Research Article Immobilization of a Pleurotus ostreatus …downloads.hindawi.com/journals/bmri/2014/308613.pdf · · 2015-11-22enzyme use in continuous, packed bed, stirred tank,

Research ArticleImmobilization of a Pleurotus ostreatus Laccase Mixture onPerlite and Its Application to Dye Decolourisation

Cinzia Pezzella1 Maria Elena Russo2 Antonio Marzocchella3

Piero Salatino3 and Giovanni Sannia1

1 Dipartimento di Scienze Chimiche Universita di Napoli Federico II via Cynthia 4 80126 Napoli Italy2 Istituto di Ricerche sulla Combustione Consiglio Nazionale delle Ricerche Piazzale V Tecchio 80 80125 Napoli Italy3 Dipartimento di Ingegneria Chimica dei Materiali e della Produzione Industriale Universita degli Studi di Napoli Federico IIPiazzale V Tecchio 80 80125 Napoli Italy

Correspondence should be addressed to Cinzia Pezzella cpezzellauninait

Received 13 January 2014 Revised 3 April 2014 Accepted 24 April 2014 Published 8 May 2014

Academic Editor Neelu Nawani

Copyright copy 2014 Cinzia Pezzella et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In the present study a crude laccase preparation from Pleurotus ostreatus was successfully immobilized on perlite a cheap poroussilica material and tested for Remazol Brilliant Blue R (RBBR) decolourisation in a fluidized bed recycle reactor Results showedthat RBBR decolourisation is mainly due to enzyme action despite the occurrence of dye adsorption-related enzyme inhibitionFine tuning of immobilization conditions allowed balancing the immobilization yield and the resulting rate of decolourisationwith the adsorption capacity of the solid biocatalyst In the continuous lab scale reactor a maximum conversion degree of 561was achieved at reactor space-time of 42 h Stability and catalytic parameters of the immobilized laccases were also assessed incomparison with the soluble counterparts revealing an increase in stability despite a reduction of the catalytic performances Botheffects are most likely ascribable to the occurrence of multipoint attachment phenomena

1 Introduction

Theever-increasing attention towards the design of industrialprocesses with low environmental impact and high sustain-ability has encouraged the search for new biocatalysts to beemployed as green tools for several industrial applicationsIn this ldquogreenerrdquo perspective laccases (p-diphenol-dioxygenoxidoreductases EC 11032) represent an interesting class ofbiocatalyst being able to oxidize a wide spectrum of aromaticcompounds along with reducing molecular oxygen to water[1] Besides the exploitation in several applicative fieldssuch as in food sector paper and pulp industry biosensingpolymer functionalization and textile industries [2] laccaseshave attracted growing interest for their successful use inbioremediation processes [3] particularly for the treatmentof dye-containing wastewaters [4 5] thanks to the structuralsimilarity ofmany textile dyes with laccase natural substrates

In particular laccases from the white rot fungus Pleurotusostreatus have been extensively studied for their decolouri-sation ability towards several classes of dyes and wastewatermodels [6ndash8] Reported data indicated that the extent of de-colourisation depends on the laccase isoform used [6] and onthe chemical class of the dye [8]

Even though laccase efficacy in dye conversion has beenextensively demonstrated [9] there are still many constraintsto their application for real wastewater treatment due to thehard conditions characterizing real wastewaters (extreme pHvalues very high ionic strength presence of surfactantscheating agents etc) and the huge volume of polluted watersdemanding remediation Both constraints clearly highlightthe need for the choice of very robust catalyst for the processEnzyme immobilization has widened the scope of laccaseapplication allowing not only reusing of the biocatalyst witha benefit in terms of costs but especially improving enzyme

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 308613 11 pageshttpdxdoiorg1011552014308613

2 BioMed Research International

performances leading to higher activity and stability at ex-treme pHs elevated temperatures or in organic solventsand improved thermostability Such enhanced features haveencouraged the use of immobilized laccases in several appli-cations as recently reviewed by Fernandez-Fernandez [10]Different methodologies have been reported for laccase im-mobilization such as adsorption entrapment encapsulationcovalent binding and self-immobilization The choice of themost suited method clearly depends on application that lac-case is devoted to Particularly covalent binding is the mostwidely applied method for exploitation of immobilized lac-cases in industrial applications [11ndash13]

Covalent immobilization represents an attractive optionto obtain enzymatic catalyst for wastewater treatment Thistechnique provides different advantages (i) it prevents en-zyme leakage even under harsh conditions (ii) it facilitatesenzyme use in continuous packed bed stirred tank and flu-idized bed reactors (iii) it causes stabilization of the enzymetertiary structure usually as a consequence of multipointattachment of the enzyme to the support providing enzymerigidity The stabilization provided by covalent bonding isusually counterbalanced by partial enzyme deactivationThisnegative effect can be mitigated by carefully optimizing theimmobilization conditions in order to maximize the ratiobetween immobilized enzyme activity and activity of theprimary enzyme solution

Decolourisation of several dyes has been achieved bymeans of laccase covalently bound to different supportssuch as alumina oxide pellets [14] controlled porosity-carrierbeads [15] and epoxy-activated supports [13] Effectivedye removal was also carried out by C unicolor laccasecovalently immobilized on mesostructured siliceous cellularfoams (MCGs) [16] or on supports functionalized with epoxygroups [2] In the latter case the degradation of differentclasses of dyes was reported along with an improvement ofstability toward pH temperature and storage of the immo-bilized enzyme Entrapment of laccase has also been used asan immobilization approach for environmental applicationsChitosan-coated alginate beads have been packed in a fixedbed reactor and employed in RBBR decolourisation achiev-ing up to 70 decolourisation [6] Alginate beads differentlymodified with chitosan or polyethylene glycol (PEG) havealso shown to effectively decolourise several different dyes[17ndash19] Entrapment in hydrogel structures has also beenapplied for example in the immobilization of a T versicolorlaccase resulting in low substrate affinity but improvement instorage stability and in the decolourisation of Acid Orange 52[20]

An important aspect to be considered in the design of anideal immobilized biocatalyst is the cost related to the supportand to the enzyme to be linked to it In this study a low-costlaccase mixture was obtained from P ostreatus culture brothafter optimization of laccase production conditions and one-step protein enrichment as described by Palmieri et al [6]On the other hand the search for a cheap solid support hasoriented our study toward the choice of a siliceous materialperlite Perlite is an amorphous aluminium silicate with morethan 70 content of silica Besides exhibiting the advantagesof an inorganic carrier with respect to organic ones such as a

greater mechanical stability and resistance toward microbialattack and organic solvents perlite turns out to be a cheaperalternative in comparison with other reported inorganic sup-ports such as silica gels alumina and zeolites [21]The choiceof such an inert support implies that its surface has to be prop-erly modified in order to offer functional groups for proteinbinding Surface modification of these materials can be easilyachieved and their reactivity may be finely tuned in the der-ivatization steps [22]

In this work covalent immobilization of laccase on acti-vated siliceous support perlite was investigated and the im-mobilized biosystem was tested for its potential exploitationin dye decolourisation using the reactive dye RBBR as modelsubstrate The immobilization process was properly opti-mized with reference to the immobilization yield and to thedye adsorption capacity of the solid biocatalyst Stability andcatalytic parameters of immobilized laccases were also as-sessed in comparison with the soluble counterpart

2 Materials and Methods

21 Fungal Culture and Crude Laccase Extraction Pleuro-tus ostreatus (type Florida ATCC number MYA-2306) wasmaintained through periodic transfer every 3 weeks at 4∘ onagar plates containing 24 gL potato dextrose and 5 gL yeastextract Medium components were supplied by Difco Labo-ratories (Detroit MI) Mycelium growth and crude laccasemixture extraction were carried out following the proceduresdescribed by Faraco et al [7]

The crude laccase mixture extract is characterized by fourisoforms (POXA1b POXA3a POXA3b and POXC) Theactivity of the POXC isoform accounts for about 99 of thetotal mixture activity [7]The laccase mixture used in the im-mobilization trials displays a specific activity of 70Umg

22 Dye The anthraquinonic dye Remazol Brilliant Blue R(RBBR) was purchased from Sigma-Aldrich Powder puritywas 50Dye concentrationwasmeasured by recording opti-cal absorbance at 592 nm The extinction coefficient (120576

592=

9000Mminus1 cmminus1) referred to total powder concentration wascorrected taking into account the purity All other reagentswere purchased from Sigma-Aldrich with a ge98 purity

Laccase Activity Assay Laccase activity was assayed at 25∘Cby monitoring the oxidation of ABTS at 420 nm (120576

420= 36 times

103Mminus1 cmminus1) [6] The assay mixture contained 2mMABTS

in 01M sodium citrate buffer (pH 30)119870119872

values were estimated using the software GraphPadPrism (GraphPad Software La Jolla CA USA httpwwwgraphpadcom) on a wide range of substrate concentrations(005ndash3mM) Enzyme activity was expressed in internationalunits (IU)

23 Protein Determination and Electrophoresis Protein con-centration was determined using the BioRad Protein Assay(Bio-Rad Laboratories Segrate (MI) Italy) with bovineSerum albumin (BSA) as standard

BioMed Research International 3

24 Perlite Pretreatment and Derivatization Perlite (SIPER-NAT 22copy) was supplied by Degussa (Hanau Germany) So-lids (density about 1026 kgm3) were sieved in the range of90ndash150120583m Perlite was pretreated with 12MHNO

3at 60∘C

for 4 hours and then extensively washed with water and driedat 60∘C Solids were vigorously fluidizedwithwater to removefinesTheminimumfluidization velocity estimated accordingto Fan [23] for the liquid-solids system is about 4sdot10minus6ms

Silanization is a crucial step with regard to subsequentreproducibility of the chemical functionalization Howeverthe surface coverage orientation and organization of reactivegroups are still a subject of controversy [24] Despite resultingin a lower surface concentration of amino groups in compar-ison with reaction in organic solvents aqueous silanizationwith APTS (aminopropyltrimethoxysilane) has been selectedfor this study since it was demonstrated to result in a morestable and uniform immobilized enzyme layer [25]

Glutaraldehyde is a bifunctionalmolecule which has beenextensively used as an enzyme immobilizing agent Althoughthere are many discussions on the composition of the glu-taraldehyde solution (monomeric and polymeric forms) andon the structures responsible for its properties it is generallyaccepted that it is capable of reacting with surface aminegroups of enzyme and carriers through the formation ofSchiff rsquos bases and Michaelrsquos adducts [26]

Carrier activation was carried out as follows (i) 02 g ofdry pretreated perlite was mixed with APTS at a concentra-tion from 04 to 4 in 5mL distilled water and incubated at80∘C for 2 h under constant mixing (ii) the suspension waswashed thoroughly with 50mM sodium phosphate (NaP)buffer pH 65 and treated with glutaraldehyde solutions anddissolved at different concentrations in the same buffer for2 h at room temperature (iii) the activated perlite was exten-sively washed with the overcited buffer and finally incubatedfor 1 h with 5mL of a solution of laccase mixture (22UmL)in 50mMNaP buffer pH 65 at room temperature Residualactive glutaraldehyde was inactivated by 1 h incubation with100mM glycine at room temperature

25 Adsorption Experiments Dye adsorption on unreactedparticles was determined by incubating 02 g of particles in aRBBR solution at preset concentrations All the experimentswere performed in the conditions usually adopted duringdye conversion tests 20mM sodium acetate (NaA) pH 45 atroom temperature Dye concentration in the liquid phase wasmeasured as optical absorbance at 592 nm The dye adsorp-tionwas highlighted by the decrease of the optical absorbancein the liquid phase as well as by an increase of particles colou-ration The procedure adopted for each experiment was (i)dispersion of particles in dye solution (ii) as the dye concen-tration in the liquid did not changed anymore (achievementof equilibrium between solids and liquid) the liquid was re-placed with fresh dye solution The cyclic operation was re-peated until no change in the initial concentration of dye wasobserved The overall amount of adsorbed dye during eachcycle was calculated from the mass balance referred to thedye

(2)

(7)

(4)

(6)

(5)

(1)

(3)

Figure 1 Apparatus equippedwith fluidized bed reactor adopted forRBBR conversion by means of immobilized laccases (1) Fluidizedbed reactor (2) peristaltic pump (3) feed tank (4) recirculationgear pump (5) flow-cell and spectrophotometer (6) data acquisitionunit (7) waste tank

26 Assay of Immobilized Enzyme Activity The activity oflaccase immobilized on perlite was estimated by measuringthe oxidation rate of 221015840-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) in a recirculating fixed bed reactorpreviously designed for the assessment of activity of enzymesimmobilized on granular solids [13]The device was equippedwith a fixed bed reactor loaded with biocatalyst particles Itwas operated by circulating the liquid phase containing thesubstrate between the fixed bed and a mixed tank accordingto the procedure reported by Russo et al [13] The increaseof optical absorbance at 420 nm was assessed by online mea-surements on the circulating liquid phase in the mixed tankThe operating conditions were purposely selected in orderto prevent mass transfer limitations during the enzymaticconversion the assaywas carried out under kinetic controlledregime The operating conditions were total reaction volume(tank tubes fittings and inert fixed bed) 72mL tubularreactor packed with 018mL solid biocatalyst and liquidcirculation rate at volumetric flow rate 20mLmin

27 Fluidized Bed Reactor for RBBR Conversion Conversionof RBBR by immobilized laccases was investigated in afluidized bed reactor continuously operated with respect tothe liquid phase A sketch of the apparatus is shown in Fig-ure 1 Biocatalyst particles were loaded in a cylindrical vessel(25 cm ID 30 cm long) Solid particles were fluidized withthe liquid stream delivered by a gear pump (VG 1000 digitVerder)Thedye-bearing liquid solutionwas fed at the reactorby means of a peristaltic pump The liquid was recirculatedthrough the reactor at volumetric flow rate (119876

119903) by means

of a peristaltic pump (Miniplus Gilson) Dye concentrationin the outlet stream was measured continuously by means ofa spectrophotometer (Cary 50 Varian Inc) equipped with


a flow-cell Optical absorbance was measured at 592 nm Dyeconversion was carried out in conditions assessed as optimalfor RBBR decolourisation 20mM sodium acetate buffer pH45 at room temperature [6] The minimum liquid flow raterequired for the solid fluidization estimated assuming thecrude solid density for example without any adsorbed spe-cies was about 010mLmin

Total liquid volume was set at 71mL The test procedurewas as follows

(i) saturation of the catalyst with the dye The reactorwas fedwith dye bearing liquid stream at 124mLmin(more than 100 times the expectedminimumfluidiza-tion rate) without stream recycling The liquid flowrate was enough to fluidized particles The reactorspace-time (120591 cong 4 min) was set to saturate the solidswith dye at a negligible enzymatic conversion solidsaturation was accomplished within 2 hours

(ii) as the solid was saturated the feeding flow rate119876 wasdecreased to the set value and the liquid was recycled(119876119903= 13mLmin) A series of steady state regimes

was investigated by setting 119876 in the interval 03ndash13mLmin The reactor was operated at the preset 119876until dye concentration in the reactor approached asteady value The dye conversion was assessed understeady state conditions

3 Results and Discussion

31 Optimization of Immobilization Process on Activated Per-lite Functionalization of perlite surface has been achieved bytwo main steps (i) silanization with a trifunctional organosi-lane agent APTS that provides the reactive amino groups sus-ceptible to the following activation (ii) reaction with a cross-linking reagent glutaraldehyde

In this section results on the optimization of immobiliza-tion of crude laccase preparation on perlite are reported

Activity of laccase immobilized on perlite was measuredadopting a specifically designed device as described inSection 2 Immobilization yield (119884) is the objective parameterassessed to optimize the immobilization protocol 119884 wasdefined as the ratio between laccase activity expressed by thesolid biocatalyst and total activity in the liquid solution at thebeginning of the immobilization processes

Process optimization has been carried out assessing theeffect of the following operating conditions on the immobi-lization yield glutaraldehyde concentration pH and buffercomposition of immobilization solution time and tempera-ture of incubation total activity and total protein contentsTable 1 reports the results in terms of immobilized activityand yield for each run The operating conditions of each runare also reported

311 Effect of Glutaraldehyde Concentration The effect of theresults reported in Table 1 indicates that at 1 and 25 glu-taraldehyde concentrations immobilization yield is higherwhen incubation is performed at RT for shorter time(R1 R3) with respect to overnight incubation at 4∘C(R2 R4) No laccase activity or proteins were detected in

the recovered supernatant at the end of incubation indicatingthat 100 of the initial protein content was bounded tothe carrier The lower immobilization yield found in R2and R4 could be ascribed to further interaction of enzymeswith glutaraldehyde molecules for prolonged incubationtime causing laccase inactivation When glutaraldehydeconcentration is raised up to 5 immobilization yields arealmost comparable at RT for 4 h or overnight at 4∘C (seeR5 and R6) It is conceivable that at higher concentra-tion glutaraldehyde-glutaraldehyde interactions prevail onglutaraldehyde-protein ones thus reducing the negative ef-fect on the enzyme observable at longer incubation timesTaken together these results indicate that the reactive groupsmade available by using glutaraldehyde concentrations in therange 1 to 5 are sufficient to bind to all proteins presentin the crudemixture resulting in amaximum immobilizationyield of about 34 When immobilization experiments werecarried out further lowering glutaraldehyde concentrationdown to 05 a comparable yield (32) was achieved (seeR10) Thus glutaraldehyde concentration was set at 05 forfurther immobilization experiments

312 Effect of pH and Buffer Composition One of the factorsthat can affect enzyme immobilization is the pH value ofthe coupling mixture The optimal pH value should be thecompromise between conditions favoring enzyme stabilityand those promoting the nucleophilic attack of proteinreactive groups to the glutaraldehyde functionalized supportThe results obtained performing laccase immobilization atpH values 55 65 and 75 are listed in Table 1 (R17ndashR22)The immobilization yield was almost constant in the neutralpH range (sim50) and decreased at 19 at low pH Exceptfor test carried out at low pH no laccase activity or proteinswere found in the recovered supernatant at high pH (65 and75) At low pH about 25 of the initial laccase activity wasdetected in the supernatant

As reported in Table 1 immobilization at pH 55 was car-ried out in a buffer containing a different counterion sodiumcitrate instead of sodium phosphate buffer at the samemolar-ity To complete the scenario of the combined pH buffereffects tests were also performed at the three investigated pHvalues adopting citrate-based buffer the McIlvaine bufferUnder these conditions (R20 through R22) a significant re-duction of immobilization yield was observed at all the testedpH with up to 28 of the initial laccase activity recovered inthe supernatants

These findings could be a consequence of the hetero-functional nature of the activatedmatrix after glutaraldehydeactivation the support may expose unreacted amino groupswhich confer some ionic exchanger features to the support[22] In such heterofunctional matrices a first ionic adsorp-tion of the protein on the amino groups of the support wasfound to occur before the covalent reaction between glu-taraldehyde activated sites and the enzymes occurs A dif-ferent counterion selectivity (citrate gt phosphate) towardsthe unreacted amino groups of the support could explainthe observed effect by assuming that most of the positivelycharged groups on the support are shielded by citrate (whose


Table 1 Effects of experimental parameters on laccase immobilization yield on perlite NaC 50mM sodium citrate buffer McI McIlvainersquosbuffer lowastIncubations were performed in 50mM sodium phosphate buffer unless otherwise is indicated Reported data are the mean values ofthree independent experiments The standard deviation of tests carried out at a given set of operating conditions was less than plusmn10

Run Initial activity(IUg)

Initial protein(mgg)

Glutaraldehyde vol pHlowast Incubation time

and temperatureImmobilizedactivity (IUg)

Immobilizationyield ()

R1 550 45 1 65 4 h RT 200 36R2 550 45 1 65 Overnight 4∘C 128 23R3 550 45 25 65 4 h RT 206 37R4 550 45 25 65 Overnight 4∘C 151 27R5 550 45 5 65 4 h RT 206 37R6 550 45 5 65 Overnight 4∘C 207 38R7 550 775 1 65 1 h RT 160 29R8 550 775 1 65 4 h RT 161 29R9 550 775 05 65 1 h RT 186 34R10 550 775 05 65 4 h RT 178 32R11 80 13 05 65 1 h RT 56 70R12 285 475 05 65 1 h RT 136 48R13 800 1125 05 65 1 h RT 199 25R14 500 425 05 65 1 h RT 173 35R15 500 12 05 65 1 h RT 173 35R16 500 225 05 65 1 h RT 184 37R17 275 225 05 NaC 55 1 h RT 52 19R18 275 225 05 65 1 h RT 137 50R19 275 225 05 75 1 h RT 120 43R20 275 225 05 McI 55 1 h RT 32 12R21 275 225 05 McI 75 1 h RT 66 24R22 275 225 05 McI 65 1 h RT 42 15

concentration increases lowering pH in McIlvaine buffercomposition) Such charge-shielding effect would hinder thefirst ionic interchange of the protein on the amino groups ofthe supports thus impairing immobilization yield

313 Temperature and Time of Incubation The effect of tem-perature on the immobilization yield was assessed comparingthe runs R1ndashR10 The immobilization yield assessed for testscarried out at incubation temperature as low as 4∘C did notchanged with respect to that assessed at room temperatureIn addition extending the incubation time to overnightincubation (at 4∘C) caused a decrease in immobilization yield(compare R1ndashR3 with R2ndashR4) or resulted in almost compara-ble yields (compare R5 to R6) with respect to 4 hours room-temperature incubation When laccase activity and totalprotein content were determined in the liquid supernatantas a function of the incubation time both measured valuesbecame negligible after 15 minutes incubation indicating theimmobilization of almost the entire protein content of thecrude mixture occurred Hence further experiments werecarried out at incubation time set at 1 h without temperaturecontrol

314 Effect of Total Activity and Total Protein Contents Someimmobilization tests were carried out at the ratio between

0

50

100

150

200

250

0

20

40

60

80

100

0 150 300 450 600 750 900

Imm

obili

zed

activ

ity (I

Ug

)

Imm

obili

zatio

n yi

eld

()

Initial activity (IUg)

Figure 2 Immobilization yield (solid line) and immobilized activity(dashed line) versus laccase initial activity

initial laccase activity andmass of dry support set in the rangeof 80ndash800 IUg (experiments R11ndashR13) For a given amountof dry support results showed that the immobilization yielddecreased with the amount of initial laccase activity whilethe immobilized activity (IUg) approached a constant value(Figure 2) This observation could be interpreted taking intoaccount protein overcrowding on the carrier surface that


may reduce substrate accessibility to the active site Howeverresults of the test series of tests R14 through R16 enabledus to rule out this hypothesis As a matter of fact testscarried out at a given activity (IUg) and at protein contentincreased by adding BSA were characterized by absence ofimmobilization yield as it would have been expected if theabove interpretationwas verifiedMoreover it isworth notingthat proteins were completely bounded to the support evenat the highest concentration investigated Both series of data(R11 through 13 and R14 through R16) can be accounted forconsidering that access of substrate to immobilized enzymewas restricted by the irreversible sealing of carriermicroporesby polymeric components contained in the crude mixture(ie ferulic acid polymer) or other contaminants whoseamount increases when a more concentrated laccase mixtureis used On the basis of the abovementioned results theoptimal parameters selected are (i) solids activation with05 glutaraldehyde (ii) 1 h incubation with laccase mixture(50mM sodium phosphate buffer pH 65 and 80 IUg sup-port) at room temperature In these conditions a maximumimmobilization yield of about 70 was achieved

As expected there is a difference between the actual lac-case activity expressed by the biocatalyst and the activity lossin the liquid supernatantThis effect may be due to the modi-fications of the enzyme structures occurring during covalentimmobilization

The analysis of POXC primary sequence coupled to theexamination of protein 3D model reveals the presence ofsix Lys residues all of them localized on the protein surfaceand potentially available for the covalent bonding withglutaraldehyde As shown in Figure 3 all Lys residues aremapped on the opposite side with respect to the activesite thus enzyme binding in nonproductive orientation isless probable On the other hand multipoint attachment ofprotein to support seems to be favored since two couples oflysine residues very close to each other are identifiable onthe protein surface (Lys 504 and Lys 70 in Figure 3(a) Lys51 and Lys 20 in Figure 3(b)) Such kind of interaction withthe support may be responsible for distortion of enzymaticconformation causing a decrease in its activity [10]

32 Assessment of Immobilized Biosystem Performances Theperformances of the immobilized biosystem have been testedby assessing its stability and catalytic parameters

321 Stability Parameters In order to have a suitable amountof immobilized activity on an easily handling amount ofsolid support an enzyme to support ratio of about 290 IUgwas used obtaining with the aforesaid optimized conditionsan immobilization yield of about 45 Storage stability ofthe immobilized laccase mixture has been monitored bothat room temperature and at 4∘C and was compared withthat observed for free laccase mixture Residual activity wascalculated and expressed as percentages of residual activityat different time intervals As showed in Figure 4 the im-mobilized mixture stored at room temperature displaysabout 7-fold increased stability with respect to the free laccase

mixture (11990512

free enzyme = 16 days 11990512

immobilized enzyme =116 days) On the contrary stability at 4∘C shows a 3-foldincrease of 119905

12(11990512


immobilizedenzyme = 61 days)

The enhanced stability of immobilized laccasemay be dueto the prevention of structural rearrangement and the lowerflexibility of the immobilized form both caused bymultipointattachment to the support [28] These results are consistentwith those described in other reports of covalent laccaseimmobilization on silica-based supports such as kaolinite ormesoporous silica nanoparticles [15 29ndash32] In most casesin which glutaraldehyde was used as crosslinking agentimmobilization process exhibits low laccase recovery butimprovements in the operational stability and stability againstdenaturing agents are evident [16] Liu et al for examplereported an improvement of both thermal and operationalstability of laccase when silanized and glutaraldehyde acti-vated silica nanoparticles were used as supports as illustratedby the retention of 61 of the residual activity after 4 h at60∘C and the retention of 55 of the activity after 10 cyclesof operation

Immobilization can provide an artificial microenviron-ment surrounding the enzyme that can alter surface-exposedhydrophilic andor charged groups and their electrostaticinteractions and thus influencing protein structure and func-tion In particular in the immobilized system described inthis study enzymes could be doubly protected by thermalinactivation thanks to the higher number of positive chargeson the surface and to the increased hydrophobicity bothprovided by silanization [33]

322 Catalytic Parameters In order to perform a kineticcharacterization of the immobilized biocatalyst purified lac-case POXC from P ostreatus has been used rather than thecrude mixture The purified enzyme has been immobilizedin the following conditions 02 g of activated perlite wasincubated for 1 h with purified POXC solution (50mM NaPbuffer pH 65 and 250 IUg of support as initial activity) atroom temperature resulting in about 40 immobilizationyield (100 IUg support) Kinetics of immobilized POXCagainst ABTS has been assessed by means of the circulatingfixed bed reactor commonly employed for activity measure-ments Results provided 119870

119872= 044mM and 119870cat = 12 sdot

104minminus1 Comparing these parameters with those charac-

teristics of POXC in liquid phase (119870119872= 003mM119870cat = 62sdot

105minminus1) both a decreased value for 119870cat and a higher

value for 119870119872

were found These differences could be a con-sequence of either the loss of conformational integrity of theimmobilized enzyme due to multipoint attachment as alsoreported for different immobilized biocatalyst [10] or of low-er accessibility of substrate to the active sites of the immo-bilized enzyme caused by enzyme overcrowding on supportsurface

33 Dye Adsorption The immobilized enzyme mixture wastested for its decolourisation potential against the modelanthraquinonic dye Remazol Brilliant Blue R (RBBR) A firstseries of tests was addressed to assess the extension of dye


(a) (b)

Figure 3 Three-dimensional model of POXC elaborated with Pymol [27] with accessible lysine residues shown as stick The protein isshowed in two opposite orientations wherein the access to the active site is indicated by a red arrow T1 copper is shown as a magenta sphereCopper atoms of the trinuclear cluster are displayed as cyano spheres

10

100

1000

0 10 20 30

Resid

ual a

ctiv

ity (

)

Time (d)

(a)

10

100

1000

Resid

ual a

ctiv

ity (

)

0 10 20 30Time (d)

(b)

Figure 4 Stability of immobilized and free laccasemixturemonitored at 4∘C (a) and at room temperature (b) Black squares and free enzymeblack triangles and immobilized enzyme

adsorption on solids surface This characterization was car-ried out to discriminate between dye conversion by immobi-lized enzyme and dye adsorption the enzymatic conversioncould be masked by a rapid adsorption kinetics andorlarge adsorption capacity of the solid support Moreover theadsorption of dyes as well as adsorption of their oxi-dized products on the immobilization support could inacti-vateinhibit the enzyme [34]The assessment of the RBBR onthe activated carrier could allow adopting strategies to mini-mize any adverse effect on dye conversion

Tests were carried out with both untreated particles andactivated particles No adsorption of RBBR was observed onthe inert particles Tests carried out with samples preparedadopting the optimal conditions defined in the previoussection reported an adsorption of about 71mgRBBRgperliteTherefore the adsorption phenomenon is related to the func-tional groups from silanization or glutaraldehyde activation

process Enzymatic assays performed after dye saturationshowed that immobilized laccase activity was completelyinhibited

The reported findings inspired modifications of theimmobilization protocol to limit dye adsorption to a negli-gible value The effect of several parameters on adsorptioncapacity and immobilization yield was investigated In par-ticular the parameters affecting the surface properties of thesupport (ie APTS concentration and pH temperature andconcentration for glutaraldehyde crosslinking) were tunedResults are summarized in Table 2 The adsorption capacityof the silanized perlite rises up to 961mgRBBRgperlite (A2in Table 2) As a matter of fact by reducing the APTSconcentration from 4 to 04 volume keeping the otherconditions constant the adsorption capacity was approxi-mately halved and an undesired drop of immobilization yieldwas observed The extent of perlite silanization determined


Table 2 Adsorption capacity of treated perlite samples obtained in different experimental conditions Data are average values of threeindependent experiments The standard deviation of each series of results was less than plusmn10

Perlite sample APTS () Glutaraldehyde activation Adsorption capacity(mg RBBRg solid) Immobilization yield ()

A1 4 05 (pH 65) 71 45A2 4 mdash 96 mdashA3 04 05 (pH 65) 28 29A4 4 05 (pH 65 2X) 64 mdashA5 04 05 (pH 8) 31 16A6 4 05 (pH 8) 64 38A7 04 1 (pH 8 60∘C) 33 51

Table 3 Immobilized enzyme samples employed in decolourisation experiments Initial and final activity refer to laccase activity measuredon solid carrier at the beginning and at the end of the decolourisation experiment

Perlite sample Immobilization conditions Adsorption capacity(mgRBBRgsolid)

Initial activity(IUmL) Immobilization yield () Final activity

(IUmL)C1 04 APTS 05 Glut pH 65 275 10 23 3C2 04 APTS 1 Glut pH 85 60∘C 33 28 51 9

the concentration of active amino groups on the carriersurface this parameter likely plays a key role in the adsorp-tion of the dye Thus the improvement of the condition forglutaraldehyde activation would provide a reliable solution tosaturate the free amino groups available for the interactionwith the anionic dye In run A4 two consecutive activationstepswith glutaraldehydewere assessedHowever adsorptionwas not significantly affected by this treatment Rising ofthe pH from 65 up to 8 during glutaraldehyde reactionprovided a further decrease of the immobilization yield downto 164 together with a negligible effect on adsorptioncapacity (compare A5 with A3) This effect was assessed oncarrier silanized with 4 or 04 APTS solutions (A5-A6)

An effective reduction of RBBR loading (329mgg) cou-pled with a satisfying immobilization yield (512) wasachieved by incubating the solid silanized with 04 APTSwith 1 glutaraldehyde at pH 8 and 60∘C [25] The latteroperating conditions were selected for further investigationof dye degradation by laccase immobilized on perlite

34 Decolourisation Experiments Preliminary tests carriedout in fixed beds filled with of enzyme-immobilized particlespointed out that this reactor system does not fit with thefeatures of the coated particles As a matter of fact the bedmade of inert particles was successfully operated even thougha high pressure drop was measured However clogging phe-nomena were recorded when particles with immobilizedenzymes were adopted liquid stream flowed very slowly not-withstanding the high pressure drop adopted across the bed

The fluidized bed was chosen to get round the fixed bedconstraint [35 36] Liquid flow rate required for particlesfluidization was supplied by the recirculating stream whilethe stream containing the dye was continuously supplied at

0

20

40

60

80

100

0 50 100 150 200 250 300

RBBR

conv

ersio

n (

)

120591 (min)

Figure 5 RBBR conversion degrees versus reactor space-time (120591)Data refer to decolourisation experiments carried out in fluidizedbed reactor operated with immobilized laccases sample C1 (dashedline) sample C2 (solid line)

the flow rate related to the residence time suitable for dyeconversion

Preliminary decolourisation experiments were carriedout adopting two batches of immobilized enzyme samplesC1 and C2 reported in Table 3 with the operating conditionsadopted for the immobilization and samples C1 and C2 areacharacterized by comparable dye adsorption capacities anddifferent amount of immobilized laccase activity The initialactivity of sample C1 was 10 IUmL and that of sample C2 was28 IUmL Table 4 reports the dye conversion degrees meas-ured under steady state conditions as a function of the reactorspace-time 120591 Dye conversion degrees versus the reactorspace-time are plotted in Figure 5


Table 4 Decolourisation experiments with laccase immobilized on perlite

Perlitesample

Inlet RBBRconcentration

(mgL)

Recirculationflow rate Q

119903

(mLmin)

Total volume(mL)

Dye-feedingrate 119876

(mLmin)

Recirculationratio

120591

(min)

Outlet RBBRconcentration

(mgL)

Dye conversion()

C1 415 13 71100903

mdash1450

7176273

415355279

mdash1433

C2 391 13 711609028

mdash1446

4476253

354218172

954456

The maximum conversion degree was measured for testscarried out with sample C1 33 at 120591 = 46 h Performancemeasured with the C2 sample were definitively satisfactory561 conversion at 120591 = 42 h Decolourisation runs withboth samples C1 and C2 lasted about 80 and 160 h with a totalvolume of treated RBBR solution of 35 and 45 L respectivelyComparing the results of the decolourisation tests carriedout with samples C1 and C2 it could be inferred that thebetter performance achieved by sample C2 was due to itshigher activity per volume unit of carrier It is remarkableto note that (i) in both decolourisation experiments residualenzyme activity was about 30 of the initial activity (ii) bothsamples are characterized by almost the same dye adsorptioncapacity (about 30mgg carrier) These results suggest thatthe biocatalyst deactivation is mainly related to adsorptionphenomena occurring at the same extent for both samplesTwofold reason suggests ruling out the effect of the extent ofconversion on biocatalyst deactivation within the operationtime investigated (about 160 h) (a) the same deactivationextent has been measured even though different conversionshave been measured (b) constant conversion was observedunder steady state conditions for long time

The analysis of reported results suggests that the pro-cedure adopted for the preparation of sample C2 (Table 3)produced a catalyst characterized by satisfactory values ofimmobilization yield for example satisfactory rate of de-colourisation and low adsorption capacity of RBBRThe lat-ter feature provides the minimum deactivation extent relatedto the fast dye adsorption phenomenon that initially com-petes with the dye conversion during decolourisation tests

4 Conclusions

In the present study a crude laccase preparation from Postreatus was successfully immobilized on perlite a cheapporous silica material Optimization of process experimentalparameters was performed Stability and catalytic parametersof the immobilized laccases were compared with those of freeenzyme Remarkable results show that the stability increasesas a consequence of enzyme immobilization but a reductionof the catalytic performances in terms of ABTS conversionkinetics was also observed These results are in agreementwith those expected phenomena occurring as consequences

of enzyme immobilization on solid supports [37] for exam-ple reduced catalytic activity andor increased stability withrespect to the soluble counterpart as results of electrostaticand partitioning effects in the immobilized enzymemicroen-vironment of conformational losses inside the pores of thesupport and external and internal mass transfer phenom-ena Moreover decreased protein flexibility resulting frommultipoint attachment andor enzyme overcrowding on thesurface of the support [38] are also responsible for impairedcatalytic performances of the immobilized system Concern-ing the optimization of immobilization protocol it can beconcluded that the large achieved yield of immobilizationthe enhanced stability of the immobilized biocatalyst togetherwith the relatively low cost of both the crude enzyme extractand the support encourage the exploitation of such biosystemin different industrial application demanding laccases

Part of the study was devoted to the assessment of theperformances of the immobilized laccase in terms of contin-uous conversion of a reference dye Accordingly the immo-bilized system was tested for RBBR conversion in a fluidizedbed recycle reactor Results obtained in this work showed thatthe observed RBBR decolourisation by immobilized laccaseis mainly due to enzyme action despite the occurrence ofdye adsorption-related enzyme inhibition In this regard finetuning of immobilization conditions has allowed balancingthe immobilization yield and the resulting rate of decolouri-sation with the adsorption capacity of the solid biocatalystIn the continuous lab scale reactor a maximum conversiondegree of 561 was achieved at reactor space-time of 42 h

In conclusion the reported datamay inspire further workconcerning the applicability of such immobilized biocatalystto the treatment of wastewaters containing dyes Kineticparameters of dye conversion as well as of deactivation pro-cesses (adsorption-related and long-term deactivation) maybe inferred in order to provide tools for the rational designof continuous decolourisation processes with immobilizedbiocatalysts

Conflict of Interests

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


Acknowledgments

This work was supported by grants from the Italian MIUR(Ministero dellrsquoUniversita e della Ricerca Scientifica Progettidi Rilevante Interesse Nazionale) PRIN 2009STNWX3 Theauthors are grateful to Mrs Serena Fiorenzano for her pre-cious assistance in the experimental work

References

[1] P Giardina V Faraco C Pezzella A Piscitelli S Vanhulle andG Sannia ldquoLaccases a never-ending storyrdquo Cellular andMolec-ular Life Sciences vol 67 no 3 pp 369ndash385 2010

[2] A Kunamneni I Ghazi S Camarero A Ballesteros F J Plouand M Alcalde ldquoDecolorization of synthetic dyes by laccaseimmobilized on epoxy-activated carriersrdquo Process Biochemistryvol 43 no 2 pp 169ndash178 2008

[3] J-AMajeau S K Brar andRD Tyagi ldquoLaccases for removal ofrecalcitrant and emerging pollutantsrdquo Bioresource Technologyvol 101 no 7 pp 2331ndash2350 2010

[4] A Piscitelli C Pezzella V Lettera P Giardina V Faraco andGSannia ldquoFungal laccases structure function and applicationrdquoin Fungal Enzymes Progress and Prospects T M P Maria deLourdes and M Rai Eds pp 113ndash151 2013

[5] D Wesenberg I Kyriakides and S N Agathos ldquoWhite-rotfungi and their enzymes for the treatment of industrial dye ef-fluentsrdquo Biotechnology Advances vol 22 no 1-2 pp 161ndash1872003

[6] G Palmieri G Cennamo and G Sannia ldquoRemazol BrilliantBlue R decolourisation by the fungus Pleurotus ostreatus and itsoxidative enzymatic systemrdquo Enzyme andMicrobial Technologyvol 36 no 1 pp 17ndash24 2005

[7] V Faraco C Pezzella AMiele P Giardina andG Sannia ldquoBio-remediation of colored industrial wastewaters by the white-rot fungi Phanerochaete chrysosporium and Pleurotus ostreatusand their enzymesrdquo Biodegradation vol 20 no 2 pp 209ndash2202009

[8] V Faraco C Pezzella P Giardina A Piscitelli S Vanhulle andG Sannia ldquoDecolourization of textile dyes by the white-rot fun-gi Phanerochaete chrysosporium and Pleurotus ostreatusrdquo Jour-nal of Chemical Technology and Biotechnology vol 84 no 3 pp414ndash419 2009

[9] S Rodrıguez Couto and J L Toca Herrera ldquoIndustrial and bio-technological applications of laccases a reviewrdquo BiotechnologyAdvances vol 24 no 5 pp 500ndash513 2006

[10] M Fernandez-Fernandez M A Sanroman andDMolde ldquoRe-cent developments and applications of immobilized laccaserdquoBiotechnology Advances vol 31 no 8 pp 1808ndash1825 2013

[11] M Arroyo ldquoInmovilizacion de enzimas fundamentos metodosy aplicacionesrdquo Ars Pharmaceutica vol 39 no 2 pp 23ndash291998

[12] D Brady and J Jordaan ldquoAdvances in enzyme immobilisationrdquoBiotechnology Letters vol 31 no 11 pp 1639ndash1650 2009

[13] M E Russo P Giardina A Marzocchella P Salatino and GSannia ldquoAssessment of anthraquinone-dye conversion by freeand immobilized crude laccase mixturesrdquo Enzyme and Micro-bial Technology vol 42 no 6 pp 521ndash530 2008

[14] S Rodrıguez Couto J F Osma V Saravia G M Gubitz andJ L Toca Herrera ldquoCoating of immobilised laccase for stabilityenhancement a novel approachrdquo Applied Catalysis A Generalvol 329 pp 156ndash160 2007

[15] P-P Champagne and J A Ramsay ldquoReactive blue 19 decoloura-tion by laccase immobilized on silica beadsrdquo Applied Microbiol-ogy and Biotechnology vol 77 no 4 pp 819ndash823 2007

[16] A Rekuc B Jastrzembska J Liesiene and J Bryjak ldquoCompar-ative studies on immobilized laccase behaviour in packed-bedand batch reactorsrdquo Journal ofMolecular Catalysis B Enzymaticvol 57 no 1ndash4 pp 216ndash223 2009

[17] L Lu M Zhao and Y Wang ldquoImmobilization of laccase by al-ginate-chitosan microcapsules and its use in dye decoloriza-tionrdquoWorld Journal of Microbiology and Biotechnology vol 23no 2 pp 159ndash166 2007

[18] P WANG X FAN L CUI Q WANG and A ZHOU ldquoDe-colorization of reactive dyes by laccase immobilized in algi-nategelatin blent with PEGrdquo Journal of Environmental Sciencesvol 20 no 12 pp 1519ndash1522 2008

[19] J Phetsom S Khammuang P Suwannawong and R Sarn-thima ldquoCopper-alginate encapsulation of crude laccase fromLentinus polychrous lev and their effectiveness in synthetic dyesdecolorizationsrdquo Journal of Biological Sciences vol 9 no 6 pp573ndash583 2009

[20] O Yamak N A Kalkan S Aksoy H Altinok and N HasircildquoSemi-interpenetrating polymer networks (semi-IPNs) forentrapment of laccase and their use in Acid Orange 52 decol-orizationrdquo Process Biochemistry vol 44 no 4 pp 440ndash4452009

[21] S-F Torabi K Khajeh S Ghasempur N Ghaemi and S-OR Siadat ldquoCovalent attachment of cholesterol oxidase andhorseradish peroxidase on perlite through silanization activitystability and co-immobilizationrdquo Journal of Biotechnology vol131 no 2 pp 111ndash120 2007

[22] L Betancor F Lopez-Gallego A Hidalgo et al ldquoDifferentmechanisms of protein immobilization on glutaraldehyde acti-vated supports effect of support activation and immobilizationconditionsrdquo Enzyme andMicrobial Technology vol 39 no 4 pp877ndash882 2006

[23] L S Fan Gas-Liquid-Solid Fluidization Engineering Butter-worths Boston Mass USA 1989

[24] A K Singh AW Flounders J V Volponi C S Ashley KWal-ly and J S Schoeniger ldquoDevelopment of sensors for direct de-tection of organophosphatesmdashpart I immobilization charac-terization and stabilization of acetylcholinesterase and organ-ophosphate hydrolase on silica supportsrdquo Biosensors and Bio-electronics vol 14 no 8-9 pp 703ndash713 1999

[25] S W Park Y I Kim K H Chung S I Hong and S W KimldquoCovalent immobilization of GL-7-ACA acylase on silica gelthrough silanizationrdquo Reactive and Functional Polymers vol 51no 2-3 pp 79ndash92 2002

[26] I Migneault C Dartiguenave M J Bertrand and K C Wal-dron ldquoGlutaraldehyde behavior in aqueous solution reactionwith proteins and application to enzyme crosslinkingrdquoBioTech-niques vol 37 no 5 pp 790ndash802 2004

[27] W L DeLanoThe PyMOLMolecular Graphics System DeLanoScientific San Carlos Calif USA 2002

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

[29] D E Dodor H-M Hwang and S I N Ekunwe ldquoOxidation ofanthracene and benzo[a]pyrene by immobilized laccase fromTrametes versicolorrdquo Enzyme and Microbial Technology vol 35no 2-3 pp 210ndash217 2004


[30] X Hu X Zhao and H-M Hwang ldquoComparative study of im-mobilized Trametes versicolor laccase on nanoparticles and ka-oliniterdquo Chemosphere vol 66 no 9 pp 1618ndash1626 2007

[31] Y Liu CGuo FWang C-Z Liu andH-Z Liu ldquoPreparation ofmagnetic silica nanoparticles and their application in laccaseimmobilizationrdquoTheChinese Journal of Process Engineering vol8 no 3 pp 583ndash588 2008

[32] A Salis M Pisano M Monduzzi V Solinas and E SanjustldquoLaccase from Pleurotus sajor-caju on functionalised SBA-15mesoporous silica immobilisation and use for the oxidationof phenolic compoundsrdquo Journal of Molecular Catalysis BEnzymatic vol 58 no 1ndash4 pp 175ndash180 2009

[33] V R Sarath Babu M A Kumar N G Karanth andM SThak-ur ldquoStabilization of immobilized glucose oxidase against ther-mal inactivation by silanization for biosensor applicationsrdquo Bi-osensors and Bioelectronics vol 19 no 10 pp 1337ndash1341 2004

[34] N Duran M A Rosa A DrsquoAnnibale and L Gianfreda ldquoAppli-cations of laccases and tyrosinases (phenoloxidases) immobi-lized on different supports a reviewrdquo Enzyme and MicrobialTechnology vol 31 no 7 pp 907ndash931 2002

[35] G Olivieri A Marzocchella and P Salatino ldquoHydrodynamicsandmass transfer in a lab-scale three-phase internal loop airliftrdquoChemical Engineering Journal vol 96 no 1ndash3 pp 45ndash54 2003

[36] G Olivieri A Marzocchella and P Salatino ldquoA novel three-phase airlift reactor without circulation of solidsrdquo CanadianJournal of Chemical Engineering vol 88 no 4 pp 574ndash5782010

[37] P W Tardioli G M Zanin and F F de Moraes ldquoCharacteri-zation ofThermoanaerobacter cyclomaltodextrin glucanotrans-ferase immobilized on glyoxyl-agaroserdquo Enzyme and MicrobialTechnology vol 39 no 6 pp 1270ndash1278 2006

[38] D S Clark ldquoI Can immobilization be exploited to modify en-zyme activityrdquo Trends in Biotechnology vol 12 no 11 pp 439ndash443 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


performances leading to higher activity and stability at ex-treme pHs elevated temperatures or in organic solventsand improved thermostability Such enhanced features haveencouraged the use of immobilized laccases in several appli-cations as recently reviewed by Fernandez-Fernandez [10]Different methodologies have been reported for laccase im-mobilization such as adsorption entrapment encapsulationcovalent binding and self-immobilization The choice of themost suited method clearly depends on application that lac-case is devoted to Particularly covalent binding is the mostwidely applied method for exploitation of immobilized lac-cases in industrial applications [11ndash13]

Covalent immobilization represents an attractive optionto obtain enzymatic catalyst for wastewater treatment Thistechnique provides different advantages (i) it prevents en-zyme leakage even under harsh conditions (ii) it facilitatesenzyme use in continuous packed bed stirred tank and flu-idized bed reactors (iii) it causes stabilization of the enzymetertiary structure usually as a consequence of multipointattachment of the enzyme to the support providing enzymerigidity The stabilization provided by covalent bonding isusually counterbalanced by partial enzyme deactivationThisnegative effect can be mitigated by carefully optimizing theimmobilization conditions in order to maximize the ratiobetween immobilized enzyme activity and activity of theprimary enzyme solution

Decolourisation of several dyes has been achieved bymeans of laccase covalently bound to different supportssuch as alumina oxide pellets [14] controlled porosity-carrierbeads [15] and epoxy-activated supports [13] Effectivedye removal was also carried out by C unicolor laccasecovalently immobilized on mesostructured siliceous cellularfoams (MCGs) [16] or on supports functionalized with epoxygroups [2] In the latter case the degradation of differentclasses of dyes was reported along with an improvement ofstability toward pH temperature and storage of the immo-bilized enzyme Entrapment of laccase has also been used asan immobilization approach for environmental applicationsChitosan-coated alginate beads have been packed in a fixedbed reactor and employed in RBBR decolourisation achiev-ing up to 70 decolourisation [6] Alginate beads differentlymodified with chitosan or polyethylene glycol (PEG) havealso shown to effectively decolourise several different dyes[17ndash19] Entrapment in hydrogel structures has also beenapplied for example in the immobilization of a T versicolorlaccase resulting in low substrate affinity but improvement instorage stability and in the decolourisation of Acid Orange 52[20]

An important aspect to be considered in the design of anideal immobilized biocatalyst is the cost related to the supportand to the enzyme to be linked to it In this study a low-costlaccase mixture was obtained from P ostreatus culture brothafter optimization of laccase production conditions and one-step protein enrichment as described by Palmieri et al [6]On the other hand the search for a cheap solid support hasoriented our study toward the choice of a siliceous materialperlite Perlite is an amorphous aluminium silicate with morethan 70 content of silica Besides exhibiting the advantagesof an inorganic carrier with respect to organic ones such as a

greater mechanical stability and resistance toward microbialattack and organic solvents perlite turns out to be a cheaperalternative in comparison with other reported inorganic sup-ports such as silica gels alumina and zeolites [21]The choiceof such an inert support implies that its surface has to be prop-erly modified in order to offer functional groups for proteinbinding Surface modification of these materials can be easilyachieved and their reactivity may be finely tuned in the der-ivatization steps [22]

In this work covalent immobilization of laccase on acti-vated siliceous support perlite was investigated and the im-mobilized biosystem was tested for its potential exploitationin dye decolourisation using the reactive dye RBBR as modelsubstrate The immobilization process was properly opti-mized with reference to the immobilization yield and to thedye adsorption capacity of the solid biocatalyst Stability andcatalytic parameters of immobilized laccases were also as-sessed in comparison with the soluble counterpart

2 Materials and Methods

21 Fungal Culture and Crude Laccase Extraction Pleuro-tus ostreatus (type Florida ATCC number MYA-2306) wasmaintained through periodic transfer every 3 weeks at 4∘ onagar plates containing 24 gL potato dextrose and 5 gL yeastextract Medium components were supplied by Difco Labo-ratories (Detroit MI) Mycelium growth and crude laccasemixture extraction were carried out following the proceduresdescribed by Faraco et al [7]

The crude laccase mixture extract is characterized by fourisoforms (POXA1b POXA3a POXA3b and POXC) Theactivity of the POXC isoform accounts for about 99 of thetotal mixture activity [7]The laccase mixture used in the im-mobilization trials displays a specific activity of 70Umg

22 Dye The anthraquinonic dye Remazol Brilliant Blue R(RBBR) was purchased from Sigma-Aldrich Powder puritywas 50Dye concentrationwasmeasured by recording opti-cal absorbance at 592 nm The extinction coefficient (120576

592=

9000Mminus1 cmminus1) referred to total powder concentration wascorrected taking into account the purity All other reagentswere purchased from Sigma-Aldrich with a ge98 purity

Laccase Activity Assay Laccase activity was assayed at 25∘Cby monitoring the oxidation of ABTS at 420 nm (120576

420= 36 times

103Mminus1 cmminus1) [6] The assay mixture contained 2mMABTS

in 01M sodium citrate buffer (pH 30)119870119872

values were estimated using the software GraphPadPrism (GraphPad Software La Jolla CA USA httpwwwgraphpadcom) on a wide range of substrate concentrations(005ndash3mM) Enzyme activity was expressed in internationalunits (IU)

23 Protein Determination and Electrophoresis Protein con-centration was determined using the BioRad Protein Assay(Bio-Rad Laboratories Segrate (MI) Italy) with bovineSerum albumin (BSA) as standard



3at 60∘C






(2)

(7)

(4)

(6)

(5)

(1)

(3)




119903) by means





























0

50

100

150

200

250

0

20

40

60

80

100

0 150 300 450 600 750 900

Imm

obili

zed

activ

ity (I

Ug

)

Imm

obili

zatio

n yi

eld

()










mixture (11990512



12(11990512






119872= 044mM and 119870cat = 12 sdot




value for 119870119872




(a) (b)


10

100

1000

0 10 20 30

Resid

ual a

ctiv

ity (

)

Time (d)

(a)

10

100

1000

Resid

ual a

ctiv

ity (

)

0 10 20 30Time (d)

(b)


















0

20

40

60

80

100

0 50 100 150 200 250 300

RBBR

conv

ersio

n (

)

120591 (min)






Perlitesample


(mgL)


119903

(mLmin)

Total volume(mL)


(mLmin)

Recirculationratio

120591

(min)


(mgL)

Dye conversion()

C1 415 13 71100903

mdash1450

7176273

415355279

mdash1433

C2 391 13 711609028

mdash1446

4476253

354218172

954456



4 Conclusions








Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3at 60∘C






(2)

(7)

(4)

(6)

(5)

(1)

(3)




119903) by means





























0

50

100

150

200

250

0

20

40

60

80

100

0 150 300 450 600 750 900

Imm

obili

zed

activ

ity (I

Ug

)

Imm

obili

zatio

n yi

eld

()










mixture (11990512



12(11990512






119872= 044mM and 119870cat = 12 sdot




value for 119870119872




(a) (b)


10

100

1000

0 10 20 30

Resid

ual a

ctiv

ity (

)

Time (d)

(a)

10

100

1000

Resid

ual a

ctiv

ity (

)

0 10 20 30Time (d)

(b)


















0

20

40

60

80

100

0 50 100 150 200 250 300

RBBR

conv

ersio

n (

)

120591 (min)






Perlitesample


(mgL)


119903

(mLmin)

Total volume(mL)


(mLmin)

Recirculationratio

120591

(min)


(mgL)

Dye conversion()

C1 415 13 71100903

mdash1450

7176273

415355279

mdash1433

C2 391 13 711609028

mdash1446

4476253

354218172

954456



4 Conclusions








Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























0

50

100

150

200

250

0

20

40

60

80

100

0 150 300 450 600 750 900

Imm

obili

zed

activ

ity (I

Ug

)

Imm

obili

zatio

n yi

eld

()










mixture (11990512



12(11990512






119872= 044mM and 119870cat = 12 sdot




value for 119870119872




(a) (b)


10

100

1000

0 10 20 30

Resid

ual a

ctiv

ity (

)

Time (d)

(a)

10

100

1000

Resid

ual a

ctiv

ity (

)

0 10 20 30Time (d)

(b)


















0

20

40

60

80

100

0 50 100 150 200 250 300

RBBR

conv

ersio

n (

)

120591 (min)






Perlitesample


(mgL)


119903

(mLmin)

Total volume(mL)


(mLmin)

Recirculationratio

120591

(min)


(mgL)

Dye conversion()

C1 415 13 71100903

mdash1450

7176273

415355279

mdash1433

C2 391 13 711609028

mdash1446

4476253

354218172

954456



4 Conclusions








Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












0

50

100

150

200

250

0

20

40

60

80

100

0 150 300 450 600 750 900

Imm

obili

zed

activ

ity (I

Ug

)

Imm

obili

zatio

n yi

eld

()










mixture (11990512



12(11990512






119872= 044mM and 119870cat = 12 sdot




value for 119870119872




(a) (b)


10

100

1000

0 10 20 30

Resid

ual a

ctiv

ity (

)

Time (d)

(a)

10

100

1000

Resid

ual a

ctiv

ity (

)

0 10 20 30Time (d)

(b)


















0

20

40

60

80

100

0 50 100 150 200 250 300

RBBR

conv

ersio

n (

)

120591 (min)






Perlitesample


(mgL)


119903

(mLmin)

Total volume(mL)


(mLmin)

Recirculationratio

120591

(min)


(mgL)

Dye conversion()

C1 415 13 71100903

mdash1450

7176273

415355279

mdash1433

C2 391 13 711609028

mdash1446

4476253

354218172

954456



4 Conclusions








Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







mixture (11990512



12(11990512






119872= 044mM and 119870cat = 12 sdot




value for 119870119872




(a) (b)


10

100

1000

0 10 20 30

Resid

ual a

ctiv

ity (

)

Time (d)

(a)

10

100

1000

Resid

ual a

ctiv

ity (

)

0 10 20 30Time (d)

(b)


















0

20

40

60

80

100

0 50 100 150 200 250 300

RBBR

conv

ersio

n (

)

120591 (min)






Perlitesample


(mgL)


119903

(mLmin)

Total volume(mL)


(mLmin)

Recirculationratio

120591

(min)


(mgL)

Dye conversion()

C1 415 13 71100903

mdash1450

7176273

415355279

mdash1433

C2 391 13 711609028

mdash1446

4476253

354218172

954456



4 Conclusions








Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)


10

100

1000

0 10 20 30

Resid

ual a

ctiv

ity (

)

Time (d)

(a)

10

100

1000

Resid

ual a

ctiv

ity (

)

0 10 20 30Time (d)

(b)


















0

20

40

60

80

100

0 50 100 150 200 250 300

RBBR

conv

ersio

n (

)

120591 (min)






Perlitesample


(mgL)


119903

(mLmin)

Total volume(mL)


(mLmin)

Recirculationratio

120591

(min)


(mgL)

Dye conversion()

C1 415 13 71100903

mdash1450

7176273

415355279

mdash1433

C2 391 13 711609028

mdash1446

4476253

354218172

954456



4 Conclusions








Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













0

20

40

60

80

100

0 50 100 150 200 250 300

RBBR

conv

ersio

n (

)

120591 (min)






Perlitesample


(mgL)


119903

(mLmin)

Total volume(mL)


(mLmin)

Recirculationratio

120591

(min)


(mgL)

Dye conversion()

C1 415 13 71100903

mdash1450

7176273

415355279

mdash1433

C2 391 13 711609028

mdash1446

4476253

354218172

954456



4 Conclusions








Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Perlitesample


(mgL)


119903

(mLmin)

Total volume(mL)


(mLmin)

Recirculationratio

120591

(min)


(mgL)

Dye conversion()

C1 415 13 71100903

mdash1450

7176273

415355279

mdash1433

C2 391 13 711609028

mdash1446

4476253

354218172

954456



4 Conclusions








Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

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