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Process Biochemistry 47 (2012) 742–748

Contents lists available at SciVerse ScienceDirect

Process Biochemistry

jo u rn al hom epa ge: www .e lsev ier .com/ locate /procbio

xtraction and purification of laccase by employing a novel rhamnolipideversed micellar system

in Penga,b, Xing-zhong Yuana,b,∗, Guang-ming Zenga,b, Hua-jun Huanga,b, Hua Zhonga,b,hi-feng Liua,b, Kai-long Cuia,b, Yun-shan Liangc, Zi-yuan Penga,b, Ling-zhi Guoa,b, Yu-kun Maa,b,ei Liua,b

College of Environmental Science and Engineering, Hunan University, Changsha 410082, PR ChinaKey Laboratory of Environmental Biology and Pollution Control, Hunan University, Ministry of Education, Changsha 410082, PR ChinaCollege of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, PR China

r t i c l e i n f o

rticle history:eceived 20 October 2011eceived in revised form 24 January 2012ccepted 7 February 2012vailable online 15 February 2012

a b s t r a c t

Biosurfactant-based reversed micellar extraction (RME) is an innovative method for separation and purifi-cation of biomolecules. In this study, rhamnolipid (RL), a kind of biosurfactant, was firstly adopted toform a novel reversed micellar system for extracting and purifying laccase from Coriolus versicolor crudeextract. Several significant factors affecting both forward and backward extraction processes were stud-ied. The appropriate conditions for forward extraction process were: 3.3 mM RL, 50 mM KCl, pH 5.5 and

eywords:hamnolipideversed micelleeversed micellar extractionurificationaccase

extracting time 40 min. As regards backward extraction process, 250 mM KCl, pH 7.0 and extracting time40 min were suggested. The corresponding activity recovery (AR) and purification fold (PF) were 92.7%and 4.79, respectively. Electrophoresis analysis indicated that the laccase was successfully purified. Afterthis reversed micellar system was reused three times, the AR and PF declined to 70.8% and 4.35, respec-tively, indicating that the reversed micellar system could be reused. Comparisons results of syntheticsurfactant-based RME and RL-based RME further verified the superiority of RL.

. Introduction

Laccase is a broad group of enzymes called polyphenol oxidaseontaining copper atoms in the catalytic center, which has receivedrowing attention for its role in lignin degradation and potentialpplications in the detoxification of phenolic pollutants as wells highly stubborn environmental pollutants [1]. Those significantunctions make laccase indispensable for its application in spe-ific biotechnological process. Up to date, lots of laccase has beenroduced from different species of microorganisms. Unfortunately,ost of them have low yield of enzyme activities and poor ther-al stability [2,3]. Only few laccase are thermostable which still

ave low purity and yield of enzyme activities [4]. This preventsaccase from industrial use on a large scale in some applicationelds. Therefore, extraction and purification of laccase are becom-

ng more and more important. However, traditional purificationethods such as column chromatography, salt or solvent precipi-

ation techniques are limited by their complicated operation, long

∗ Corresponding author at: College of Environmental Science and Engineering,unan University, Changsha 410082, PR China. Tel.: +86 731 88821413;

ax: +86 731 88821413.E-mail address: yxz@hnu.edu.cn (X.-z. Yuan).

359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2012.02.006

© 2012 Elsevier Ltd. All rights reserved.

separation time required, and little recovery of activity [5]. In addi-tion, those methods share a common problem in scale-up, whichmakes them uneconomical and impractical [6,7].

Reversed micellar extraction (RME) is an innovative method forseparation and purification of proteins [8]. Reversed micelles arewater-in-oil microemulsion droplets stabilized by surfactants inapolar solvents, in which the surfactant molecules assemble them-selves with the polar head to the inner side and the apolar tail incontact with the organic solvent. The surfactant molecules’ self-aggregation only occurs when the surfactant concentration is abovethe critical micelle concentration (CMC) [9]. RME has attractedmuch attention for its energy-saving feature and the possibilityof sequential operations. RME consists of two processes: (i) theforward extraction process, i.e. the target biomolecules transferfrom crude extract into reversed micelle; (ii) the backward extrac-tion process, i.e. the target biomolecules release from reversedmicelle and transfer into a fresh aqueous phase. Many researchershave tried the RME method which was mainly carried out by syn-thetic surfactant [10–13]. However, synthetic surfactant has beenrestricted by its low dissolvability and less environmental compati-

bility [14]. In view of the weakness of synthetic surfactants and as analternative, biosurfactant arises at the historic moment [15,16]. Bio-surfactant has low toxicity, high biodegradability, and outstandingantiviral activity.

X. Peng et al. / Process Biochemistry 47 (2012) 742–748 743

tion vi

heuppalRpvccictts

2

2

tawnd

2

SwCo

2

ssom

Fig. 1. The scheme of laccase purifica

So far, few studies focusing on applying biosurfactant in RMEave been reported. The extraction and purification of laccase bymploying a novel biosurfactant reversed micellar system wasnprecedented. Rhamnolipid (RL), a kind of anion biosurfactant, isroduced from Pseudomonas aeruginosa. RL-based reversed micelleresented good feature and provided a great potential for industrialpplication of RME technology [14,17]. In addition, the activity ofaccase can be improved after contacting with RL [18]. In this paper,L-based reversed micellar system was applied to extracting andurifying laccase produced from Coriolus versicolor. The effect ofarious key factors on both forward and backward extraction pro-esses were investigated, including the concentration of RL, pH, theontent of salt and extracting time, etc. Extra ethanol was addedn the backward extraction process to improve the extraction effi-iency. Meanwhile, electrophoresis experiments were carried outo ascertain the purity of the separated laccase. For a compara-ive purpose, the purification of laccase was also conducted viaynthetic surfactant-based RME.

. Materials and methods

.1. Microorganism

P. aeruginosa (ATCC 9027) was obtained from the Center of Type Culture Collec-ion (Wuhan, China). The strain was maintained on 277.15 K peptone agar slantsnd transferred monthly. The white rot fungal strain C. versicolor (ACCC 51171)as supplied by the Institute of Agricultural Resources and Regional Planning, Chi-ese Academy Agricultural Science. The strain was maintained on 277.15 K potatoextrose agar slants and transferred monthly.

.2. Chemicals

2, 2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) was supplied byigma Company, USA. Sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and Tween-80ere acquired from Haialading Reagent Company and Tianjin Bodi Chemical Co. Ltd,hina, respectively. All other chemicals used for the experiments and analyses weref AR grade.

.3. Rhamnolipid and laccase production

Slant-grown P. aeruginosa (ATCC 9027) was inoculated into a seed medium con-isting of 3 g/L beef extract, 5 g/L peptone, and 5 g/L NaCl with a pH value of 7.0. Theubsequent cultivation lasted for 24 h at 310.15 K and 200 rpm in an orbital shaker tobtain seed culture. After then, a seed culture (3% (v/v))was added to the productionedium composed of 18 g/L glucose, 1 g/L MgSO4·7H2O, 1.5 g/L Na2HPO4·12H2O,

a RL-based reversed micellar system.

1.5 g/L KH2PO4, 2 g/L NaNO3, and 0.1 g/L FeSO4·7H2O with a pH value of 6.5. Thecultures were carried out at 310.15 K and 200 rpm in an orbital shaker. After 48 h offermentation, the broth was adjusted to pH 8.0. After then, the cells were removed bycentrifugation at 8000 rpm. The suspension was gathered with a pH value of 2.0 andstored at 277.15 K for 12 h. The crude RL was obtained via liquid-liquid extraction.During the liquid-liquid extraction, the volume ratio of fermentation broth, chloro-form and methanol was 3: 2: 1. The mixture was rotate-evaporated at 313.15 K. Afterthen the crude RL biosurfactant was further purified using the method reported byZhong [16].

C. versicolor was cultivated in liquid medium in shake flasks. The mediumconsisted of 2 g/L glucose, 1.28 g/L ammonium tartrate, 2 g/L KH2PO4, 0.5 g/LMgSO4·7H2O, 0.076 g/L CaCl2·7H2O, 0.001 g/L Vitamin B1, 1 g/L Tween80, 7 mL/Ltrace elements liquid, and 10 mM acetic acid buffer (pH 4.5). The trace elementsliquid was prepared as described by Maria [19]. The cultures were carried out at301.15 K and 150 rpm on a rotary shaker for six days. After incubation, the brothwas filtered and then centrifuged at 8000 rpm for 10 min to remove mycelia. Afterthen the supernatant was stored at 277.15 K for further experiments.

2.4. Critical micelle concentration of RL

Critical micelle concentration (CMC) is defined as the concentration of surfac-tants above which micelles are spontaneously formed. The CMC of RL solubilized inisooctane was detected through the method of fluorescence (Perkin Elmer instru-ment, LS55, US) [14].

2.5. Forward and backward extraction processes

The scheme of laccase purification via RL-based reversed micellar system isshown in Fig. 1. The forward extraction process was carried out by mixing equalvolumes (5 mL) of organic and aqueous solution in the stoppered conical flask at298.15 K and 200 rpm for a series of time. The organic phase was a mixture ofisooctane/n-hexanol (1:1 (v/v)) containing RL at different concentrations. The aque-ous phase was a crude extract with varied pH and ionic strength. In the process ofbackward extraction, 5 mL of reversed micellar organic solution containing laccasewas mixed with 5 mL of stripping aqueous solution in the stoppered conical flaskat 298.15 K and 200 rpm in the process. Ethanol was added to the organic phaseat 10 vol.%. The stripping aqueous solution used in this study was 0.01 M citric acidbuffer solution. The pH value and KCl concentration in the stripping aqueous solutionwere prepared similarly to the case of aqueous solution in the forward extraction.After the backward extraction process, the mixture was centrifuged at 4800 rpmfor 10 min and then stood in the separating funnel until those two phases wereseparated clearly.

2.6. Protein and laccase activity assay

Spectrophotometric method was used for the measurement of laccase activ-ity by using ABTS as substrate at 420 nm using UV-Visible spectrophotometer

744 X. Peng et al. / Process BiochemF

luore

scen

ce i

nte

nsi

ty (

a.u.)

650

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(a

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wVi

(d

2

A

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P

ecPtbo

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Aislor[

3

etaT

Concentration of RL (mM)

Fig. 2. Changes in the fluorescence intensity.

Shimadzu, Model UV-200S, Japan). One unit of laccase activity was defined as themount of enzyme required to liberate 1 �mol of ABTS per minute.

= 106 × Vt × �A

V × ε × �t(1)

here A is activity of laccase, Vt is the total volume in colorimetric determination, is the volume of enzyme used in assay (mL), �t is the incubation time (min), �A

s the absorbency increase value, and ε is the absorption factor value (3.6 × 104).Protein was determined by the Lowry–Folin method with bovine serum albumin

BSA) as standard [20]. Each sample was analyzed in triplicate while the standardeviations of all analyses were less than 5%.

.7. Definition

R = Aa

Aˇ× 100% (2)

EE = Paf

Pbf× 100% (3)

EE = Pab

Paf× 100% (4)

F = SAa

SAb× 100% (5)

In these above equations, AR refers to activity recovery; FEE refers to forwardxtraction efficiency; BEE refers to backward extraction efficiency; PF refers to purifi-ation fold. Aa and Ab are activity values of laccase after and before RME, respectively.af , Pbf and Pab are protein concentrations in organic phase after forward extrac-ion process, in crude extract before forward extraction and in aqueous phase afterackward extraction process, respectively. SAa and SAb are specific activity valuesf laccase after and before RME, respectively.

. Results and discussion

.1. Critical micelle concentration of RL

The changes in the fluorescence intensity have been studied.s seen from Fig. 2, above the CMC (0.055 mM), the fluorescence

ntensity showed a mutation. The change of fluorescence inten-ity contained two processes: (i) when the concentration of RL wasower than CMC, fluorescent probes existed mainly in the formf monomer; (ii) when the concentration of RL reached CMC, theeversed micelles formed and the monomers began to assemble14].

.2. Factors in forward extraction process

When investigating the influence of key parameters on forward

xtraction process, the operation conditions of backward extrac-ion process were fixed as follows. The value of pH in strippingqueous solution was 7.0 and the concentration of KCl was 250 mM.he extracting time was set at 40 min.

istry 47 (2012) 742–748

3.2.1. Effect of the concentration of RLThe nature of surfactant and its concentration were the major

factors affecting the extraction efficiency of RME. The effect ofRL concentration on the forward extraction process is shown inFig. 3(A). With the RL concentration increasing from 2.7 to 3.3 mM,the AR, FEE and PF were boosted up to their peaks at 85.4%, 57.5%and 4.46, respectively. However, the further increasing content ofRL resulted in decreases in AR, FEE and PF.

The increase in RL concentration would elevate the number ofaggregating RL and reversed micelles, which in turn enhanced theextraction of laccase. Further increment in RL concentration causedmicellar clustering, which decreased the interfacial area availableto the biomolecule resulting in a decrease in the extraction capacityof reversed micelles [21]. In addition, the intermicellar collisionsoccurred more frequently as their large population at a higherRL concentration, which resulted in de-assembling/deformationof reversed micelles and then led to decreased extraction effi-ciency [22]. Similar results have been reported on extractingnattokinase [23], lysozyme [24], and lipase [6] by using reversedmicelle.

3.2.2. Effect of KCl concentration in aqueous phaseThe changes of AR, FEE and PF in the presence of KCl

(25–200 mM) are illustrated in Fig. 3(B). The AR, FEE and PFimproved with the KCl concentration increasing from 25 to 50 mM.When the KCl concentration was elevated beyond 50 mM, decliningtrends in the AR, FEE and PF were observed. With KCl at optimumconcentration of 50 mM, AR, FEE and PF were 89.8%, 52.0% and 4.22,respectively.

The ionic strength of aqueous phase was also an importantfactor in RME. The content of KCl determined the extent of interac-tion between biomolecules and RL head groups. The concentrationof KCl in aqueous phase would be optimum, when the addi-tion dosage of KCl was just sufficient to enhance the interactionbetween biomolecules and RL head groups, and hence to reducethe repulsive forces between RL heads groups. The balance of theseforces would result in the formation of stable reversed micelles.The reversed micelles were unstable at lower KCl concentrations,due to the absence of adequate electrostatic forces between RLheads and biomolecules as well as dominance of repulsive forcesbetween RL heads. Therefore, the AR and FEE were lower at 25 mMof KCl. Many researchers have proposed that the absence of saltwould cause cloudy organic phase due to the transfer of largeamount of water, resulting in lower extraction of biomolecule[6].

Nevertheless, the biomolecule intake capacity of reversedmicelles was decreased when the KCl concentrations in aque-ous solution further increased from 50 to 200 mM. This mightbe explained by two reasons as following: (i) the Debye lengthdecreased when the KCl concentration increased, thereby theelectrostatic interaction between the charged biomolecules andthe charged RL heads groups of reversed micelles reduced; (ii)the electrostatic repulsion between the charged head groupsof RL reduced as the KCl concentration increased, thereby thesize of reversed micelle decreased [25]. The smaller reversedmicelles had larger curvature, which led to a gradual expul-sion of the biomolecules residing inside reversed micelles. Theprocess was termed as a squeezing-out effect [21]. Similar phe-nomena have also been observed previously by other researchers[6,7].

3.2.3. Effect of pH in aqueous phase

The influence of pH in aqueous phase on forward extraction pro-

cess was studied, too. Specific results are depicted in Fig. 3(C). Theresults were unstable when the values of pH were lower than 4.0or in the alkaline environment (data not shown). Therefore, the

X. Peng et al. / Process Biochemistry 47 (2012) 742–748 745

(A) Concentration of RL (mM)

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50 mM, KCl , pH 5.5 and extracting time 40 min in forward extraction.

(B) Concentration of KCl (mM)

20017515012510075502530

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3.3 mM RL , 50mM KCl, and pH 5.5 in forward extraction

Fig. 3. (A) Effect of RL concentration on AR, FEE and PF in forward extraction process. (B) Effect of KCl concentration on AR, FEE and PF in forward extraction process. (C) Effecto extracp

pttr

iru4atompdwhdosaa

f aqueous phase pH on AR, FEE and PF in forward extraction process. (D) Effect of

rocess, pH: 7.0, KCl: 250 mM and extracting time: 40 min.

H values in aqueous phase were set at the range of 4.0–6.0. Bothhe AR and FEE increased with the pH values increasing from 4.0o 5.5. The maximum values of AR and FEE were 91.8% and 61.4%,espectively.

Nascimento [12] proposed that the optimum pH was lower thansoelectric point (pI) of target biomolecule in an anionic surfactanteversed micelle. However, the AR and FEE reached their peak val-es at pH 5.5 which is higher than the pI of fungi laccase (around pH.0) [26], indicating that hydrophobic interactions between laccasend RL and/or organic solvent were also important in the extrac-ion process of laccase. Similar results have also been shown byther authors. Aires-Barros and Cabral [27] reported that approxi-ately 50% of lipase B was solubilized at pH values higher than its

I in a micellar solution of an anionic surfactant (AOT). Zhao [28]iscovered an interesting phenomenon that when the pH valueas equal to its pI, the extraction of soybean protein achieved aigher efficiency by employing AOT-based reversed system. Theecline of AR and FEE was probably caused by the denaturation

f laccase and changes in the ionization state of RL at inappo-ite pH values. Least protein impurities were extracted and thectivity of laccase retained to a large extent when pH was fixedt 5.5.

ting time on AR, FEE and PF in forward extraction process. In backward extraction

3.2.4. Effect of extracting time in forward extraction processThe effect of extracting time on the forward extraction process is

demonstrated in Fig. 3(D). It was found that the AR improved firstly,reaching the highest value of 86.1% at 30 min, and then decreasedtardily. However, the FEE sharply increased from the start, and thendecreased from 10 min. The PF showed an increase from 3 to 40 minand decreased slowly thereafter. The highest PF achieved 4.09 afterextracting for 40 min.

The biomolecules extracted from crude extract were mixture oflaccase and protein impurities. Both AR and FEE increased with theextracting time increasing from 3 to 10 min. Possible explanationsfor this fact may be that as the extractiong time was prolonged, theextraction took place gradually and a mass of laccase and proteinimpurities transferred into the reversed micelles at a high speedfrom 3 to 10 min. So the extraction efficiencies (AR, FEE and PF)sharply increased. A large number of biomolecules trapped intothe reversed micelle, while the ratio of laccase in the biomoleculeswas lower compared with that after longer extracting time. This

was the reason that led to the increase in AR and the decreasein FEE from 10 to 30 min. Previous researchers have reported thatenzymes entrapped in AOT reversed micelle showed some changesof conformation and loss of activity, while longer extracting time

746 X. Peng et al. / Process Biochemistry 47 (2012) 742–748

(A) Stripping aqueous phase pH

9.08.07.06.05.04.00

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250 mM KCl and pH 7.0 in backward extraction

Fig. 4. (A) Effect of stripping aqueous phase pH on AR, FEE and PF in backward extraction process. (B) Effect of KCl concentration on AR, FEE and PF in backward extractionp ess. In

cear

3

caliec

weoT

rocess. (C) Effect of extracting time on AR, FEE and PF in backward extraction proc

aused much more loss of activity [22,29]. Meanwhile, too longxtracting time might vibrate out the biomolecules which werelready hosted in the reversed micelle. As a consequence, the FEEeduced.

.3. Factors in backward extraction process

Backward extraction process was studied in order to constitute aomplete RME. Ethanol was added in backward extraction processt 10 vol.% because the activity recovery of laccase appeared to beower without ethanol. N-hexanol was dispersed in the surround-ng solvent and probably incorporated into the micellar shell, whilethanol was trapped in the water pool of reversed micelle [30]. Thiso-solvent facilitated the backward extraction process.

When investigating the influence of key parameters on back-

ard extraction process, the operation conditions of forward

xtraction process were fixed as follows. The value of pH in aque-us solution was 5.5 while the concentration of KCl was 50 mM.he extracting time was set at 40 min.

forward extraction process, pH: 5.5, KCl: 50 mM and extracting time: 40 min.

3.3.1. Effect of pH in stripping aqueous phaseThe influence of the pH value in stripping aqueous phase on the

backward extraction process is illustrated in Fig. 4(A). It could befound that AR and BEE increased with the pH value elevating from4.0 to 7.0, but both decreased later. The highest value of AR and BEEwere boosted up to 92.7% and 29.4%, respectively. The PF reacheda maximum value of 4.12 at pH 7.0.

Generally, during the extraction of biomolecule by an anionicsurfactant-based reversed micelle, the stripping aqueous phase pHshould be elevated above the pI to recover the biomolecule inbackward extraction process. Unlike forward extraction process,the aqueous phase pH should generate least electrostatic interac-tion for releasing the biomolecule into fresh aqueous solution. Inthe present study, the appropriate pH in stripping aqueous phasefor the backward extraction process was higher than the pI valueof fungi laccase. A strong interaction between the biomolecules

and reversed micelles induced the micelle-micelle interaction ormicellar cluster formation, resulting in a decrease of back-extractedfractions. Therefore, the control of micelle-micelle interaction maybecome a key factor for back extraction of laccase [31].

chemistry 47 (2012) 742–748 747

3

pKKaK4c

eFpbesfam

3

ifwPta

trartrASrlbiat

3

wewtpstt

3

eofiitMi

Fig. 5. SDS-PAGE analysis of purified laccase from C. versicolor. Lane 1: crude extract;lane 2: after reversed micellar extraction and lane 3: marker.

Table 1AThe reuse of reversed micelle.

Extraction times Activity recovery (%) Purification fold

1 91.64 ± 1.03 4.75 ± 0.042 85.34 ± 2.35 4.53 ± 0.033 80.19 ± 3.31 4.35 ± 0.034 70.75 ± 3.57 4.35 ± 0.04

Table 1BComparison of synthetic surfactant-based RME and RL-based RME.

Sufactant Activity recovery (%) Purification fold

AOT 84.35 + 2.21 3.28 + 0.03

X. Peng et al. / Process Bio

.3.2. Effect of the concentration of KCl in stripping aqueous phaseThe effect of the KCl concentration on the backward extraction

rocess is shown in Fig. 4(B). The AR improved with the increase ofCl concentration and reached a maximum of 89.9% (with 250 mMCl). Meanwhile, the curve of BEE went gently from 50 to 200 mM,nd then presented a surge which approached 37.9% (with 250 mMCl). The PF seemed to be consistent with AR and BEE, and reached.31 (with 250 mM KCl). However, further increasing the KCl con-entration resulted in a reduction of AR and BEE.

Improvement of ionic strength would lead to the decrease oflectrostatic interaction and the promotion of backward transfer.urthermore, higher content of KCl during backward extractionrocess would destabilize reversed micelles, and transfer theiomolecule into the stripping aqueous phase [25]. Meanwhile, thelectrostatic repulsion between the RL heads groups decreased asalt concentration increased. This favored the exclusion of laccaserom the reverse micellar core. With the ionic strength increasingbove 250 mM, the ions formed an electrostatic shield around theicelles, resulting in the decrement of extraction efficiency [32].

.3.3. Effect of extracting time in backward extraction processThe effect of extracting time on the backward extraction process

s presented in Fig. 4(C). When the extracting time was prolongedrom 10 to 40 min, the AR was raised from 36.3% to 91.1%. Mean-hile, the BEE was elevated from 22.8% to 36.5%. Accordingly, the

F was improved from 2.75 to 4.31. However, when the extractingime exceeded 40 min, the AR, BEE and PF decreased to 28.3%, 33.5%nd 1.46, respectively.

Rational extraction time could provide enough time for extrac-ion, resulting in an increase in the number of biomolecules whicheleased from reversed micelle. As with the time prolonged, morend more laccase transferred into the stripping aqueous phase, butelatively minor protein impurities released. This might be due tohe difference between the structures of laccase and protein impu-ities [2]. As a result, the AR and BEE were both increased. TheR showed a shape of bell reaching maximum of 91.1% at 40 min.imilar phenomena have also been observed previously by otheresearchers [10]. However, the excessive extracting time mightead to the damage of laccase. As BEE increased, the number ofiomolecules released from reversed micelles improved accord-

ngly, resulting in the increase of AR. The AR, BEE and PF reached plateau at the time of 50 min, indicating that the transfer of bothhe laccase and protein impurities reached equilibrium [23].

.4. SDS-PAGE analysis

The purity of laccase extracted from RL-based reversed micelleas further confirmed by SDS-PAGE profiles (Fig. 5). The crude

xtract and extracted laccase samples were loaded to 12% gel alongith the marker. As shown in Fig. 5, the crude extract contained

hree bands, indicating the existence of protein impurities. Theurified laccase showed a single band at 60 kDa which was con-istent with previous literature [33]. Meanwhile, the reduction ofhe number of bands in the extracted laccase sample just provedhat laccase was successfully separated and purified.

.5. The reuse of reversed micellar system

The recycling of reversed micellar solution after backwardxtraction process was expected to improve the cost-effectivenessf this process. The reversed micellar system was gathered afterrst extraction and contacted with fresh crude extract, proceed-

ng second extraction. The AR and PF are listed in Table 1A. Withhe increase of reuse times, the AR decreased from 91.6% to 70.8%.

eanwhile, the PF showed a slight decline from 4.79 to 4.35, whichndicated the reuse possibility of reversed micellar system. The

Tween-80 83.44 + 1.33 3.14 + 0.04RL 91.64 ± 1.03 4.75 ± 0.04

RL may precipitate with target biomolecules during the extractionprocess, resulting in a loss of biosurfactant. This was responsiblefor the decrease of AR.

3.6. Comparison of synthetic surfactant-based RME and RL-basedRME

For a comparative purpose, the laccase was also purified viasynthetic surfactant-based RME. Comparison results between syn-thetic surfactant (AOT and Tween-80) and RL are shown in Table 1B.In terms of RL-based RME, the AR and PF of laccase were higheras compared to other two synthetic surfactant-based RME. Theseresults further verified the superiority of RL.

4. Conclusion

Rhamnolipid (RL)-based reverse micellar extraction could besuccessfully applied for the extraction and purification of laccase.RL-based RME found to be more efficient compared to syn-thetic surfactant-based RME. The extraction efficiency was greatlyaffected by the processing conditions, emphasizing the need foroptimizing the processing conditions for improved extraction. Theoptimized conditions for extraction resulted in purification fold (PF)of 4.79 with an activity recovery (AR) of 92.7%. The PF was found

to be 4.35 and AR declined to 70.8% after the reversed micellar sys-tem was reused three times. SDS-PAGE profile further confirmedthe purity of extracted laccase using RL-based reversed micelles.

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48 X. Peng et al. / Process Bio

cknowledgments

This work was supported by the National Natural Scienceoundation of China (50978087, 51009063 and 50978088), theunan Provincial Natural Science Foundation of China (10JJ7005),

he Hunan Key Scientific Research Project (2009FJ1010), theunan University Graduate Education Innovation Project

531107011019), and the Hunan Provincial Innovation Foundationor Postgraduate (CX2010B157).

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