research article preparation of laccase immobilized...

Hindawi Publishing CorporationJournal of ChemistryVolume 2013 Article ID 387181 7 pageshttpdxdoiorg1011552013387181

Research ArticlePreparation of Laccase Immobilized Cryogels andUsage for Decolorization

Murat Uygun

Kocarlı Vocational and Training School Adnan Menderes University 09970 Aydın Turkey

Correspondence should be addressed to Murat Uygun muygunaduedutr

Received 29 May 2013 Revised 15 July 2013 Accepted 15 July 2013

Academic Editor Tanaji Talele

Copyright copy 2013 Murat UygunThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Poly(methyl methacrylate-co-glycidyl methacrylate) (poly(MMA-co-GMA)) cryogels were synthesized by radical cryopolymer-ization technique Then laccase enzyme was covalently attached to the cryogel and characterized by using swelling studies andSEM and EDX analyses Kinetic properties and optimum conditions of the immobilized and free laccase were studied and it wasfound that 119881max of the immobilized laccase was lower than that of free laccase 119870

119898of the immobilized laccase was increased upon

immobilization Optimum pH was found to be 40 for each type of laccase while optimum temperature was shifted to the warmerregion after the immobilization It was also found that thermal stability of the immobilized laccase was higher than that of freelaccase Immobilized laccase could be used for 10 times successive reuse with no significant decrease in its activity Also theselaccase immobilized cryogels were successfully used for the decolorization of seven different dyes

1 Introduction

Laccase (benzenedioloxygen oxidoreductase EC 11032) isa copper bearing enzyme in multicopper family which isproduced by various plants and secreted by diverse fungispecies which have lignin degradation capability [1] Recentlysome bacterial species which demonstrate laccase activitywere also characterized Laccase oxidize a wide variety of thephenolic compounds and aromatic amines by catalyzing thereaction of the one electron oxidation of phenols anilinesand aromatic thiols to their radicals with parallel reductionof oxygen to water [2] Substrate specificity of the laccaseis broad and it can oxidize not only these compounds butalso nonphenolic substances [3] Laccase can be used invarious industrial applications such as pulp delignificationwood fiber modification dye or stain bleaching chemicalor medicinal synthesis and contaminated water or soilremediation due to its wide specificity behavior against itsnonspecific substrates [4] Besides all these one of the uniqueproperties of laccase is the capability for degradation ofdyes The enzyme can decolorize various dyes such as azoanthraquinone and triphenylmethane through nonspecific

free radical mechanismwith creation of phenolic compounds[5 6]

Using the laccase in industrial applications has somelimitations such as low stability and productivity and its highproduction cost [7] In order to improve the reusability ofthe enzymes increase the enzyme stability and reduce thecost laccase has been immobilized successfully on varioussupport materials such as magnetic chitosan microspheres[1] nonporous poly(GMAEGDMA) beads [8] polyamide66 fibers [9] poly(glycidyl methacrylate) brush graftedpoly(hydroxyethyl methacrylate) films [10] methylene bluemodified mesoporous silica MCM-41PVA [11] magneticmesoporous silica nanoparticle [7] alginategelatin blentwith PEG [12] epoxy-activated Sepabeads EC-EP3 and Dil-beads NK [4] alginate-chitosan microcapsules [13] and PVAcryogel [14]

There is a great interest in the area of the improvementand development of new materials which are used for thebioseparation processes due to the ever growing demandsfor the biologically active pure compounds (low molecularweight compounds biopolymers such as DNA and proteins

2 Journal of Chemistry

viruses cell organelles and cells) [15] For this reason devel-opment of macroporous polymeric materials has attractedgreat attention especially in biomedical biotechnologicalandmedical purposes [16] One of the new types of polymericmaterials which have significant potential in biotechnology iscryogelsThey are synthesized at frozen temperature and thusare named as cryogel (in Greek frozen or ice) Cryogels areextremely porous polymericmaterials and can be synthesizedin various morphology and porosity by using any of thegel producing precursors [15] Nowadays cryogels find toomany usages in various biotechnological applications as achromatographic material a support material for immobi-lization of molecules and cells a matrix for electrophoresisand immunodiffusion and a gel support for solid culturemedia [17] Basic application areas of the cryogels are bio-catalysis with immobilized enzymes and cells biosepara-tion for purification of target molecules chromatographyof cell organelles viruses microbial and mammalian cellsand three-dimensional matrix for mammalian cell culture[16]

Dyes are widely used in textile paper cosmetic pharma-ceutical and dyeing and printing industries and the effluentsfrom these industries were released to the environment Thiseffluent contains environmentally hazardous polluting agentssuch as pesticides heavy metals pigments and dyes Thedegradation of these dye bearing effluents is not too easyand conventional treatment techniques (such as activatedsludge trickling filters adsorption coagulation-flocculationion-exchange oxidation electrochemical methods etc) doesnot effectively degrade them due to the complex structureof the effluent besides these techniques may also generatehazardous byproducts [3 18] These treatment techniquesare highly expensive and because of this their application inindustrial waste treatment is also limited In recent yearsthere is a great attention to the use of microbial enzymesfor dye decolorization applications due to their effectivetreatment behavior [19] For these purposes laccase has beenwidely used for decolorization of variety of synthetic dyessuch as Remazol Brilliant Blue [4 20ndash22] Acid Green 27Acid Violet 7 and Indigo Carmine [23] Neolane YellowMaxilon Blue Neolane Pink Basacryl Yellow Neolane Blueand Bezaktiv Yellow [3] Remazol Black-5 [19 21] Acid Blue62 Acid Blue 40 Reactive Blue 81 Direct Black 22 Acid Red27 [24] Reactive Red 251 Reactive Orange 122 [21] ReactiveBlack 5 Acid Blue 25 Methyl Orange Methyl Green AcidGreen 27 [4] Direct Red 28 Acid Orange 74 Reactive Blue15 Acid Blue 74 Reactive Blue 19 Azure B [25] BromophenolBlue Naphthol Blue and Methyl Red [26]

In this paper decolorization efficiency of the immobilizedlaccase was studied For these purposes poly(methyl meth-acrylate-co-glycidyl methacrylate) (poly(MMA-co-GMA))cryogels were synthesized with cryopolymerization tech-nique Prepared cryogels were characterized by using SEMand EDX analyses and swelling studies Then laccase wascovalently immobilized onto these newly synthesized cryo-gels Kinetic parameters of free and immobilized laccasewere also investigated and optimum pH and temperatureprofiles were studied Also thermal stability of the freeand immobilized form of laccase was investigated Finally

decolorization capability of the immobilized laccase wasstudied with various dyes

2 Experimental

21 Materials Laccase (from Trametes versicolor) methylmethacrylate glycidyl methacrylate NN1015840-methylene-bisa-crylamide (MBAAm) ammonium persulfate (APS) and NNN1015840N1015840-tetramethylene diamine (TEMED) were suppliedfrom Sigma (St Louis USA) All other chemicals wereof reagent grade and purchased from Aldrich (SteinheimGermany)

22 Synthesis of Poly(MMA-co-GMA) Cryogel Poly(MMA-co-GMA) cryogel was synthesized with free radical cryopoly-merization technique [27] Polymerization procedure wasexplained as follows Firstly 0283 g ofMBBAmwas dissolvedin 100mL of distilled water and mixed with 50mL of MMAandGMA solution which was prepared by dissolving 107mLof MMA and 100 120583L of GMA in 100mL of distilled waterThen 200mgofAPS and 25120583Lof TEMEDwere added to thismixture and polymerization reaction was initiated Resultingsolution was immediately poured to the syringe and wasfrozen at minus12∘C for 24 h After this polymerization periodprepared cryogel was washed with distilled water in orderto remove unreacted monomers and initiators Chemicalstructure of the poly(MMA-co-GMA) cryogel was shown inFigure 1

23 Immobilization of Laccase onto Poly(MMA-co-GMA)Cryogel Laccase immobilization experiments were carriedout according to the literature described [28] Brieflypoly(MMA-co-GMA) cryogel was equilibrated with pH 80phosphate buffer (50mM) for 2 h Then 200mL of laccasesolution (20mgmL in pH 80 phosphate buffer) was passedthrough the cryogel column (0335 g-dry weight) by usinga peristaltic pump at 22∘C for 18 h Covalent attachmentbetween the cryogel and laccase molecule was carried out bythe help of epoxy groups of theGMAmonomer in the cryogelstructure Immobilization of the laccase onto poly(MMA-co-GMA) cryogel was schematically demonstrated in Figure 2Immobilized amount of laccase was determined by mea-suring the initial and final laccase concentrations with themethod of Bradford [29]

24 Characterization of Poly(MMA-co-GMA) Cryogel Ener-gy Dispersive X-Ray (EDX) analysis of cryogel was carriedout by using an EDX instrument (LEO EVO 40 CarlZeiss NTS USA) Pore size and cryogel morphology wereinvestigated with Scanning Electron Microscopy (SEM) Forthis cryogel was dried and coated with one layer of goldand then SEM photograph of cryogel was taken by using anSEM device (Philips XL-30S FEGThe Netherland) Swellingdegree (S) of poly(MMA-co-GMA) and laccase immobilizedpoly(MMA-co-GMA) cryogels were also determined Forthis cryogels were dried at 60∘C for 3 days and weighed (119898

119889)

Then cryogels were placed in 500mL of distilled water at

Journal of Chemistry 3

CH3

CH3

CH3

C

OOC O

C O

CH2

CH2

CH3

CH3

C

OC O

O

CH2C CH2

CH3

OC O

CH2

O

C CH2

n

Poly(methyl methacrylate-co-glycidyl methacrylate)

+

Methylmethacrylate

Glycidylmethacrylate

Figure 1 Chemical structure of poly(MMA-co-GMA) cryogel

Poly

(MM

A-co

-GM

A) c

ryog

el

Poly

(MM

A-co

-GM

A) c

ryog

el

Laccase

+

OH

OH

OH

O

O

O

Figure 2 Schematic presentation of the immobilization of laccaseonto cryogel structure

25∘C for 2 h Swelled cryogels were taken out from water andweighed (119898

119904) and the swelling degree was calculated as [27]

119878 =119898119904minus 119898119889

119898119889

(1)

25 Laccase Activity Studies Laccase activity was deter-mined by using the 221015840-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as a substrate [30] For free enzyme 01mLof ABTS solution (100mM)wasmixed with 880120583L of pH 40acetate buffer solution (100mM) and incubated at 25∘C for20minThen enzymatic reaction was initiated with additionof 20120583L of enzyme solution Activity of the immobilizedlaccase was determined in continuous system For this 10mLof ABTS (100mM) in acetate buffer (pH 40 100mM) wasused as a substrate solution and passed through the laccaseimmobilized cryogelThe activity of the free and immobilizedform of laccase was measured by using the increase inabsorbance at 420 nm One unit of laccase activity is definedas the required enzyme amount for the oxidation of 10120583molof ABTS per min at 25∘C

26 Determination of Kinetic and Optimal Properties of Freeand Immobilized Laccase In order to determine the 119870

119898and

119881max values of the free and immobilized form of laccaseinitial ABTS concentrations were changed between 10 and100mM in pH 40 acetate buffer (100mM) at 25∘C Activitystudies of the free and immobilized form of laccase wereperformed in the pH range of 30ndash60 by using 01M ofacetate buffer (for pH 30ndash45) and 01M of phosphate buffer

Figure 3 SEM photograph of the poly(MMA-co-GMA) cryogel

(for pH 50-60) in order to determine the optimum pHprofiles of two laccase forms In order to determine theoptimum temperature of the laccase medium temperaturewas changed between 40 and 60∘C Thermal stability offree and immobilized laccase was also determined at 55 and65∘C For this enzyme preparations were incubated at 55and 65∘C and activities of the enzymes were measured withthe above mentioned method with defined time intervals for5 h In order to investigate the operational stability of theimmobilized laccase activity measurements were repeatedfor 10 times Storage stability of free and immobilized laccasewas also investigated for 30 days For this purpose enzymepreparationswere stored at+4∘Cand activities of the enzymeswere determined at the beginning and at the end of 30days of storage After each activity experiments immobilizedcryogels were washed with water and equilibrated with pH40 acetate buffer (100mM) for next activity study

27 Decolorization Studies Seven different dyes Procion Red(120582max 536 nm) Reactive Green 5 (120582max 674 nm) ReactiveBrown 10 (120582max 526 nm) Reactive Green 19 (120582max 631 nm)Cibacron Blue F3GA (120582max 605 nm) Alkali Blue 6B (120582max587 nm) and Brilliant Blue 6 (120582max 607 nm)) were used forthe investigation of the decolorization efficiency of the laccaseimmobilized poly(MMA-co-GMA) cryogel For this 100mLof dye solution (01mgmL) was passed through the laccaseimmobilized cryogel column by using a peristaltic pump at


14

12

10

8

6

4

2

0

(keV)2 4

SSC O

N

CPS

(eV

)

Figure 4 EDX spectrum of the laccase immobilized poly(MMA-co-GMA) cryogel

0

20

40

60

80

100

2 3 4 5 6

Activ

ity (

)

pH

Immobilized laccaseFree laccase

Figure 5 Effect of pH on the activity of free and immobilized formof laccase

the flow rate of 05mLmin Decolorization activity wasmon-itored photometrically by using a UV-Vis spectrophotometer(Shimadzu 1601 Japan) for 10min

All measurements were repeated three times and theaverage values were used for all calculations

3 Results and Discussion

31 Synthesis and Characterization of Poly(MMA-co-GMA)Cryogel Synthesized poly(MMA-co-GMA) cryogel hadsponge-like morphology and was elastic and opaque Whencompressed by hand cryogel lost all water accumulatedinside the pores This cryogel exhibited fast swellingproperties and when dried cryogel was submerged in water

0

20

40

60

80

100

120

Activ

ity (

)


0 10 20 30 40 50 60Temperature (∘C)

Figure 6 Effect of temperature on the activity of free and immobi-lized form of laccase

Table 1 Kinetic constants of the free and immobilized laccase

Enzyme type 119870119898(mM) 119881max (120583molmin)

Free 526 0016Immobilized 1111 0013

it swelled rapidly and restored its original shape and sizewithin 1-2min Internal structure and morphology of thepoly(MMA-co-GMA) cryogel was shown in Figure 3 Asshown in the figure cryogel had a macroporous structureand pore diameter was found in the range of 10ndash100 120583mEDX analysis of the laccase immobilized poly(MMA-co-GMA) cryogel was demonstrated in Figure 4 As seen heresynthesized laccase immobilized cryogel composed ofCONand S atoms As clearly seen here that while the poly(MMA-co-GMA) cryogel contained only C and O atoms additionalN and S atoms appeared here due to the incorporation ofprotein structured laccase onto the cryogenic structureImmobilized amount of laccase was also investigated andit was found to be 517mgg cryogel Specific activities offree and immobilized form of laccase were determinedas 89 times 10minus3Umg and 77 times 10minus3Umg respectivelyAs stated here activity of laccase decreased slightly uponimmobilization and this decrease is carried out probablydue to the certain conformational changes which weretaken place upon immobilization The equilibrium swellingdegree of the poly(MMA-co-GMA) and laccase immobilizedpoly(MMA-co-GMA) cryogels were calculated as 821 gH2Og cryogel and 993 g H

2Og cryogel respectively It can

be concluded from this result that swelling degree of thecryogel increased with incorporation of laccase onto thecryogel structure

32 Kinetic and Optimal Properties of Free and ImmobilizedLaccase The kinetic constants of free and immobilized formof laccase were summarized in Table 1 As seen in thetable 119881max value of laccase decreased upon immobilizationfrom 0016 to 0013120583molmin The 119870

119898value of immobilized


Activ

ity (

)


0102030405060708090

100

0 1 2 3 4 5Time (h)

(a)


Activ

ity (

)

0102030405060708090

100

0 1 2 3 4 5Time (h)

(b)

Figure 7 Thermal stability profile of the free and immobilized laccase at 55∘C (a) and 65∘C (b)

laccase (1111mM) was about 2 times higher than that offree laccase (526mM) This increase in 119870

119898value indicated

that affinity of the laccase to its substrate decreased withimmobilizationThese decreases in the activity were probablydue to the steric hindrances caused by the support ordecrease in the enzyme flexibility or diffusional limitationsof substrate [1] Effect of pH on the activity of the free andimmobilized laccase was demonstrated in Figure 5 As seenin figure maximum activity was observed at pH 40 forboth free and immobilized laccase Above and below this pHvalue enzymatic activity of the laccase decreased dramati-cally Optimum temperature profile of free and immobilizedlaccase was shown in Figure 6 As seen here optimumtemperatures of free and immobilized laccase were found tobe 25 and 45∘C respectively This shift towards the highertemperature brings about very important property to theimmobilized form of laccase Dyeing and painting processgenerally carried out at high temperatures and effluentsfrom these industries are also protecting their temperaturesCooling is often time consuming and it is essential to treatthem even if they are already hot Immobilized laccase withoptimum temperature at 45∘Cmay be successfully applicablefor the decolorization of such wastes The same thermalproperty of the immobilized laccase was also monitoredwith the thermal stability studies Thermal stability profilesof the free and immobilized laccase were investigated at 55and 65∘C and findings were demonstrated in Figures 7(a)and 7(b) respectively As seen in the figure while freelaccase protected 64 of its initial activity at the end of5 h incubation at 55∘C immobilized laccase showed 77 ofinitial activity The same finding also monitored at 65∘Cimmobilized laccase demonstrated 67 of its initial activityat the end of 5 h incubation while free laccase showed only34 From these results it can be concluded that thermalstability and resistance of the laccase were increased withimmobilization process These findings can also enhance theusability of the immobilized form laccase in waste water

Activ

ity (

)

0102030405060708090

100

0 2 4 6 8 10Reuse number

Figure 8 Operational stability of the immobilized laccase

management Operational stability profile of the immobilizedlaccase was demonstrated in Figure 8 As demonstrated infigure operational stability of the immobilized laccase wasfound to be very high At the end of the 10th reuse activity ofthe immobilized laccase decreased only about 67 Storagestability of the free and immobilized form of laccase wasalso determined and it was found that while immobilizedenzyme protected 855 of its initial activity free preparationprotected 528 of its initial activity at the end of the 30 days

33 Decolorization Studies Decolorization efficiency ofimmobilized laccase was demonstrated in Figure 9 As seenhere immobilized laccase decolorized the studied sevendyes effectively All dyes decolorized by using immobilizedlaccase at the rate of 50 at the end of 10min Decolorizationpercentage of the dyes were also given in Table 2 Murugesanet al [19] used laccase from Ganoderma lucidum fordecolorization of Remazol Brilliant Blue R and they foundthat Remazol Brilliant Blue R was decolorized by 774within 2 h Peralta-Zamora et al [21] investigated that the


Table 2 Decolorization percentage of dyes at the end of 10min

Dyes ProcionRed

ReactiveGreen 5

ReactiveBrown 10

ReactiveGreen 19

Cibacron BlueF3GA

AlkaliBlue 6B

BrilliantBlue 6

Decolorization 8153 5981 7359 6633 6295 5971 6168

Time (min)0 2 4 6 8 10

0

10

20

30

40

50

60

70

80

Dec

olor

izat

ion

()

Procion RedReactive Green 5Reactive Brown 10Reactive Green 19

Cibacron Blue F3GAAlkali Blue 6BBrilliant Blue 6

Figure 9 Decolorization efficiency of immobilized laccase ontopoly(MMA-co-GMA) cryogel

decolorization of Remazol Brilliant Blue R Remazol BlackB Reactive Orange 122 and Reactive Red 251 dyes by usingimmobilized laccase within 30min and decolorizationcapacities were found to be 35ndash45 10 10ndash30 and5ndash55 respectively Kunamneni et al [4] used immobilizedform of laccase in order to decolorize the synthetic dyesand they found 61ndash82 decolorization rates within 6 hClaus et al [25] used laccase from Trametes versicolor fordecolorization of azo dyes and they reached 30ndash82decolorization efficiency within 16 h Murugesan et al [6]purified laccase enzyme from Pleurotus sajor-caju and usedfor the decolorization of three azo dyes Investigators found70ndash90 decolorization yield within 24 h One of the mostimportant features of laccase immobilized poly(MMA-co-GMA) cryogel was its speed It reached high decolorizationvalues only within 10min and it can be concluded form theseresults that immobilized from of laccase was successfullyused for the decolorization of dyes by using a continuoussystem and this system can be adapted to the industrial wastewater management system as a decolorization agent

4 Conclusion

Dye effluents cause serious environmental pollution andmanagement of these effluents is difficult due to the complexstructure of the dye wastes and used techniques for thesepurposes are very expensive For these purposes new decol-orization techniques have been developed and used for man-agement of the dye effluents Laccase has been used extremely

for the decolorization process due to its unique enzymaticproperties Its immobilized form especially has been usedin various decolorization studies One of the new polymericmaterials which are used intensively in biotechnological areais cryogel Preparation of these polymeric materials is easyand can be produced in desired shape size and functionali-ties In this presented work laccase was successfully immobi-lized onto poly(MMA-co-GMA) cryogel and decolorizationproperties of this preparation was investigated It was shownthat this new immobilized laccase preparation was used fordecolorization of seven different dyes and decolorized allstudied dyes effectively It can be concluded from these resultsthat this new laccase immobilized cryogenic medium canbe used for the decolorization of the dye and paint industryeffluents and the other dye bearing waste waters

Conflict of Interests

No conflict of interests was declared

References

[1] D-S Jiang S-Y Long J Huang H-Y Xiao and J Y ZhouldquoImmobilization of Pycnoporus sanguineus laccase onmagneticchitosan microspheresrdquo Biochemical Engineering Journal vol25 no 1 pp 15ndash23 2005

[2] S Camarero D Ibarra M J Martınez and A T MartınezldquoLignin-derived compounds as efficient laccase mediators fordecolorization of different types of recalcitrant dyesrdquo Appliedand Environmental Microbiology vol 71 no 4 pp 1775ndash17842005

[3] H Zouari-Mechichi T Mechichi A Dhouib S Sayadi AT Martınez and M J Martınez ldquoLaccase purification andcharacterization from Trametes trogii isolated in Tunisia decol-orization of textile dyes by the purified enzymerdquo Enzyme andMicrobial Technology vol 39 no 1 pp 141ndash148 2006

[4] 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

[5] A Zille B Gornacka A Rehorek and A Cavaco-PauloldquoDegradation of azo dyes by Trametes villosa laccase over longperiods of oxidative conditionsrdquo Applied and EnvironmentalMicrobiology vol 71 no 11 pp 6711ndash6718 2005

[6] K Murugesan M Arulmani I-H Nam Y-M Kim Y-SChang and P T Kalaichelvan ldquoPurification and characteriza-tion of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential indecolorization of azo dyesrdquo Applied Microbiology and Biotech-nology vol 72 no 5 pp 939ndash946 2006

[7] F Wang C Guo L-R Yang and C-Z Liu ldquoMagneticmesoporous silica nanoparticles fabrication and their laccaseimmobilization performancerdquo Bioresource Technology vol 101no 23 pp 8931ndash8935 2010


[8] M Y Arica B Altintas and G Bayramoglu ldquoImmobilizationof laccase onto spacer-arm attached non-porous poly(GMAEGDMA) beads application for textile dye degradationrdquo Biore-source Technology vol 100 no 2 pp 665ndash669 2009

[9] C Silva C J Silva A Zille G M Guebitz and A Cavaco-Paulo ldquoLaccase immobilization on enzymatically functional-ized polyamide 66 fibresrdquo Enzyme and Microbial Technologyvol 41 no 6-7 pp 867ndash875 2007

[10] G Bayramoglu and M Y Arica ldquoImmobilization of laccaseonto poly(glycidylmethacrylate) brush grafted poly(hydrox-yethylmethacrylate) films enzymatic oxidation of phenoliccompoundsrdquo Materials Science and Engineering C vol 29 no6 pp 1990ndash1997 2009

[11] X Xu P Lu Y Zhou Z Zhao and M Guo ldquoLaccase immo-bilized on methylene blue modified mesoporous silica MCM-41PVArdquoMaterials Science and Engineering C vol 29 no 7 pp2160ndash2164 2009

[12] PWang X Fan L Cui QWang and A Zhou ldquoDecolorizationof reactive dyes by laccase immobilized in alginategelatin blentwith PEGrdquo Journal of Environmental Sciences vol 20 no 12 pp1519ndash1522 2008

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

[14] M D Stanescu M Fogorasi B L Shaskolskiy S Gavrilas andV I Lozinsky ldquoNew potential biocatalysts by laccase immobi-lization in PVA cryogel type carrierrdquo Applied Biochemistry andBiotechnology vol 160 no 7 pp 1947ndash1954 2010

[15] M B Dainiak I Y Galaev A Kumar F M Plieva and BMattiasson ldquoChromatography of living cells using supermacro-porous hydrogels cryogelsrdquo Advanced Biochemical Engineeringand Biotechnology vol 106 pp 101ndash127 2007

[16] F M Plleva I Y Galaev and B Mattiasson ldquoMacroporousgels prepared at subzero temperatures as novel materials forchromatography of particulate-containing fluids and cell cul-ture applicationsrdquo Journal of Separation Science vol 30 no 11pp 1657ndash1671 2007

[17] V I Lozinsky I Y Galaev F M Plieva I N Savina H Jungvidand B Mattiasson ldquoPolymeric cryogels as promising materialsof biotechnological interestrdquoTrends in Biotechnology vol 21 no10 pp 445ndash451 2003

[18] S R Couto M Sanroman and G M Gubitz ldquoInfluence ofredoxmediators andmetal ions on synthetic acid dye decolour-ization by crude laccase from Trametes hirsutardquo Chemospherevol 58 no 4 pp 417ndash422 2005

[19] K Murugesan I-H Nam Y-M Kim and Y-S Chang ldquoDecol-orization of reactive dyes by a thermostable laccase producedby Ganoderma lucidum in solid state culturerdquo Enzyme andMicrobial Technology vol 40 no 7 pp 1662ndash1672 2007

[20] H Hou J Zhou J Wang C Du and B Yan ldquoEnhancementof laccase production by Pleurotus ostreatus and its use for thedecolorization of anthraquinone dyerdquo Process Biochemistry vol39 no 11 pp 1415ndash1419 2004

[21] P Peralta-Zamora C M Pereira E R L Tiburtius et alldquoDecolorization of reactive dyes by immobilized laccaserdquoApplied Catalysis B vol 42 no 2 pp 131ndash144 2003

[22] G M B Soares M Costa-Ferreira and M T Pessoa deAmorim ldquoDecolorization of an anthraquinone-type dye usinga laccase formulationrdquo Bioresource Technology vol 79 no 2 pp171ndash177 2001

[23] Y Wong and J Yu ldquoLaccase-catalyzed decolorization of syn-thetic dyesrdquoWater Research vol 33 no 16 pp 3512ndash3520 1999

[24] A Michniewicz S Ledakowicz R Ullrich and M HofrichterldquoKinetics of the enzymatic decolorization of textile dyes bylaccase from Cerrena unicolorrdquo Dyes and Pigments vol 77 no2 pp 295ndash302 2008

[25] H Claus G Faber andH Konig ldquoRedox-mediated decoloriza-tion of synthetic dyes by fungal laccasesrdquo Applied Microbiologyand Biotechnology vol 59 no 6 pp 672ndash678 2002

[26] M Nagai T Sato H Watanabe K Saito M Kawata andH Enei ldquoPurification and characterization of an extracellularlaccase from the edible mushroom Lentinula edodes and decol-orization of chemically different dyesrdquo Applied Microbiologyand Biotechnology vol 60 no 3 pp 327ndash335 2003

[27] M Uygun D A Uygun E Ozcaliskan S Akgol and ADenizli ldquoConcanavalin A immobilized poly(ethylene glycoldimethacrylate) based affinity cryogel matrix and usability ofinvertase immobilizationrdquo Journal of Chromatography B vol887-888 pp 73ndash78 2012

[28] G Bayramoglu S Akgol A Bulut A Denizli and M YArica ldquoCovalent immobilisation of invertase onto a reactivefilm composed of 2-hydroxyethyl methacrylate and glycidylmethacrylate properties and application in a continuous flowsystemrdquo Biochemical Engineering Journal vol 14 no 2 pp 117ndash126 2003

[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[30] M D Stanescu S Gavrilas R Ludwig D Haltrich and VI Lozinsky ldquoPreparation of immobilized Trametes pubescenslaccase on a cryogel-type polymeric carrier and application ofthe biocatalyst to apple juice phenolic compounds oxidationrdquoEuropean FoodResearch andTechnology vol 234 no 4 pp 655ndash662 2012

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ElectrochemistryInternational Journal of



CatalystsJournal of


viruses cell organelles and cells) [15] For this reason devel-opment of macroporous polymeric materials has attractedgreat attention especially in biomedical biotechnologicalandmedical purposes [16] One of the new types of polymericmaterials which have significant potential in biotechnology iscryogelsThey are synthesized at frozen temperature and thusare named as cryogel (in Greek frozen or ice) Cryogels areextremely porous polymericmaterials and can be synthesizedin various morphology and porosity by using any of thegel producing precursors [15] Nowadays cryogels find toomany usages in various biotechnological applications as achromatographic material a support material for immobi-lization of molecules and cells a matrix for electrophoresisand immunodiffusion and a gel support for solid culturemedia [17] Basic application areas of the cryogels are bio-catalysis with immobilized enzymes and cells biosepara-tion for purification of target molecules chromatographyof cell organelles viruses microbial and mammalian cellsand three-dimensional matrix for mammalian cell culture[16]

Dyes are widely used in textile paper cosmetic pharma-ceutical and dyeing and printing industries and the effluentsfrom these industries were released to the environment Thiseffluent contains environmentally hazardous polluting agentssuch as pesticides heavy metals pigments and dyes Thedegradation of these dye bearing effluents is not too easyand conventional treatment techniques (such as activatedsludge trickling filters adsorption coagulation-flocculationion-exchange oxidation electrochemical methods etc) doesnot effectively degrade them due to the complex structureof the effluent besides these techniques may also generatehazardous byproducts [3 18] These treatment techniquesare highly expensive and because of this their application inindustrial waste treatment is also limited In recent yearsthere is a great attention to the use of microbial enzymesfor dye decolorization applications due to their effectivetreatment behavior [19] For these purposes laccase has beenwidely used for decolorization of variety of synthetic dyessuch as Remazol Brilliant Blue [4 20ndash22] Acid Green 27Acid Violet 7 and Indigo Carmine [23] Neolane YellowMaxilon Blue Neolane Pink Basacryl Yellow Neolane Blueand Bezaktiv Yellow [3] Remazol Black-5 [19 21] Acid Blue62 Acid Blue 40 Reactive Blue 81 Direct Black 22 Acid Red27 [24] Reactive Red 251 Reactive Orange 122 [21] ReactiveBlack 5 Acid Blue 25 Methyl Orange Methyl Green AcidGreen 27 [4] Direct Red 28 Acid Orange 74 Reactive Blue15 Acid Blue 74 Reactive Blue 19 Azure B [25] BromophenolBlue Naphthol Blue and Methyl Red [26]

In this paper decolorization efficiency of the immobilizedlaccase was studied For these purposes poly(methyl meth-acrylate-co-glycidyl methacrylate) (poly(MMA-co-GMA))cryogels were synthesized with cryopolymerization tech-nique Prepared cryogels were characterized by using SEMand EDX analyses and swelling studies Then laccase wascovalently immobilized onto these newly synthesized cryo-gels Kinetic parameters of free and immobilized laccasewere also investigated and optimum pH and temperatureprofiles were studied Also thermal stability of the freeand immobilized form of laccase was investigated Finally

decolorization capability of the immobilized laccase wasstudied with various dyes

2 Experimental

21 Materials Laccase (from Trametes versicolor) methylmethacrylate glycidyl methacrylate NN1015840-methylene-bisa-crylamide (MBAAm) ammonium persulfate (APS) and NNN1015840N1015840-tetramethylene diamine (TEMED) were suppliedfrom Sigma (St Louis USA) All other chemicals wereof reagent grade and purchased from Aldrich (SteinheimGermany)

22 Synthesis of Poly(MMA-co-GMA) Cryogel Poly(MMA-co-GMA) cryogel was synthesized with free radical cryopoly-merization technique [27] Polymerization procedure wasexplained as follows Firstly 0283 g ofMBBAmwas dissolvedin 100mL of distilled water and mixed with 50mL of MMAandGMA solution which was prepared by dissolving 107mLof MMA and 100 120583L of GMA in 100mL of distilled waterThen 200mgofAPS and 25120583Lof TEMEDwere added to thismixture and polymerization reaction was initiated Resultingsolution was immediately poured to the syringe and wasfrozen at minus12∘C for 24 h After this polymerization periodprepared cryogel was washed with distilled water in orderto remove unreacted monomers and initiators Chemicalstructure of the poly(MMA-co-GMA) cryogel was shown inFigure 1

23 Immobilization of Laccase onto Poly(MMA-co-GMA)Cryogel Laccase immobilization experiments were carriedout according to the literature described [28] Brieflypoly(MMA-co-GMA) cryogel was equilibrated with pH 80phosphate buffer (50mM) for 2 h Then 200mL of laccasesolution (20mgmL in pH 80 phosphate buffer) was passedthrough the cryogel column (0335 g-dry weight) by usinga peristaltic pump at 22∘C for 18 h Covalent attachmentbetween the cryogel and laccase molecule was carried out bythe help of epoxy groups of theGMAmonomer in the cryogelstructure Immobilization of the laccase onto poly(MMA-co-GMA) cryogel was schematically demonstrated in Figure 2Immobilized amount of laccase was determined by mea-suring the initial and final laccase concentrations with themethod of Bradford [29]

24 Characterization of Poly(MMA-co-GMA) Cryogel Ener-gy Dispersive X-Ray (EDX) analysis of cryogel was carriedout by using an EDX instrument (LEO EVO 40 CarlZeiss NTS USA) Pore size and cryogel morphology wereinvestigated with Scanning Electron Microscopy (SEM) Forthis cryogel was dried and coated with one layer of goldand then SEM photograph of cryogel was taken by using anSEM device (Philips XL-30S FEGThe Netherland) Swellingdegree (S) of poly(MMA-co-GMA) and laccase immobilizedpoly(MMA-co-GMA) cryogels were also determined Forthis cryogels were dried at 60∘C for 3 days and weighed (119898

119889)

Then cryogels were placed in 500mL of distilled water at


CH3

CH3

CH3

C

OOC O

C O

CH2

CH2

CH3

CH3

C

OC O

O

CH2C CH2

CH3

OC O

CH2

O

C CH2

n


+

Methylmethacrylate



Poly

(MM

A-co

-GM

A) c

ryog

el

Poly

(MM

A-co

-GM

A) c

ryog

el

Laccase

+

OH

OH

OH

O

O

O




119878 =119898119904minus 119898119889

119898119889

(1)



119898and






14

12

10

8

6

4

2

0

(keV)2 4

SSC O

N

CPS

(eV

)


0

20

40

60

80

100

2 3 4 5 6

Activ

ity (

)

pH







0

20

40

60

80

100

120

Activ

ity (

)


0 10 20 30 40 50 60Temperature (∘C)











Activ

ity (

)


0102030405060708090

100

0 1 2 3 4 5Time (h)

(a)


Activ

ity (

)

0102030405060708090

100

0 1 2 3 4 5Time (h)

(b)





Activ

ity (

)

0102030405060708090

100







Dyes ProcionRed

ReactiveGreen 5

ReactiveBrown 10

ReactiveGreen 19

Cibacron BlueF3GA

AlkaliBlue 6B

BrilliantBlue 6

Decolorization 8153 5981 7359 6633 6295 5971 6168

Time (min)0 2 4 6 8 10

0

10

20

30

40

50

60

70

80

Dec

olor

izat

ion

()





4 Conclusion





References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


CH3

CH3

CH3

C

OOC O

C O

CH2

CH2

CH3

CH3

C

OC O

O

CH2C CH2

CH3

OC O

CH2

O

C CH2

n


+

Methylmethacrylate



Poly

(MM

A-co

-GM

A) c

ryog

el

Poly

(MM

A-co

-GM

A) c

ryog

el

Laccase

+

OH

OH

OH

O

O

O




119878 =119898119904minus 119898119889

119898119889

(1)



119898and






14

12

10

8

6

4

2

0

(keV)2 4

SSC O

N

CPS

(eV

)


0

20

40

60

80

100

2 3 4 5 6

Activ

ity (

)

pH







0

20

40

60

80

100

120

Activ

ity (

)


0 10 20 30 40 50 60Temperature (∘C)











Activ

ity (

)


0102030405060708090

100

0 1 2 3 4 5Time (h)

(a)


Activ

ity (

)

0102030405060708090

100

0 1 2 3 4 5Time (h)

(b)





Activ

ity (

)

0102030405060708090

100







Dyes ProcionRed

ReactiveGreen 5

ReactiveBrown 10

ReactiveGreen 19

Cibacron BlueF3GA

AlkaliBlue 6B

BrilliantBlue 6

Decolorization 8153 5981 7359 6633 6295 5971 6168

Time (min)0 2 4 6 8 10

0

10

20

30

40

50

60

70

80

Dec

olor

izat

ion

()





4 Conclusion





References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


14

12

10

8

6

4

2

0

(keV)2 4

SSC O

N

CPS

(eV

)


0

20

40

60

80

100

2 3 4 5 6

Activ

ity (

)

pH







0

20

40

60

80

100

120

Activ

ity (

)


0 10 20 30 40 50 60Temperature (∘C)











Activ

ity (

)


0102030405060708090

100

0 1 2 3 4 5Time (h)

(a)


Activ

ity (

)

0102030405060708090

100

0 1 2 3 4 5Time (h)

(b)





Activ

ity (

)

0102030405060708090

100







Dyes ProcionRed

ReactiveGreen 5

ReactiveBrown 10

ReactiveGreen 19

Cibacron BlueF3GA

AlkaliBlue 6B

BrilliantBlue 6

Decolorization 8153 5981 7359 6633 6295 5971 6168

Time (min)0 2 4 6 8 10

0

10

20

30

40

50

60

70

80

Dec

olor

izat

ion

()





4 Conclusion





References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Activ

ity (

)


0102030405060708090

100

0 1 2 3 4 5Time (h)

(a)


Activ

ity (

)

0102030405060708090

100

0 1 2 3 4 5Time (h)

(b)





Activ

ity (

)

0102030405060708090

100







Dyes ProcionRed

ReactiveGreen 5

ReactiveBrown 10

ReactiveGreen 19

Cibacron BlueF3GA

AlkaliBlue 6B

BrilliantBlue 6

Decolorization 8153 5981 7359 6633 6295 5971 6168

Time (min)0 2 4 6 8 10

0

10

20

30

40

50

60

70

80

Dec

olor

izat

ion

()





4 Conclusion





References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Dyes ProcionRed

ReactiveGreen 5

ReactiveBrown 10

ReactiveGreen 19

Cibacron BlueF3GA

AlkaliBlue 6B

BrilliantBlue 6

Decolorization 8153 5981 7359 6633 6295 5971 6168

Time (min)0 2 4 6 8 10

0

10

20

30

40

50

60

70

80

Dec

olor

izat

ion

()





4 Conclusion





References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article preparation of laccase immobilized...

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