COMMUNICATION TO THE EDITOR

Ionic Liquids as Alternative Co-Solvents forLaccase: Study of Enzyme Activity and Stability

Ana Paula Mora Tavares, Oscar Rodriguez, Eugenia A. Macedo

Laboratory of Separation and Reaction Engineering (LSRE),

Departamento de Engenharia Quımica, Faculdade de Engenharia, Universidade do Porto,

Rua do Dr. Roberto Frias, 4200-465, Porto, Portugal; telephone: þ351-22-508-1653;

fax: þ351-22-508-1674; e-mail: eamacedo@fe.up.pt

Received 14 November 2007; revision received 18 February 2008; accepted 20 February 2008

Published online 7 March 2008 in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/bit.21866

ABSTRACT: The activity and stability of commercial laccase(DeniLite base) in three different water soluble ionic liquids(ILs) (1-ethyl-3-methylimidazolium 2-(2-methoxyethoxy)ethylsulfate, [emim][MDEGSO4], 1-ethyl-3-methylimida-zolium ethylsulfate, [emim][EtSO4], and 1-ethyl-3-methy-limidazolium methanesulfonate, [emim][MeSO3]) havebeen studied and compared to that in two organic solvents(acetonitrile and dimethyl sulfoxide). Initial enzyme acti-vities were similar among the ILs if the same conditions wereused. A high reduction on initial enzyme activity was foundwith acidic pH (5.0). The effect of pH and solvent concen-tration on enzyme stability were investigated in more detailfor 1 week. The enzyme maintained a high stability at pH 9.0for all ILs tested. [emim][MDEGSO4] was the most promis-ing IL for laccase with an activity loss of about 10% after7 days of incubation. The kinetic studies in the presenceof ABTS as substrate allowed to calculate the Michaelis–Menten parameters. Good agreement was found betweenexperimental data and calculated values using the Michaelis–Menten mechanism, with a total average relative deviationof 2.1%.

Biotechnol. Bioeng. 2008;101: 201–207.

� 2008 Wiley Periodicals, Inc.

KEYWORDS: ionic liquids; laccase; kinetic parameters;organic solvents; stability

Introduction

Ionic liquids (ILs) are commonly defined as substancescomposed only by ions which are liquid at or close to roomtemperature. They are getting increasing attention fromboth industry and academics, and research involving ILs isexpanding to many different areas of knowledge. Despite

Correspondence to: E.A. Macedo

Contract grant sponsor: LSRE

Contract grant number: FEDER/POCI/2010

Contract grant sponsor: Fundacao para a Ciencia e a Tecnologia (FCT, Portugal)

Contract grant number: SFRH/BPD/22697/2005; SFRH/BPD/24271/2005

� 2008 Wiley Periodicals, Inc.

their properties are not yet deeply understood, publicationsusually highlight their negligible vapor pressure, thermaland chemical stability, and the possibility to ‘‘design’’ theirphysicochemical properties by suitable choice of theanion and the cation (Earle and Seddon, 2000; Seddon,1997). Because of their negligible vapor pressure, ILs areclaimed as ‘‘green’’ alternatives for volatile organic solvents(VOCs).

The vast amount of publications regarding ionic liquidscovers many different topics (Rogers and Seddon, 2002,2003, 2005a,b), with the use as reaction media or as solventsfor separation processes (Brennecke and Maginn, 2001;Rogers et al., 2002; Wasserscheid and Welton, 2003; Welton,1999) being the most important. In recent years, ILs havealso been applied as solvents for biocatalysis: several studieshave been published regarding the activity and the stabilityof proteins in ionic liquids (Diego et al., 2005; Fujita et al.,2005, 2006; Lau et al., 2004; Lozano et al., 2001; Park andKazlauskas, 2003) and the performance of ILs as solvents forenzymatic reactions (Barahona et al., 2006; Baumann et al.,2005; Chiappe et al., 2007; Kragl et al., 2002; Lutz-Wahlet al., 2006; Sgalla et al., 2007). An excellent review hasrecently been published covering these issues (Rantwijk andSheldon, 2007). It can be seen from the above references thatsome ILs may be a suitable media (either as solvent orco-solvent) for enzymatic reactions (as they enhance theenzyme activity), or for enzyme storage (as they improve theenzyme stability). However, many aspects of relevance inbioprocesses have still been poorly studied, such as the effectof pH. An optimal pH is fundamental to maximize enzymeactivity. It has been demonstrated that enzyme activity withorganic solvents is very dependent on pH (Zaks andKlibanov, 1985) and also that the pH may be changed by thesolvent (Rantwijk and Sheldon, 2007). According to somestudies (Matsumoto et al., 2001; Persson and Bornscheuer,2003), enzyme pretreated with organic solvents led to anincrease in enzyme activity and stability. Therefore, it isexpected that a similar behavior should be obtained in ILs.

Biotechnology and Bioengineering, Vol. 101, No. 1, September 1, 2008 201

Thus, the study of this variable is essential to successfulapplication of ILs to enzymatic processes.

Most of the research conducted on biocatalysis in ILsrefers to lipases. Also data for proteases and peroxidasescan be found. Nevertheless, to our best knowledge, thereare very few data available concerning laccase. Laccase(benzenediol: oxygen oxidoreductase, EC 1.10.3.2) is one ofthe most interesting enzymes studied for degradation ofphenolic compounds. Phenols such as hydroquinone,catechols, guaiacol and 2,6-dimethoxyphenol are goodsubstrates for laccase in aqueous media. However, mostphenolic compounds present a low solubility in water, suchas bisphenol A (Okazaki et al., 2002). The presence of a co-solvent can increase the solubility of phenolic compounds,thus raising the capacity of a bioreactor and avoiding theappearance of two phases.

The oxidation by laccase is a one-electron reaction withthe reduction of oxygen to water (Riva, 2006). Fungal laccasehas been intensively studied in aqueous media becauseof their large industrial application in pulp delignification(Gamelas et al., 2005), bioremediation (Chiacchierini,2004), decolourization of dyes (Amaral et al., 2004) andothers. Although there are also several studies about the useof laccase in organic solvents (Mustafa et al., 2005; Nowakand Wilkołazka, 2007), they can affect the enzyme activityand so limit their application.

In this work, we describe for the first time the study oflaccase activity and stability in three different ILs: 1-ethyl-3-methylimidazolium 2-(2-methoxyethoxy) ethylsulfate,[emim][MDEGSO4], 1-ethyl-3-methylimidazolium ethyl-sulfate, [emim][EtSO4], and 1-ethyl-3-methylimidazoliummethanesulfonate), [emim][MeSO3]. The effect of pH onlaccase activity and stability was also evaluated.

Materials and Methods

Enzymes and Chemicals

The ionic liquids 1-ethyl-3-methylimidazolium 2-(2-methox-yethoxy) ethylsulfate, [emim][MDEGSO4], 1-ethyl-3-methy-limidazolium ethylsulfate, [emim][EtSO4], and 1-ethyl-3-methylimidazolium methanesulfonate, [emim][MeSO3], wereprovided by Solvent Innovation GmbH and were used withoutfurther purification. 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) (98%) was obtainedfrom Sigma (Madrid, Spain). Acetonitrile (HPLC-gradientgrade,>99.8%) and dimethyl sulfoxide (DMSO, GR,>99.9%)were obtained from VWR-Prolabo and Merck, respectively. Allreagents were used without further purification. The watercontent of the ionic liquids used was measured by Karl Fishertitration (Metrohm 756 Karl Fisher Coulometer). The resultsobtained were 0.113 %wt for [emim][MDEGSO4], 0.178 %wtfor [emim][EtSO4], and 0.279 %wt for [emim][MeSO3].

Commercial laccase (EC 1.10.3.2) (Denilite base II; 800 U/g)from genetically modified Aspergillus was kindly provided byNovozymes.

202 Biotechnology and Bioengineering, Vol. 101, No. 1, September 1, 2008

Measurements of Activity and Stability

The laccase activity was assayed spectrophotometrically(Thermo Electron, model UV1 spectrophotometer). TheABTS substrate solution was prepared mixing 5 mL of ABTS(0.4 mM) with 19 mL of 0.05 mM citrate/0.1 mM phosphatebuffer at pH 4.5. To measure the laccase activity, 0.1 mL ofthe incubated enzyme solution (containing ILs, buffer and/or organic solvents) was added to 1.9 mL of the ABTSsolution at 408C (Ander and Messner, 1998). The changein absorbance at 420 nm (e¼ 36� 103 M�1 cm�1) wasrecorded for 30 s and the catalytic activity was determined bymeasuring the slope of the initial linear portion of the kineticcurve. One unit was defined as the amount of enzyme thatoxidized 1 mmol of ABTS per min and the activities wereexpressed in U/L.

Laccase stability in the presence of ionic liquids wasdetermined by incubating the enzyme solution in a (ILþbuffer) solution, with 10%, 25%, 50%, and 75% (v/v) IL. Thewater content of the ILs is negligible (see Enzymes andChemicals Section) and does not affect the study of this essay.A sample incubated with only phosphate buffer (50 mM,pH 7.0) was used as reference (named control in tablesand figures). Laccase stability in the presence of acetonitrileand DMSO was measured for comparison, at the sameconcentrations. The laccase initial activity was measured afterthe addition of the enzyme solution (final concentration of2,000 U/L) into the incubation media. The residual activitieswere determined at regular time intervals, up to 7 days, usingthe standard procedure described previously.

Effect of pH on Laccase Stability With Ionic Liquids

Laccase stability at different pH values was determined byincubating the enzyme solution in phosphate buffer (controlsample, 50 mM) or in 10%, 25%, 50%, and 75% (v/v) of IL/buffer solutions at pH values of 5.0, 7.0, and 9.0 for 7 days.pH buffer was adjusted by adding small amounts ofphosphoric acid and checking the value with a pH-meter.Again, the phosphate buffer solution was used as referencefor a given pH (named control sample, as above). The initiallaccase activity was measured after the addition of theenzyme solution and the residual activities were determinedat regular time intervals, using the standard proceduredescribed above.

Enzyme Kinetics

The kinetics of the process were evaluated spectrophoto-metrically in 50 mM phosphate buffer (pH 7.0) and in 25%(v/v) IL/buffer solution at pH 7.0, with ABTS as substrate.The rate of ABTS oxidation was studied at 420 nm(e¼ 36� 103 M�1 cm�1) at ABTS concentrations in therange 0.01–0.1 mM. Experimental data obtained wereanalyzed by regression analysis using the Michaelis–Mentenequation.

Results and Discussion

Effect of pH on Initial Laccase Activity in the Presenceof Ionic Liquids and Organic Solvents

The initial laccase activity was determined in presenceof three 1-ethyl-3-methyl imidazolium ILs (with anions[MDEGSO4], [EtSO4] and [MeSO3]) at concentrationsvarying from 10% to 75% (v/v), at three different pH values:5.0, 7.0, and 9.0. Experiments were also performed in twoorganic solvents (DMSO and acetonitrile) for comparison,using the same concentration range but only at pH 7.0. Theresults are presented in Figure 1. Laccase was most active atpH 7.0–9.0. The different solvents have little effect in laccaseactivities at pH 7.0 or 9.0 (both ILs and organic solvents)at the lower concentrations (10% and 25% (v/v)). Whensolvent concentration is raised to 50% (v/v), laccaseactivity decreases in the presence of acetonitrile and, to alower extent, in [emim][MDEGSO4]. Laccase activity in thepresence of all other solvents is maintained even at this

Figure 1. Effect of pH on the initial laccase activity at different co-solvent

concentrations.

concentration. At these concentrations, the laccase activityaverages, considering the results from all solvents was653� 33 U/L and for ILs was 627� 7.8 U/L.

At pH 5.0 laccase activity is clearly reduced for all samplesexamined, including the control (buffer) sample. Never-theless, the presence of the ILs lowers this deactivation,specially using [emim][EtSO4] and [emim][MDEGSO4]. Atthis pH, maximum laccase activity was obtained for[emim][EtSO4] with no significant differences observedfor the three concentrations studied. Similar behavior wasobtained for [emim][MDEGSO4], despite deactivation ishigher at the lowest concentration. For [emim][MeSO3],deactivation is dependent on concentration, lowering asthe IL concentration is raised. Due to the higher acidity,the pH 5.0 may partially inactivate the enzyme. Even so,the activities using ILs were higher when compared to thecontrol sample probably due to a certain increase inpH from 5.0 to 5.5–6.0 when the ILs were added to thesystem.

No laccase activity was obtained at 75% (v/v) concentra-tion of ILs or organic solvents. In all cases the enzymeprecipitates. After re-dissolving the precipitate in the samebuffer, almost full enzyme activity was recovered, thusshowing that enzyme structure remained unchanged.A similar behavior has also been observed for laccase using4-methyl-N-butylpyridinium tetrafluoroborate, [4-MBP][BF4], at concentrations above 50% (v/v) (Hinckley et al.,2002). The precipitation on the ILs probably occurs due tosalting-out effect promoted at high concentration.

Effect of ILs Concentration on the Laccase Stability

The stability of laccase was studied in the presence of theseILs and organic solvents for the same concentration range,0–50% (v/v) and pH conditions (5.0, 7.0, and 9.0) for 7 days.Experiments with 75% (v/v) solvent concentration were notperformed because of enzyme precipitation (see Effect ofpH on Initial laccase Activity in the Presence of Ionic Liquidsand Organic Solvents Section). Figure 2 shows the results forall solvents used (both ILs and organic) at pH 7. The resultsin Table I indicate that enzyme stability in ILs is comparableto that of the control sample and in conventional organicsolvents. In general, the stability of laccase in ILs was higherat pH 9.0 than at pH 7.0 or 5.0. At this pH (9.0) the activityloss in [emim][MDEGSO4] was lower than in the controlsample for 7 days. This same behavior was obtained forthe other pH values studied. The activity loss using[emim][EtSO4] was also lower than for the control sampleat pH 9.0 and 5.0, but higher at pH 7.0. The behavior using[emim][MeSO3] was the opposite: the activity loss washigher at pH 5.0 and 9.0, but lower at pH 7.0. As a generaltrend, increasing the IL content results in a decrease of theenzyme activity, at least among the ILs studied here. This canbe clearly observed from Figure 2, which corresponds to pH7.0. But at pH 9.0 the concentration effect is not significativefor [emim][MDEGSO4] and [emim][EtSO4]. From all the

Tavares et al.: Laccase in Ionic Liquids 203

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Figure 2. Effect of co-solvent concentration on laccase stability at pH 7.

above, [emim][MDEGSO4] seems to be the most tolerableIL for laccase, with better performance at pH 9.0. Underthese conditions, the highest activity was obtained (activityloss< 10% after 1 day incubation time and <20% after7 days). The loss of activity in [emim][EtSO4] was also betterthan in the control sample, thus this IL may also be a suitableco-solvent for laccase. The highest enzyme activity wasobtained at 10% of ILs (highest water content) in thereaction medium. Bell et al. (1997) studied the laccaseactivity in various organic solvents and showed thatenzymatic activity is affected by water content. They showedthat the variation of water content promotes changes in thenature and polarity of the medium, thus causing changes onenzyme behavior. They related the reduction of enzyme

204 Biotechnology and Bioengineering, Vol. 101, No. 1, September 1, 2008

activity with the decreasing of water activity. It is known thatresidual water and enzyme activity are related. The enzymestructure ‘‘needs’’ water molecules to be active (Halling,2004).

Kinetic Study

The effect of ILs (at 25% (v/v) concentration) on enzymekinetics with ABTS as substrate was investigated in moredetail. The selected pH for initial laccase activities was 7.0, asthis value provided optimal conditions (Fig. 1). The initialreaction rates as a function of substrate concentration areshown in Figure 3.

Table I. Residual activity and activity loss of laccase in ionic liquid (IL) or

organic solvents at different conditions.

Co-solvent

Ca

(%,

v/v) pH

Residual

activity (U/L) Activity loss (%)

0b 1 2 4 7 1 2 4 7

Control 5 228 214 207 169 146 6.2 9.2 25.6 35.9

[MeSO3] 10 5 264 238 227 180 174 9.7 13.9 31.9 34.0

25 5 350 338 320 265 235 3.4 8.6 24.4 32.9

50 5 414 348 323 260 225 16.0 22.0 37.1 45.6

[EtSO4] 10 5 477 415 402 403 350 13.1 15.8 15.6 26.7

25 5 510 480 457 382 391 5.9 10.3 25.0 23.2

50 5 493 447 430 371 9.6 13.0 24.9

[MDEGSO4] 10 5 320 318 317 284 250 0 0.25 10.8 21.5

25 5 406 406 406 377 368 0 0 7.3 9.6

50 5 445 411 438 362 344 7.7 9.1 18.8 22.7

Control 7 713 633 474 399 341 11.3 33.5 44.0 52.2

[MeSO3] 10 7 636 478 421 362 349 24.9 33.7 43.0 45.1

25 7 689 503 479 424 384 27.0 30.5 38.4 44.3

50 7 661 461 426 376 348 30.3 35.6 43.1 47.4

[EtSO4] 10 7 620 507 355 318 273 18.3 42.8 48.8 56.1

25 7 636 516 346 311 276 18.8 45.5 51.1 56.7

50 7 581 373 290 244 234 35.8 50.0 58.0 59.7

[MDEGSO4] 10 7 625 534 397 367 329 14.7 36.6 41.3 47.4

25 7 625 517 403 382 345 17.2 35.4 38.9 44.8

50 7 646 368 305 283 256 32.6 44.2 48.1 53.1

DMSO 10 7 690 655 548 433 368 5.1 20.6 37.2 46.7

25 7 708 672 594 508 488 5.1 16.2 28.3 31.1

Acetonitrile 10 7 653 498 430 370 325 23.7 34.1 43.3 50.2

25 7 569 367 332 290 248 35.4 41.6 49.1 56.4

50 7 381 177 178 146 142 53.5 53.3 61.6 62.8

Control 9 718 658 634 529 542 8.3 11.7 26.3 24.6

[MeSO3] 10 9 577 532 475 411 410 7.7 17.6 28.7 29.0

25 9 572 486 447 401 366 15.1 21.8 30.0 36.0

50 9 568 448 386 365 345 21.2 32.1 39.2 46.8

[EtSO4] 10 9 642 632 554 497 517 1.4 13.7 22.6 19.4

25 9 629 610 561 483 487 3.01 10.8 23.2 22.7

50 9 614 610 610 507 501 0.64 10.9 17.5 18.4

[MDEGSO4] 10 9 686 627 614 580 595 8.6 10.5 15.5 13.3

25 9 645 599 595 555 575 7.2 7.8 14.0 10.9

50 9 610 588 558 499 555 3.6 8.5 18.2 9.0

aC: co-solvent concentration.bDay(s).

Figure 3. Effect of ILs on ABTS oxidation rate by laccase: points (experimental),

lines (Michaelis–Menten equation). Lineweaver–Burk plot (inner figure).

Kinetic parameters of laccase both in the presence of ILsand in aqueous buffer were calculated according to theclassical Michaelis–Menten equation:

V ¼ Vm½S�Km þ ½S� (1)

where V is the velocity of the reaction (mM/min); Vm is themaximum velocity (mM/min) and [S] is the ABTSconcentration (mM).

The Michaelis–Menten parameters Vm and Km wereobtained by nonlinear least-squares regression using SigmaPlot v5.0 (SPSS Inc.). The parameters are given in Table II

together with the regression coefficient (R2) and the averagerelative deviation (%ARD) between experimental andcalculated initial velocities. %ARD were calculated as

%ARD ¼ 100

Ndata

XNdata

i¼1

j xexpi � xcalc

i

xexpi

j (2)

Good agreement was found between the experimental initialvelocities and those calculated from Equation (1), asindicated both by the regression coefficient (R2) and thelow average relative deviation (Table II). The same values ofVm and very close values of Km were obtained in all casesusing the Lineweaver–Burk graphical approach, which isshown in Figure 3. Among the ILs studied, the effect of IL onVm and Km values increased in the order [emim][MDEG-SO4]< [emim][MeSO3]< [emim][EtSO4]. A reduction onVm was obtained in the presence of [emim][EtSO4] (86%),[emim][MeSO3] (67%) and [emim][MDEGSO4] (65%)respect to the buffer control sample (20.5 mM/min). Alsoa decrease on Km values of 10%, 16%, and 17% for[emim][EtSO4], [emim][MeSO3] and [emim][MDEGSO4],respectively, was observed when compared to Km value inbuffer solution (8.53 mM). The lower value of Vm in thepresence of ILs corresponds to a lower maximum reactionrate. This is clearly observed in Figure 3. The lower Km valuesobtained in ILs should mean that the enzyme attains itsmaximum catalytic efficiency at a lower substrate concen-tration, however the Vm was lower in their presence. Thus,it should depend on the substrate concentration if thepresence of any of the ILs improves or not the enzymaticoxidation of ABTS. A recent study of the effect of other ILson the laccase kinetics for syringaldazine oxidation(Hinckley et al., 2002) has also shown a decrease on theVm value when compared to that in aqueous buffer. Thelower values of Km in the presence of ILs are in agreementwith the conclusions of Bell et al. (1997) for enzyme activityin organic solvents: Km increases with water content. The

Tavares et al.: Laccase in Ionic Liquids 205

Biotechnology and Bioengineering

Table II. Kinetic parameters of ABTS oxidation by laccase in reaction systems with different ILs and the respective correlation coefficient (R2).

Kinetic parameters Control (phosphate buffer) [emim][MeSO3] [emim][EtSO4] [emim][MDEGSO4]

Vm (mM/min) 20.5 13.9 17.7 13.4

Km (mM) 8.53 7.19 7.67 7.08

R2 0.9730 0.9922 0.9774 0.9845

%ARD 2.92 2.42 1.12 1.90

same conclusions were pointed out by Halling (2004) in arecent review.

Conclusions

ILs showed to be an interesting co-solvent for laccase interms of enzyme activity and stability. When compared topure buffer solution, the presence of ILs at optimizedconditions stabilized the enzyme, decreasing the activity lossafter 7 days of incubation. Among the ILs used here,[emim][MDEGSO4] was the most promising IL for laccasewith an activity loss of about 10% after 7 days of incubation.As a general trend, increasing the IL content results ina decrease of the enzyme activity and at high IL content(75%, v/v) the enzyme precipitates. From the obtainedresults, ILs have an effective potential as co-solvents and area good alternative to organic solvents for laccase enzymaticreactions.

Financial support for this work was in part provided by LSRE

financing by FEDER/POCI/2010, for which the authors are thankful.

A.P.M. Tavares and O. Rodriguez acknowledge the financial support

(Grants SFRH/BPD/22697/2005 and SFRH/BPD/24271/2005, respec-

tively) from Fundacao para a Ciencia e a Tecnologia (FCT, Portugal).

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