Thermokinetic Comparison of Trypan Blue Decolorizationby Free Laccase and Fungal Biomass

N. N. A. Razak & M. S. M. Annuar

Received: 8 February 2013 /Accepted: 6 January 2014# Springer Science+Business Media New York 2014

Abstract Free laccase and fungal biomass from white-rot fungi were compared in thethermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue.The decolorization in both systems followed a first-order kinetics. The apparent first-order rateconstant, k1′, value increases with temperature. Apparent activation energy of decolorizationwas similar for both systems at ∼22 kJ mol−1, while energy for laccase inactivation was18 kJ mol−1. Although both systems were endothermic, fungal biomass showed higherenthalpy, entropy, and Gibbs free energy changes for the decolorization compared to freelaccase. On the other hand, free laccase showed reaction spontaneity over a wider range oftemperature (ΔT=40 K) as opposed to fungal biomass (ΔT=15 K). Comparison of entropychange (ΔS) values indicated metabolism of the dye by the biomass.

Keywords Azo dye . Decolorization . Laccase . Thermodynamic . Kinetics . White-rot fungi

Introduction

Synthetic dyes are used in industries such as textile, paper, pharmaceutical, cosmetics, andfood industries [1]. Due to large-scale production and extensive applications, synthetic dyescan cause considerable environmental pollution and serious health-risk factors [2]. They areretained on the substrates by physical adsorption, chemical interactions with metals and salts,mechanical retaining, and solution or via covalent bonding. Decolorization of these dyes byphysical or chemical adsorption and precipitation methods are usually time-consuming andmostly ineffective [3]. Furthermore, these methods cause accumulation of the dye as sludgeand create a disposal problem later on. Hence, extensive efforts are being focused on biologicalprocesses, as they are relatively cost-effective and environmentally friendly provided that thedyes are metabolizable.

Appl Biochem BiotechnolDOI 10.1007/s12010-014-0731-7

N. N. A. Razak :M. S. M. Annuar (*)Institute of Biological Sciences, Faculty of Science, University Malaya, 50603 Kuala Lumpur, Malaysiae-mail: suffian_annuar@um.edu.my

M. S. M. AnnuarCentre for Research in Biotechnology for Agriculture (CEBAR), University of Malaya, 50603 KualaLumpur, Malaysia

Laccase (EC 1.10.3.2; benzenediol/oxygen oxidoreductase) has been the focus of manystudies due to its reaction versatility and potential biotechnological applications. It is amultinuclear copper-containing enzyme which uses molecular oxygen to oxidize a widevariety of aromatic compounds [4]. They are widely distributed in nature, originating fromplants, insects, bacteria, and especially fungi. In fact, they are primarily found in fungi andinvolved in lignin degradation, pigment biosynthesis, and detoxification of lignin-derivedproducts. White-rot fungi produce three main extracellular enzymes involved in ligninolysisviz. laccase, lignin peroxidase, and manganese peroxidase [5].

Laccase has been indicated as capable of oxidizing amines, phenolic and nonphenolic lignin-related compounds, and also highly recalcitrant environmental pollutants such as synthetic dyesby a complex ligninolytic enzymatic system [6, 7]. Due to their versatility and adaptability,bioremediation using laccase has been extensively studied for water remediation. Studies haveshown that laccase from Tramates versicolor [8], Trametes hirsuta [9], Trametes villosa [10],Trametes trogii [11], Trametes modesta [12], Coriolus versicolor [13], Pcynoporus sanguineus[14], Pycnoporus cinnabarinus [15], and Pleurotus ostreatus [16] could be used for the degra-dation of a diverse chemical structure of dyemolecules. Azo dyes are the major group of syntheticdyes, which are produced in large quantities and have become a major source of concern forchromophoric pollution. Besides causing esthetic damage, they are also toxic and carcinogenic[17]. The decolorization of azo dyes by white-rot fungi has been reported [18–23]. However,comparative studies on the thermodynamics and kinetics aspects of azo dyes decolorization byfree laccase and fungal biomass from white-rot fungi are lacking. Understanding these aspects isimportant for rational design of treatment process for synthetic dyes run offs. Therefore, in thiswork, the decolorization thermokinetics of Trypan Blue, an azo dye by free laccase enzyme wasinvestigated, and this was subsequently compared to the thermokinetics of decolorization of thesame dye by fungal biomass of a white-rot fungus, i.e., Pycnoporus sanguineus.

Materials and Methods

Optimization Parameters for Dye Decolorization

The first part of this study investigated the effects of selected variables on the decolorization ofTrypan Blue by free laccase, i.e., enzyme concentration, reactant concentration, pH, andtemperature.

Commercial laccase from Trametes versicolor (Sigma-Aldrich) with specific activity0.8 U mg−1 was used in this experiment without further purification. Laccase was preparedby dissolving the enzyme in 50 mM sodium citrate buffer (pH 4.8). The desired laccaseconcentrations (0.008, 0.01, 0.03, 0.05, 0.07, 0.10 U ml−1) were obtained by stock dilution.Syringaldazine (4-hydroxy-3,5-methoxybenzaldehyde; Sigma) was used as the substrate forthe enzyme activity assay. Of the syringaldazine, 1 mM stock was prepared by dissolving it in99.5 % ethanol and the solution was kept at 4 °C. Syringaldazine concentrations (0.02, 0.06,0.10, 0.20, 0.30, 0.40, and 0.50 mM) were prepared from the stock solution by dilution with50 % ethanol. The syringaldazine stock solution was warmed to room temperature before use.To investigate the effect of pH, 50 mM sodium citrate buffer was prepared for a pH range of 3to 6. Selection of different pH of sodium citrate buffer was based on the nearest pKa values ofthe citric acid. By referring to the pKa values of citric acid (3.13, 4.76, and 6.40), pH of sodiumcitrate buffer chosen were 3, 4, 4.8, 5, and 6. Similarly, a series of experiments were performedto investigate the effect of temperature within the range of 4 to 80 °C on laccase activities.Three independent replicates were made for every experiments conducted.

Appl Biochem Biotechnol

Enzyme Assay

Laccase activity was measured by monitoring the rate of oxidation of syringaldazine by theenzyme at 25±1 °C. Of the laccase solution, 0.2 mL was mixed with 3.0 ml of 50 mM citratebuffer (pH 4.8) in a cuvette. Of the syringaldazine, 0.2 mL of 0.1 mM was added and gentlymixed, and the absorbance was measured immediately at 525 nm for 10 min using UV/VISscanning spectrophotometer Jasco V-630 (Japan). Total reaction volume was 3.4 mL. Laccaseactivity was calculated as shown in Eq. (1),

Laccase activity U�L

� � ¼ ΔAbs

t� l

ε� total assay enzyme

enzyme sampleð1Þ

whereΔAbs is the change of absorbance at 525 nm, t is the incubation time (10 min), ε is theextinction coefficient for syringaldazine (ε525=65,000 M−1 cm−1), and l cm is the light pathlength (1 cm). One unit activity is defined as the amount of laccase that oxidizes 1 μmol ofsyringaldazine per minute.

Trypan Blue Decolorization Assay

Trypan Blue (C34H24N6O14S4Na4, molar mass of 961 Da) was used throughout the experi-ment. A stock solution of Trypan Blue was prepared by dissolving 60 mg of the dye in 1 L of50 mM of sodium citrate buffer (pH 4.8) and stored in an amber bottle to protect it from directsunlight. The solution was kept at 4 °C and was diluted in appropriate concentrations (10, 20,30, 40, and 50 mg L−1) before use.

Batch decolorization process was initiated by adding laccase to 100 mL dye solution inErlenmeyer flasks, which contained different concentrations of Trypan Blue. Initial laccaseactivity was determined at room temperature (25±1 °C) to ensure that all flasks had similarlevel of enzyme activities. The final enzyme concentration was 30±1 U L−1. The reactionflasks were incubated on a rotary shaker incubator at 150 rpm and were monitored at intervalstime for 48 h at different temperatures (288, 298, 303, 308, 318, and 328 K). Controlexperiments with heat-denatured enzyme (100 °C, 30 min) were also conducted in parallel.The assays were done in triplicates.

Dye concentration was routinely measured using spectrophotometer at 597 nm. Thecalibration of Trypan Blue concentration fitted the following equation:

A597 ¼ 0:0244Cdye ð2Þ

where A597 was the absorbance of the solution at 597 nm and Cdye was the concentration of thedye in milligram per liter. Equation (2) had a regression coefficient of 0.9999, which indicatedthat the assay model was reliable to determine the dye concentration with acceptable precision.Equation (2) was applied over a concentration range of 0 to 60 mg L−1.

Calculations

Trypan Blue Decolorization

Residual dye concentration was measured at regular intervals for up to 48 h. The fraction of thedye decolorized was calculated using Eq. (3).

Appl Biochem Biotechnol

Mdecolorized ¼ M initial−M residual

M initialð3Þ

where Mdecolorized is the fractional percentage of Trypan Blue decolorized, Minitial is the initialdye concentration, and Mresidual is the remaining dye concentration in the solution.

Rate of Dye Decolorization

Volumetric rate of dye decolorization (rvol, in milligram per liter per hour) was calculated usingEq. (4):

rvol ¼ ΔC

Δtð4Þ

where ΔC (in milligrams per milliliter) is the change in dye concentration over the timeinterval Δt (in hours).

Activation Energy

The activation energy (Ea, in joule per mole) of decolorization process was calculated using thelinearized Arrhenius equation as shown in Eq. (5):

lnk01 ¼ lnA−

Ea

RTð5Þ

where A is the frequency factor, R is the gas constant (8.3145 J mol−1 K−1), and T is theabsolute temperature (in Kelvin). To calculate k1′, the volumetric rate of dye decolorization(Eq. 4) was plotted against different initial dye concentrations for each temperature tested (288,298, 303, 308, 318, and 328 K). The rate constant k1′ was obtained from the slope of theresulting linear plot.

Thermodynamic Parameters

The decolorization reaction is assumed to be at equilibrium state when the remaining dye insolution showed no further changes in concentration over time at a particular temperature. Thetransition of the dye color was also considered a one-step process, thus the apparent equilib-rium constant Kapp was calculated as follows:

Kapp ¼P½ �eqSð Þeq

ð6Þ

where Kapp is the apparent equilibrium constant, [P]eq is the concentration of dye that has beendecolorized at equilibrium ([P]eq=initial dye concentration−dye concentration remaining atequilibrium), and [S]eq is the residual dye concentration at equilibrium.

Kapp was measured at various temperatures. The temperature dependence of Kapp isexpressed according to van’t Hoff equation as follows:

lnKapp ¼ −ΔH

RTþ ΔS

Rð7Þ

where apparent enthalpy (ΔH) is van’t Hoff enthalpy (in joule per mole) and entropy (ΔS) isentropy (in joule per mole per Kelvin).

Appl Biochem Biotechnol

At constant pressure and temperature, Gibbs free energy change (ΔG, in joule per mole) forthe reaction at nonstandard conditions was calculated using the following equation:

ΔG ¼ ΔH−TΔS ð8ÞGibbs free energy at standard condition (ΔG°, in joule per mole) was calculated as follows:

ΔG ¼ ΔG−RT lnKapp ð9Þwhere Kapp is the apparent equilibrium constant at standard conditions.

Results and Discussion

Optimized Parameters for Dye Decolorization

Laccase Concentration

A linear relationship was observed between initial rate of reaction and the enzyme concentra-tion within 8 to 70 U L−1 ranges. Within 10 s from the start of the assay, the percentage ofsubstrate (dye) converted to product was calculated so that only 5 % or less of dye decolorizedwithin this period. This was done to ensure that at any enzyme concentration tested, thesubstrate was still in excess amount and thus give accurate estimation for the initial rate ofreaction. From the straight line obtained, 30 U L−1 laccase was chosen as the optimum laccaseconcentration for decolorization.

Effect of Reactant Concentration

A rectangular hyperbolic graph was obtained when initial rate of reaction was plotted againstsyringaldazine concentration (Fig. 1). From the graph, it was clear that at lower syringaldazineconcentrations ranging from 0.02 to 0.10 mM, the rate of reaction is directly proportional tothe syringaldazine concentration. However, at higher substrate concentrations (0.20–0.50 mM)the initial rate of reaction was constant. This indicated that the oxidation activity was at themaximum and the active sites of the enzyme were virtually saturated with the substrates. Anyfurther addition of the substrate will not alter the rate of reaction. Thus, 0.20 mM of

Concentration of Syringaldazine (mM)

0.0 0.2 0.4 0.6

Initi

al r

ate

of r

eact

ion

(µm

ol m

Lm

in-1

-1)

0.00

0.01

0.02

0.03

0.04

0.05Fig. 1 Initial rate of reaction fordifferent syringaldazineconcentrations

Appl Biochem Biotechnol

syringaldazine was taken to be the optimum reactant concentration for laccase activity assay.

Effect of Buffer pH

As shown in Fig. 2, it was clear that pH significantly influenced the laccase activity. Within apH range of 4.8 and 5, the laccase activities were very similar. However, sharp decline inactivities were observed at pH 3 and 6 (Fig. 2), which might be resulted from improper ionicform of the laccase’s active site and the substrate. pH stability of an enzyme depends on manyfactors including ionic strength and chemical nature of the buffer. Based on the ANOVA test,all pH showed significant differences except for pH 4.8 and 5. Both pHs exhibited highestinitial rate of reaction, so either pH (i.e., pH 4.8 or 5) is expected to be similarly suitable for theenzyme. For decolorization experiment, 50 mM sodium citrate buffer at pH 4.8 was employed.

Effect of Temperature on Laccase Activities

Comparable laccase activities were observed within temperature range of 4 to 40 °C, i.e.,124 U L−1 (±<1 %). However, the enzyme activities started to decrease at 60 °C, i.e., 68 U L−1

(±<1 %), and no observable activity was recorded at 80 °C. Thus, the temperature range of 15–55 °C applied for the Trypan Blue decolorization in this study were reasonable in which theenzyme was thermally stable and able to retain its activities.

Effect of Temperature on Dye Decolorization

The amount of Typan Blue decolorized at equilibrium over its initial concentration at differenttemperatures is shown in Fig. 3. At any given temperature, the amount of dye decolorizedincreased linearly with increasing initial concentration of Trypan Blue. The magnitude of dyedecolorization increased with increasing temperature (288–308 K) but a decline in the amountof dye decolorized was evident at 318 and 328 K, which indicated that at this temperature theenzyme may have been thermally inactivated. Thus, dye decolorization will increase with theincrease in temperature but only up to a certain degree, above which the dye decolorizationwill decrease due to the denaturation of the enzyme. No decolorization occurred at anytemperature in control experiments (data not shown).

pH

3 4 5 6

Initi

al r

ate

of r

eact

ion

(µm

ol m

L-1m

in-1

)

0.000

0.005

0.010

0.015

0.020

0.025

0.030Fig. 2 Laccase activity at differentpH of 50 mM sodium citratebuffer

Appl Biochem Biotechnol

Table 1 showed the maximum decolorization (63 %) was observed after 48 h of incubation.Hence, the optimum temperature for Trypan Blue decolorization by laccase was observed to bewithin the range of 298 to 308 K since comparable results were obtained. According toSadhasivam et al. [24], decolorization of Trypan blue by laccase from Trichoderma harzianumreached about 33 % with laccase alone and 67 % in the presence of synthetic redox mediator 1-hydroxybenzotriazole at lower dye concentrations than were used in this study. In otherstudies, D’Sauza et al. [25] and Baldrian et al. [26] reported that Trypan Blue was decolorizedonly up to 25 % by marine fungus NIOCC #2a and 42 % by a white-rot fungus Daedaleaquercina. It is clear that the maximum percentage of Trypan Blue decolorization in this studywas considerably higher than in the previously reported literature. This was despite the higherconcentration of Trypan Blue used in this study, and the absence of any type of added syntheticredox mediator(s), which could be used to enhance the rate of decolorization.

The lowest percentage of Trypan Blue decolorization was observed at the highest temper-ature used, i.e., 328 K (Table 1). It was observed that for any dye concentration investigated atthis temperature, decolorization activities could be sustained only for the first 6 h. After that,the activities decreased and no further change in the concentration of the residual dye wasobserved. These results were in accordance with Monteiro et al. [27] which found laccase fromT. versicolor was stable for 6 h at 55 °C (328 K).

Table 1 Percentage of Trypan Blue decolorization at different temperatures

Concentration of TrypanBlue (mg L−1)

Percentage of decolorization (%) at equilibrium at different temperatures (K)

288 298 303 308 318 328

10 53 62 62 60 57 23

20 55 62 63 63 57 30

30 53 62 63 62 58 32

40 53 62 62 60 59 30

50 53 61 62 61 59 28

Standard deviation of the triplicate measurements was <5 %

Initial concentration of Trypan Blue (mg/L)

Dye

dec

olor

ized

at e

quili

briu

m (

mg/

L)0

0 10 20 30 40 50 60

5

10

15

20

25

30

35

288 K298 K303 K308 K318 K328 K

Fig. 3 Amount of Trypan Bluedecolorized at equilibrium as afunction of its initial concentrationand temperature

Appl Biochem Biotechnol

Apparent First-Order Rate Constant, k1′

For the determination of apparent rate constant (k1′), the graph of the amount of dyedecolorized against 48 h of incubation time at various concentration of dye was plotted foreach temperature tested (data not shown). Then, from the slope of the graph, volumetric rate ofdecolorization was calculated using Eq. (4). Then, the volumetric rates of decolorization wereplotted against initial concentrations of Trypan Blue in order to estimate the k1′ of the dyedecolorization (Table 2).

It was observed that the volumetric decolorization rate is directly proportional to the initialdye concentration (10–50 mg L−1) at any given temperature (288–328 K; Fig. 4). For the freelaccase system studied, the volumetric rate for every initial dye concentration tested showed alinear increase with temperature. Similar observation was made in the fungal biomass (pellet)system investigated earlier [28]. For the free enzyme system, highest volumetric rate wascalculated at 14.1 mg L−1 h−1 for the highest initial dye concentration (50 mg L−1) at 308 K.The apparent kinetics of the decolorization followed a first-order behavior. As the temperatureincreases, the slope of the line became steeper, which reflected the increase in k1′ values(Table 2). A higher k1′ values implies faster dye decolorization while a decrease in k1′ value isattributed to inactivation of laccase enzyme at higher temperatures (318–328 K).

Decolorization of dyes by Trametes spp. is shown to be following a first-order kinetics inprevious studies. For example, T. modesta showed an apparent first-order kinetics for thedecolorization of various azo dye including CI Acid Orange 5, CI Acid Orange 52, CI DirectBlue 71, CI Reactive Black 5, Orange 16, and CI Reactive Orange 107 [18]. First-orderdecolorization kinetics are also observed during the decolorization of an azo dye, Amaranth byT. versicolor [29], in decolorization of both azo and anthraquinone dye by laccase andmanganese peroxidase from T. versicolor [30] and in the decolorization of Amaranth, ReactiveBlack 5, Reactive Blue 19, and Direct Black 22 by alginate-immobilized T. versicolor [8].

Apparent Activation Energy of Decolorization

Based on k1′ values obtained at different temperatures (Table 2), the apparent activation energy(Ea) for the decolorization was estimated using Arrhenius plot (Eq. 5). A plot of the ln k1′ vs.reciprocal temperature was obtained with a regression coefficient of 0.9874 (data not shown).From the slope of the line, the Ea value was calculated at 21 kJ mol−1, which is consistent withactivation energy of enzyme-catalyzed reactions, i.e., 16–84 kJ mol−1. This value is almost

Table 2 First-order rate constants for the decolorization at different temperatures

Temperature (K) Volumetric rate (mg L−1 h−1) at different concentrations ofTrypan Blue (mg L−1)

Apparent first-order rateconstant, k1′ (h

−1)

10 20 30 40 50

288 1.4 3.2 4.7 6.4 8.1 0.16±0.009

298 1.7 4.0 5.9 8.2 10.2 0.20±0.007

303 2.3 4.9 7.2 10.0 12.3 0.25±0.003

308 2.8 5.6 8.3 11.1 14.1 0.28±0.005

318 2.0 4.2 6.2 8.6 10.8 0.21±0.003

328 1.9 3.7 6.0 7.4 7.7 0.17±0.007

Standard deviation of the triplicate measurements was<5 %

Appl Biochem Biotechnol

identical to the decolorization of Trypan Blue using self-immobilized fungal biomass ofP. sanguineus previously reported with Ea value 23 kJ mol−1 [28]. Other reported activationenergy for laccase was 21.175 kJ mol−1 for phenol polymerization [31], 57 kJ mol−1 for 1-napthol polymerization [32], and 44.8 kJ mol−1 for chlorophenol degradation [33], respective-ly. In this study, laccase-mediated decolorization activities were significantly reduced at 318 to328 K. Thus, using Eq. (5), apparent inactivation energy for laccase catalyzed decolorizationof Trypan Blue, Ed was calculated at 18 kJ mol−1.

Thermodynamics of Decolorization

Employing Van’t Hoff equation (Eq. 7),ΔH andΔS were determined from the slope and the yintercept of the straight line (Fig. 5). A plot of ln Kapp against reciprocal temperature fitted astraight line with a regression coefficient of 0.9446 (Fig. 5).

Kapp refers to the apparent equilibrium constant (Eq. 6) for decolorization process, whichwas calculated for the highest dye concentration, i.e., 50 mg L−1 at each temperature tested

Initial Concentration of Trypan Blue (mg L-1)

0 10 20 30 40 50 60

Rat

e of

dec

olor

izat

ion

(mg

L-1

h-1)

0

2

4

6

8

10

12

14

16

298 K298 K303 K308 K318 K328 K

Fig. 4 Apparent first-order rate constant, k1′ (in hours) as a function of its initial concentration and temperature

1/T (K)0.00320 0.00325 0.00330 0.00335 0.00340 0.00345 0.00350

ln K

app

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.9446R

7044.45.13202

xy ==

+−

Fig. 5 Van’t Hoff plot fordecolorization of Trypan Blueby free laccase

Appl Biochem Biotechnol

(Table 3). The Kapp value increases with the increase in temperature until 318 K and abovewhere it decreased.

From this work, ΔH and ΔS were calculated at 11 kJ mol−1 and 39 J mol−1 K−1, respec-tively. The positive value of ΔH for the processes implies the endothermic nature of thedecolorization system, whereas the positive value ofΔS reflects a decolorization system that isbecoming increasingly disordered as the temperature increases.

The values of enthalpy and entropy changes obtained subsequently were used in thecalculation of Gibbs free energy change, ΔG using Eq. (8). The ΔG value indicates thedegree of spontaneity of the decolorization process. A strong negative value reflects a moreenergetically favorable process [34]. As shown in Table 3, decolorization reaction occursspontaneously for all temperatures studied (288–328 K). Thus, the Trypan Blue decolorizationcatalyzed by laccase is an energetically favorable process and spontaneous in nature. More-over, it may also be noted that as more heat is supplied to the system, the greater the tendencyof the system to move towards decolorization. This is shown by strong increase in the valueΔG° as temperature increases. This could be explained as follow; as temperature increases,more heat is absorbed (ΔH) during the process resulting in the increase of thermal energy ofthe system which causes more energetically favorable interaction between laccase and dyeresulting in increased chances of successful decolorization.

From Table 3, the value of (ΔG−ΔG°) indicates the differences between the energy changeoccurring during the reaction under the conditions used and the energy change that wouldoccur if the reaction took place under standard conditions (1 atm, 1 M, 298 K). A graph ofΔG−ΔG° as a function of absolute temperature (in Kelvin) and initial dye concentration (inmilligram per liter) is shown in Fig. 6. An increase in energy surplus is clearly seen attemperature range of 288–308 K and a slight decrease at 318 K but is still positive in thevalue of (ΔG−ΔG°). In contrast, huge energy deficit was observed at the highest temperature,i.e., 328 K as indicated by the steep plunge (Fig. 6).

A plot of theΔG for the decolorization reaction at various temperatures is shown in Fig. 7.The ΔG values were calculated using Eq. (8) with the assumption that the ΔH and ΔScalculated earlier for free laccase system are constant within the temperature range examined.It showed ΔG=0 at approximately 280 K (or 7.28 °C), which means that at this temperature,the energy of the products and reactants are at the equilibrium. As described before, values ofΔG were negative for all temperatures studied (288–328 K) showing that Trypan Bluedecolorization by laccase is favored when temperature is greater than 7.28 °C. Meanwhile,extrapolated graph showed that the reaction is nonspontaneous (i.e., ΔG is positive) below7.28 °C (280 K).

Energetics of Trypan Blue decolorization using fungal biomass (pellets) of Pycnoporoussanguineus [28] was compared with the results obtained in this study. P. sanguineus is known toproduce laccase as its sole lignin degradation enzyme (phenoloxidase). According to Fig. 7, a

Table 3 Gibbs free energy (ΔG) of decolorization at 50 mg L−1

Temp (°C) Absolute temperature, T (K) Apparent Kapp ΔG (J mol−1) ΔG−ΔG° (J mol−1)

15 288 1.15 −299 +339

25 298 1.29 −685 +640

30 303 1.37 −879 +795

35 308 1.57 −1,072 +1,162

45 318 1.46 −1,459 +1,009

55 328 0.40 −1,846 −2,471

Appl Biochem Biotechnol

linear function of ΔG with temperature was observed for both free laccase and fungal pelletsystems. For fungal pellet system, the ΔH and ΔS values used in the calculation of ΔG weretaken from the study of [28]. It also illustrated that free laccase system has wider temperaturerange of spontaneity (ΔT=40 K) relative to fungal biomass system (ΔT=15 K). Moreover, freelaccase system was shown to be less sensitive to temperature variation as compared to fungalbiomass system. This implies that free laccase system can withstand relatively huge perturba-tion in temperature with minimal effects on the reaction spontaneity. In contrast, for fungalbiomass system huge perturbation in temperature may drastically affect the spontaneity of the

-4000

-3000

-2000

-1000

0

1000

2000

290295

300305

310315

320325

10

20

30

40

G -

G°

(J)

Temperature (K)

Concentration (m

g L -1)

-2500 -2000 -1500 -1000 -500 0

Fig. 6 Differences between Gibbs free energy change (ΔG) and Gibbs free energy change at standard condition(ΔG°) as a function of initial dye concentration (in milligrams per liter) and temperature (in Kelvin)

Temperature (K)

240 260 280 300 320 340

Gib

bs F

ree

Ene

rgy

(J)

-3000

-2000

-1000

0

1000

2000

3000

Free laccaseFungal pellet

(spontaneous)

G < 0

spontaneous)-(non

G > 01R²

1097539.11x -y

0G

Δ

Δ

Δ =

==

+

==+

Fig. 7 Comparison of Gibb free energy change as a function of temperature between free laccase and fungalpellet systems

Appl Biochem Biotechnol

reaction. ΔH value for fungal biomass system (ΔH=46 kJ mol−1) was shown to be fourfoldhigher as compared to free laccase system (ΔH=11 kJ mol−1). HugeΔH reflects more energyneed to be transferred to the system to make it favorable. Furthermore, due to highΔS value forfungal biomass system (ΔS=146 J mol−1 K−1), it is hypothesized that Trypan Blue wasmetabolized to byproduct(s) such as carbon dioxide, thus explaining the high ΔS value in thefungal biomass system compared to the free enzyme system (ΔS=39 J mol−1 K−1).

The study presented a strong support and rationalization for the technical utilization ofbiological catalysts such as laccase and/or its whole biomass system in the decolorization ofchromophore(s) contaminated water bodies. While the free enzyme system is able to toleraterelatively high temperature perturbation range as compared to the whole biomass system in thedecolorization process, the latter could be effectively use to metabolize the dye at the same time.

Acknowledgments The authors acknowledge University of Malaya for providing the research grants PG033-2013A, RP024-2012A, and UM.C/625/1/HIR/MOHE/05.

Conflict of Interests All the authors of the submission declare and clarify that we do not have a direct financialrelation with the commercial identities mentioned in the paper that might lead to a conflict of interest for any ofthe authors.

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3. Erkurt, E. A., Ünyayar, A., & Kumbur, H. (2007). Decolorization of synthetic dyes by white rot fungi,involving laccase enzyme in the process. Process Biochemistry, 42(10), 1429–1435. doi:10.1016/j.procbio.2007.07.011.

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