive dyes such as Reactive Black 5, Reactive Red 239,Reactive Yellow 15, and Reactive Blue 114 [16,17].In the present study, a D-optimal model for RSM as a

very useful design method was applied to obtain max-imal removal of Acid Blue 92 assisted by laccase. Fur-thermore, the kinetic and thermodynamic parameters oflaccase-mediated dye removal reaction were investigated.The microtoxicity experiments were also performed inorder to evaluate the toxicity of untreated and laccase-treated dye.

Materials and methodsChemicals2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulphonate) (ABTS)was purchased from Sigma-Aldrich (St. Louis, MO,USA). Acid Blue 92 (Figure 1) was kindly donated byAlvan Sabet Co. (Tehran, Iran). All other chemicals andreagents were of the highest purity available. The extra-cellular laccase of Paraconiothyrium variabile (PvL) waspurified using the method previously described by For-ootanfar et al. [20].

Laccase assayLaccase activity was determined using ABTS as the sub-strate [21]. The reaction mixture was prepared by adding0.5 mL ABTS (5 mM) dissolved in 100 mM citratebuffer (pH 4.5) and 0.5 mL of enzyme solution followedby incubation at 40C and 120 rpm. Oxidation of ABTSwas monitored by increase in absorbance at 420 nm(420 = 36000 M

1 cm1) using a UV/vis Spectrophotom-eter (UVD 2950, Labomed, Culver City, USA). One unit

of laccase activity was defined as amount of the enzymerequired to oxidize 1 mol of ABTS per min [22].Dye decolorization experimentsAfter preparation of dye solution (concentration rangeof 100200 mg/L) in citrate-phosphate buffer (0.1 M,pH range of 3.0 8.0), the purified PvL (final activityof 12.5 U/L) was added to the reaction mixture andincubated at desired temperature (3070C) for 3090 min followed by measuring the absorbance of the takensamples using a UV/visible spectrophotometer at max-imum absorbance of applied dye (571 nm). Decolorizationpercentage was then calculated using the following equa-tion: decolorization (%) = [Ai At/Ai] 100; where Ai isthe initial absorbance of the reaction mixture and At is theabsorbance after incubation time [1]. The negative control(reaction mixture containing the heat-inactivated enzyme)was prepared and incubated at the same conditions. Allexperiments were performed in triplicate and the meansof decolorization percentages were reported.

Experimental design and statistical analysisScreening studyThe screening experiment was designed based on thefractional factorial design method, which is a definite

Figure 1 Chemical structure of Acid Blue 92.The D-optimal design developed to select the designpoints in a method that minimizes the variance associated

Rezaei et al. Journal of Environmental Health Science & Engineering (2015) 13:31 Page 3 of 9with the estimates of coefficient in a specified model. Thenumber of runs in a D-optimal design are less and do notrise as fast as the classical design with an increasing num-ber of factors. The D-optimal design try to minimize thedeterminant of the (XX)1 matrix which lead to minimizethe volume of the confidence for the coefficients and max-imizes the determinant information matrix (XX); whereX defined as a matrix containing the designed pointsgenerated by the computer to fulfill the D-optimally. TheD-optimal designs, as an optimization method based on achosen optimality criterion, used the determinant of theinformation matrix XX. Maximizing the determinant ofthe information matrix (XX) lead to minimizing the deter-minant of the matrix (XX)1 which beneficially keep thelater calculations as short as possible. Finally, the Fisherstest with P-values below 0.05 employed to evaluate thestatistical significance of the effect of independent vari-ables on the response using analysis of variance (ANOVA).While the multiple correlation coefficient (R2) and ad-justed R2 used as quality indicators for the fit of second-order polynomial model equation, contour plots, andthree-dimensional surface plots were used to graphicallyshow the relationship and interactions between the codedvariables and responses. The optimal condition for enzym-atic decolorization process determined by solving theequation derived from the final quadratic model as well asgrid search of the three-dimensional surface plots.

Dye removal kinetics and energeticsKinetics of decolorizationAfter performing of decolorization reaction in the pres-ence of the dye concentrations (10500 M) at optimal

Table 1 Level of independent variables in fractionalfactorial design

Variables Symbol Unit Low level (1) High level (+1)

pH A - 3 8

Temperature B C 30 70

Enzyme C U/mL 1 2.5

Dye D mg/L 100 200

Incubation time E min 30 90pH and temperature, the velocity for different concen-trations of dye was determined. Michaelis-Mentencurve was then drawn by plotting the obtained initialvelocity against dye concentrations. Calculation of Kmand Vmax values were performed by fitting the data tothe Lineweaver-Burk plot, resulting of the Michaelis-Menten plot conversion [1,2].

Thermodynamics of decolorizationIn order to evaluate the effect of temperature on re-moval of Acid Blue 92, decolorization experiment wasperformed at temperature range of 1050C and theobtained velocities (for each temperature) were plottedagainst initial dye concentrations. Thereafter, apparentfirst-order rate constant (K) for each temperature werecalculated from the slope of each straight plot. The valueof activation energy (Ea) (kJ/mol) was then estimatedfrom the slope of the linearized Arrhenius curveachieved by drawing the ln (K) versus 1/T (103 K1):slope of Arrhenius plot = (Ea/R); where R is the gasconstant (8.3145 J/mol K), and T is the absolutetemperature (K). The enthalpy (H) and entropy (S) ofdecolorization reaction were estimated by using of VantHoff plot which was drawn by plotting the ln (Keq)against 1/T (103 K1). Keq is the apparent equilibriumconstant and was calculated from difference of initialdye concentration and residual dye concentration atequilibrium state, when the decolorization percentagebecome constant and remained dye have no longerchanges with passing the time [23]. Finally, the Gibbsfree energy (G) was calculated using the equation ofG = H TS.

Microtoxicity studiesIn order to evaluate the toxicity of produced meta-bolite(s) following laccase treatment, a series of micro-toxicity study was performed. Firstly, a preculture ofthree Gram-positive bacterial strains (Micrococcus luteusATCC 10240, Staphylococcus aureus ATCC 6538, andBacillus subtilis ATCC 6633) and three Gram-negativebacterial strains (E. coli ATCC 25922, Pseudomonasaeruginosa ATCC 9027, and Salmonella typhi ATCC19430) was prepared by incubating of each bacterial strainin Muller-Hinton broth at 37C and 150 rpm to reach theOD600 of 0.2. Consequently, the untreated dye solution(final concentration of 100 mg/L) and the sample obtainedfrom enzymatic treatment of applied dye (performed atthe optimized condition) was separately added to the pre-pared bacterial culture media and incubated at 37C.Changes in the OD600 of each bacterial strain were thenrecorded every 2 h for 10 h. A negative control (cultivatedbacterial strain in the absence of dye) was designed foreach experiment. The percentage of growth inhibition(GI%) was defined as [(1 D600S/OD600C) 100]; whereOD600S is the OD600 of sample and OD600C is the OD600of control. All experiments were performed in triplicateand mean of the obtained results was reported.

Results and discussionFractional factorial design for screening of importantvariablesIn a preliminary study, fractional factorial design wasused to evaluate the influence of five factors includingpH (A), temperature (B), the enzyme activity (C), dye

concentration (D), and incubation time (E) on thedecolorization yield in order to select the most effective

variables on laccase-catalyzed decolorization of AcidBlue 92. The respective high (+1) and low (1) levels foreach coded factor defined in Table 1. A total of 20 runsand the related responses with combination of five men-tioned variables were presented in Table 2. The half-normal probability plot employed as a graphical tool forestimation the effect of each factor alone and in combin-ation with other factors on decolorization percentage isillustrated in Figure 2. The large distance of pH (A),enzyme activity (C), and dye concentration (D) from zeroin half-normal plot, indicated that these factors signifi-cantly affected the decolorization process (Figure 2). Inaddition, considerable interaction between enzyme activity(C) and dye concentration (D) emerged from half-normalprobability plot (Figure 2). In the study of Khouni et al.[24], who applied central composite design (CCD) matrixand RSM for optimization of laccase-mediated decolo-rization of Black Novacron R and Blue Bezaktiv S-GLD150, two variables including pH and temperature posi-tively affected decolorization process while the third par-ameter (laccase activity) did not influence dye removal.Daassi et al. [25] reported the significant effect of fourfactors (laccase activity,1-hydroxybenzotriazole concentra-

D-optimal design response surface methodologyBased on the results of the fractional factorial design,three variables, including pH (A), enzyme activity (C),and dye concentration (D) were found to have a greaterinfluence on the decolorization of Acid Blue 92 assistedby laccase. Therefore, the D-optimal RSM exploitedto optimize the affecting decolorization factors. Theresponses generated from performing a total of 20 runsare presented at Table 3. Table 4 demonstrates thequadratic model as the most suitable model for theenzymatic decolorization of Acid Blue 92. The analysisof variance for the quadratic model is shown in Table 5.The model F-value (16.08) shows the significance ofthe model. In addition, Table 5 indicated pH (A), dyeconcentration (D), and their interactions; AC, CD, A2

and, C2 as significant model terms (P-value < 0.05). Thequadratic model explained the statistical relationshipbetween the selected variables and the response interms of coded factors was best fitted with the followingequation.

Y 18:27 3:99A 21:55C13:5D 8:59 AC5:16 AD12:37CD6:17A2

re

en

Rezaei et al. Journal of Environmental Health Science & Engineering (2015) 13:31 Page 4 of 9tion, dye concentration, and reaction time) on decolori-zation of Acid Orange 51 assisted by crude laccase fromTrametes trogii using a three-level Box-Behnken factorialdesign combined with response surface methodology toreach 87.87 1.27 decolorization percent.

Table 2 Fractional factorial design matrix and their observed

Run no. pH Temperature (C) Enzyme activity (U/mL) Dye conc

1 5.5 50 1.00 100

2 5.5 50 1.00 100

3 8.0 30 2.50 50

4 8.0 70 2.50 200

5 8.0 30 0.25 200

6 3.0 70 2.50 200

7 3.0 30 2.50 200

8 3.0 30 2.50 50

9 8.0 30 2.50 200

10 3.0 70 0.25 50

11 8.0 30 0.25 50

12 3.0 70 2.50 50

13 3.0 30 0.25 50

14 3.0 70 0.25 200

15 3.0 30 0.25 200

16 8.0 70 0.25 50

17 8.0 70 2.50 5018 8.0 70 0.25 200

19 5.5 50 1.00 100 11:42C2 1:56D2 1

where Y is the response (yield of decolorization) and A,C, and D are pH, enzyme activity, and dye concen-tration, respectively. In order to investigate the relation

sponses for laccase-assisted decolorization of Acid Blue 92

tration (mg/L) Incubation time (min) Response (decolorization%)

60 13.62

60 16.60

90 16.28

90 4.64

90 6.94

30 10.55

90 6.65

30 92.66

30 5.27

30 22.90

30 13.31

90 92.45

90 14.57

90 5.89

30 7.87

90 3.81

30 32.4030 3.65

60 22.25

between the independent variables and the responses,contour plots generated by RSM using the Design-

level. Surface plots demonstrate an increase in dyedecolorization with raising the pH value. Figure 3a and

Figure 2 Half-normal probability plot for statistical analysis of fractional factorial design. A: pH; C: Enzyme; D: Dye.

ion

Rezaei et al. Journal of Environmental Health Science & Engineering (2015) 13:31 Page 5 of 9Expert software. The response surface plots (Figure 3)present the decolorization of Acid Blue 92 as functionof two variables, while the third one kept at a constant

Table 3 D-optimal design matrix containing various conditRun no. pH Enzyme activity (U/mL)

1 8.00 0.75

2 1.30 1.63

3 5.50 3.10

4 3.00 0.75

5 5.50 1.63

6 5.50 1.63

7 3.00 0.75

8 5.50 1.63

9 3.00 2.50

10 3.00 2.50

11 5.50 1.63

12 5.50 1.63

13 5.50 1.63

14 8.00 2.50

15 9.70 3.25

16 8.00 2.50

17 5.50 1.63

18 5.50 1.63

19 8.00 0.75

20 5.50 0.15Figure 3c clearly show that the decolorization per-centage influenced by small alterations of the enzymeactivity.

s and related responsesDye concentration (mg/L) Response (decolorization%)

75.00 4.4

137.50 2.1

137.50 99.3

200.00 5.3

137.50 19.2

137.50 14.4

75.00 11.8

137.50 15.5

200.00 10.3

75.00 42.2

32.39 58.1

137.50 16.3

137.50 17.3

200.00 14.4

137.50 6.1

75.00 92.1

242.61 4.3

137.50 18.7

200.00 0.8

137.50 4.4

OptimizationBy solving the equation (1) and analyzing three dimen-sional response surface graphs (Figure 3), the optimallevels for pH, enzyme activity, and dye concentrationwere found to be 8.0, 2.5 U/L, and 75 mg/L, respectively.

Table 4 Sequential model sum of squares for D-optimal desig

Source Sum of squares Df

Mean vs Total 10503.78 1

Linear vs Mean 9046.07 3

2FI vs Linear 2028.02 3

Quadratic vs 2FI 2671.26 3

Cubic vs Quadratic 820.89 4

Residual 128.66 6

Total 25198.68 20

Rezaei et al. Journal of Environmental Health Science & Engineering (2015) 13:31 Page 6 of 9The yield of laccase-catalyzed decolorization of AcidBlue 92 in optimal condition was 94.1% 2.61. Theobtained results introduced pH as the most relevantfactor for the enzymatic decolorization of Acid Blue 92.Low yield of decolorization achieved at acidic pH, whilepH 8.0 was the optimal pH where maximum deco-lorization occurred. In general, most of fungal laccasesoptimally work at acidic pH levels [26]. For example, thecrude laccase of Ganoderma lucidum preferred theacidic range for dye elimination and its decolorizationactivity decreased significantly at pH levels above 6.0[16]. However, Tavares et al. [17] reported that morethan 80% decolorization of Reactive Blue 114 occurredat pH 7.07.5. The maximum removal of Azure B usingthe purified laccase of Trametes trogii BAFC 463 medi-ated by 1-hydroxybenzotriazole was achieved at pH 7.0[18]. In contrast, the laccases originated from bacterial

Table 5 Analysis of variance for D-optimal design

Source Sum of squares Df Mean squares F-value P-value

Model 13745.35 9 1527.26 16.08

twe

Rezaei et al. Journal of Environmental Health Science & Engineering (2015) 13:31 Page 7 of 9as monoazo, diazo, and triazo, have been identified asrecalcitrant colorants for oxidation using laccases com-pared to that of anthraquinone-based dyes [1,3].Figure 3 Response surface plot indicating the effects of interactions bec) the enzyme activity and the dye concentration.Energetic studyEnergetic results are illustrated in Figure 5. A significanteffect of temperature on decolorization of Acid Blue 92was observed, as decolorization rate increased from 10to 50C. The activation energy (obtained from the slopeof Arrhenius plot, Figure 5a) was 39 kJ/mol, which istypical for the decolorization reaction. The calculatedvalues for H and S were 40 kJ/mol and 131 J/mol K,respectively. The positive sign of H (Figure 5b) or the

Figure 4 Kinetic study. a) Michaelis-Menten plot, b) Lineweaver-Burk plot.negative slope of vant Hoff plot (Figure 5b) implies thatthe reaction is endothermic.

Dye toxicity

en a) pH and the enzyme activity, b) pH and the dye concentration,The obtained results of toxicity evaluation of untreatedand laccase-treated dye solution showed that when AcidBlue 92 was used, the calculated GI% in the presenceof M. luteus, S. aureus, B. subtilis, E. coli, P. aeruginosa,and S. typhi was found to be 60 1.9, 49 1.4, 51 1.3,42 1.1, 30 1.1, and 37 0.8, respectively. However,the GI% for laccase treated dye solution was 31 3.9, 34 2.8, 27 2.5, 20 2.3, 17 0.4, and 14 0.9 in in the pres-ence of M. luteus, S. aureus, B. subtilis, E. coli, P. aerugi-nosa, and S. typhi, respectively, which was significantly

Rezaei et al. Journal of Environmental Health Science & Engineering (2015) 13:31 Page 8 of 9lower for all bacterial strains. Due to the probable tox-icity of produced metabolite following physicochemicalor enzymatic treatment of synthetic dyes, many proto-cols have been developed for determination the toxicityof dyes and related compounds based on the growthinhibition of bacterial (B. megaterium and E. coli), yeast(Saccaromyces cerevisiae), or animal cells (human cer-vix cells; HeLa), and phytotoxicity studies on plantseeds (Oryza sativa or Triticum aestivum) [1,3]. In thepresent work, the toxicity of laccase-treated dye samplewas significantly reduced compared to that of parentdye. Same results was reported by Palmieri et al. [28]who determined 95% viability for B. cereus in the pres-ence of sample obtained from enzymatic elimination ofRemazol Brilliant Blue R (an anthraquinone dye) usingthe purified laccase of Pleurotus ostreatus. The growthof B. megaterium and E. coli were reported to be 99%and 94%, respectively, in the presence of laccase-treated Malachite Green (a triphenylmethane dye). Ingeneral, azo dyes have been shown to be more resistantto enzymatic removal compared to other classes ofsynthetic dyes (like anthraquinone dyes) and providehigher toxicity after enzymatic removal [3,29]. How-ever, Champagne and Ramsay [30] reported lower tox-icity for azo dyes (Acid red 27 and Reactive black 5)

Figure 5 Energetic Study. a) dependence of decolorization rate on tempeenergy changes plot.compared to anthraquinone dyes (Reactive blue 19 andDispersed blue 3) after treatment using free and immo-bilized laccases.

ConclusionResponse surface methodology is an important tool tooptimize the conditions for textile dye wastewater treat-ment. Such statistical approach reduces the number ofruns and provides valuable information on possibleinteractions between the variables and response. Thepresent study was designed to optimize the reactioncondition for laccase- catalyzed decolorization of AcidBlue 92. The results indicated pH, enzyme activity, anddye concentration as the most important variables inthe enzymatic decolorization of Acid Blue 92. Furtherstudies should be performed on the decolorization ofdyes from different families such as triphenylmethan,indigo, and anthraquinone dyes, extensively employingin the textile and chemical industries.

Competing interestsThe authors declare that they have no competing interests.

Authors informationCorrespondence for statistical experimental design; Mohammad RezaKhoshayand.

rature (1050C), b) Arrhenius plot, c) vant Hoff plot and, d) Gibbs free

Rezaei et al. Journal of Environmental Health Science & Engineering (2015) 13:31 Page 9 of 9Authors contributionsSR and HT carried out the decolorization studies of Acid Blue 92. Productionand purification of laccase from P. variabile culture and detoxification studieswas performed by HF and AA. MM contributed in writing of the manuscript.MRK involved in design of removal experiments, analyzing of data andreviewing of the manuscript. MAF involved in purchasing of requiredmaterials and instruments, designing of removal experiments, analyzing ofdata and reviewing of the manuscript. All authors read and approved thefinal manuscript.

AcknowledgementThis work was supported financially by the grant No. 92-03-33-24540 fromTehran University of Medical Sciences, Tehran, Iran to M.A.F.

Author details1Department of Pharmaceutical Biotechnology, Faculty of Pharmacy andBiotechnology Research Center, Tehran University of Medical Sciences, P.O.Box 141556451, Tehran 1417614411, Iran. 2Pharmaceutical SciencesResearch Center, Tehran University of Medical Sciences, Tehran 1417614411,Iran. 3Department of Medicinal Chemistry, Faculty of Pharmacy, KermanUniversity of Medical Sciences, Kerman, Iran. 4Department of PharmaceuticalBiotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences,Kerman, Iran. 5Department of Drug and Food Control, Faculty of Pharmacyand Pharmaceuticals Quality Assurance Research Center, Tehran University ofMedical Sciences, Tehran 1417614411, Iran.

Received: 14 November 2014 Accepted: 7 April 2015

References1. Ashrafi SD, Rezaei S, Forootanfar H, Mahvi AH, Faramarzi MA. The enzymatic

decolorization and detoxification of synthetic dyes by the laccase from asoil-isolated ascomycete, Paraconiothyrium variabile. Int Biodeter Biodegr.2013;85:17381.

2. Mirzadeh SS, Khezri SM, Rezaei S, Forootanfar H, Mahvi AH, Faramarzi MA.Decolorization of two synthetic dyes using the purified laccase ofParaconiothyrium variabile immobilized on porous silica beads. J EnvironHealth Sci Eng. 2014;12:9.

3. Saratale RG, Saratale GD, Chang JS, Govindwar SP. Bacterial decolorizationand degradation of azo dyes: a review. J Taiwan Ins Chem Eng.2011;42:13857.

4. Khlifi R, Belbahri L, Woodward S, Ellouz M, Dhouib A, Sayadi S.Decolourization and detoxification of textile industry wastewater by thelaccase-mediator system. J Hazard Mater. 2010;175:8028.

5. Aghaie-Khouzani M, Forootanfar H, Moshfegh M, Khoshayand MR, FaramarziMA. Decolourization of some synthetic dyes using optimized culture brothof laccase producing ascomycete Paraconiothyrium variabile. Biochem Eng J.2012;60:915.

6. Gholami-Borujeni F, Mahvi AH, Nasseri S, Faramarzi MA, Nabizadeh R,Alimohammadi M. Enzymatic treatment and detoxification of acid orange 7from textile wastewater. Appl Biochem Biotechnol. 2011;165:127484.

7. Gholami-Borujeni F, Faramarzi MA, Nejatzadeh-Barandozi F, Mahvi AH.Oxidative degradation and detoxification of textile azo dye by horseradishperoxidase enzyme. Fresenius Environ Bull. 2013;22:73944.

8. Gholami-Borujeni F, Mahvi AH, Naseri S, Faramarzi MA, Nabizadeh R,Alimohammadi M. Application of immobilized horseradish peroxidase forremoval and detoxification of azo dye from aqueous solution. Res J ChemEnviron. 2011;15:21722.

9. Asadgol Z, Forootanfar H, Rezaei S, Mahvi AH, Faramarzi MA. Removal ofphenol and bisphenol-A catalyzed by laccase in aqueous solution. J EnvironHealth Sci Eng. 2014;12:93.

10. Mogharabi M, Faramarzi MA. Laccase and laccase-mediated systems in thesynthesis of organic compounds. Adv Synt Catal. 2014;356:897927.

11. Forootanfar H, Movahednia MM, Yaghmaei S, Tabatabaei-Sameni M,Rastegar H, Sadighi A, et al. Removal of chlorophenolic derivatives by soilisolated ascomycete of Paraconiothyrium variabile and studying the role ofits extracellular laccase. J Hazard Mater. 2012;209210:199203.

12. Ostadhadi-Dehkordi S, Tabatabaei-Sameni M, Forootanfar H, Kolahdouz S,

Ghazi-Khansari M, Faramarzi MA. Degradation of some benzodiazepines bya laccase-mediated system in aqueous solution. Bioresour Technol.2012;125:3447.13. Mogharabi M, Nassiri-Koopaei N, Bozorgi-Koushalshahi M, Nafissi-Varcheh N,Bagherzadeh G, Faramarzi MA. Immobilization of laccase in alginate-gelatinmixed gel and decolorization of synthetic dyes. Bioinorg Chem Appl.2012;2012:6.

14. Jiang M, Ten Z, Ding S. Decolorization of synthetic dyes by crude andpurified laccases from Coprinus comatus grown under different cultures: Therole of major isoenzyme in dyes decolorization. Appl Biochem Biotechnol.2013;169:66072.

15. Roriz MS, Osma JF, Teixeira JA, Rodrguez-Couto S. Application of responsesurface methodological approach to optimise Reactive Black 5decolouration by crude laccase from Trametes pubescens. J Hazard Mater.2009;169:6916.

16. Murugesan K, Dhamija A, Nam IH, Kim YM, Chang YS. Decolourization ofreactive black 5 by laccase: optimization by response surface methodology.Dyes Pigm. 2007;75:17684.

17. Tavares APM, Cristvo RO, Loureiro JM, Boaventura RAR, Macedo EA.Application of statistical experimental methodology to optimize reactive dyedecolourization by commercial laccase. J Hazard Mater. 2009;162:125560.

18. Grassi E, Scodeller P, Filiel N, Carballo R, Levin L. Potential of Trametes trogiiculture fluids and its purified laccase for the decolorization of differenttypes of recalcitrant dyes without the addition of redox mediators. IntBiodeter Biodegr. 2011;65:63543.

19. Myers RH, Montgomery D. Response surface methodology: Process andproduct optimization using designed experiments. 2nd edition, Wiley, 2002.

20. Forootanfar H, Faramarzi MA, Shahverdi AR, Tabatabaei Yazdi M. Purificationand biochemical characterization of extracellular laccase from theascomycete Paraconiothyrium variabile. Bioresour Technol. 2011;102:180814.

21. Faramarzi MA, Forootanfar H. Biosynthesis and characterization ofgoldnanoparticles produced by laccase from Paraconiothyrium variabile.Colloids Surf, B. 2011;87:237.

22. Alberts JF, Gelderblom WCA, Botha A, Vanzyl WH. Degradation of aflatoxinB1 by fungal laccase enzymes. Int J Food Microbiol. 2009;135:4752.

23. Annuar MSM, Adnan S, Vikineswary S, Chisti Y. Kinetics and energetics ofazo dye decolorization by Pycnoporus sanguineus. Water Air Soil Pollut.2009;202:17988.

24. Khouni I, Marrot B, Amar RB. Decolourization of the reconstituted dye batheffluent by commercial laccase treatment: Optimization through responsesurface methodology. Chem Eng J. 2010;156:12133.

25. Daassi D, Zouari-Mechichi H, Frikha F, Martinez JM, Nasri M, Mechichi T.Decolorization of the azo dye Acid Orange 51 by laccase produced in solidculture of a newly isolated Trametes trogii strain. 3. Biotech. 2013;3:11525.

26. Baldrian P. Fungal laccases occurrence and properties. FEMS MicrobiolRev. 2006;30:21542.

27. Guan ZB, Song CM, Zhang N, Zhou W, Xu CW, Zhou LX, et al. Overexpression,characterization, and dye-decolorizing ability of a thermostable, pH-stable, andorganic solvent-tolerant laccase from Bacillus pumilus W3. J Mol Catal B: Enzym.2013;101:16.

28. Palmieri G, Cennamo G, Sannia G. Remazol Brilliant Blue R decolourisationby the fungus Pleurotus ostreatus and its oxidative enzymatic system.Enzyme Microb Technol. 2005;36:1724.

29. Couto SR. Decolouration of industrial azo dyes by crude laccase fromTrametes hirsute. J Hazard Mater. 2007;148:76870.

30. Champagne P-P, Ramsay JA. Dye decolorization and detoxification by laccaseimmobilized on porous glass beads. Bioresour Technol. 2010;101:22305.

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AbstractBackgroundResultsConclusions

IntroductionMaterials and methodsChemicalsLaccase assayDye decolorization experimentsExperimental design and statistical analysisScreening studyOptimization study

Dye removal kinetics and energeticsKinetics of decolorization

Thermodynamics of decolorizationMicrotoxicity studies

Results and discussionFractional factorial design for screening of important variablesD-optimal design response surface methodologyOptimizationValidation of modelKinetics and energetics of decolorizationKinetic studyEnergetic studyDye toxicity

ConclusionCompeting interestsAuthors informationAuthors contributionsAcknowledgementAuthor detailsReferences