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Research Article Effects of Temperature and pH on Immobilized Laccase Activity in Conjugated Methacrylate-Acrylate Microspheres Siti Zulaikha Mazlan 1 and Sharina Abu Hanifah 1,2,3 1 School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia 2 Polymer Research Center, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia 3 Center for Water Research and Analysis, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia Correspondence should be addressed to Sharina Abu Hanifah; [email protected] Received 12 July 2017; Revised 7 September 2017; Accepted 17 September 2017; Published 18 October 2017 Academic Editor: Matthias Schnabelrauch Copyright © 2017 Siti Zulaikha Mazlan and Sharina Abu Hanifah. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Immobilization of laccase on the functionalized methacrylate-acrylate copolymer microspheres was studied. Poly(glycidyl methacrylate-co-n-butyl acrylate) microspheres consisting of epoxy groups were synthesized using facile emulsion photocuring technique. e epoxy groups in poly(GMA-co-nBA) microspheres were then converted to amino groups. Laccase immobilization is based on covalent binding via amino groups on the enzyme surface and aldehyde group on the microspheres. e FTIR spectra showed peak at 1646 cm −1 assigned to the conformation of the polymerization that referred to GMA and nBA monomers, respectively. Aſter modification of the polymer, intensity of FTIR peaks assigned to the epoxy ring at 844 cm −1 and 904 cm −1 was decreased. e results obtained from FTIR exhibit a good agreement with the epoxy content method. e activity of laccase- immobilized microspheres increased upon increasing the epoxy content. Furthermore, poly(GMA-co-nBA) microspheres revealed uniform size below 2 m that contributes to large surface area of the microspheres to be used as a matrix, thus increasing the enzyme capacity and enzymatic reaction. Immobilized enzyme also shiſted to higher pH and temperature compared to free enzyme. 1. Introduction e selection of a suitable carrier material is a key fac- tor in enzyme covalent immobilization [1]. Poly(glycidyl methacrylate) (PGMA) containing epoxy group has been widely applied due to its attractive properties and discovered as an ideal support for enzyme immobilization [2]. It has the ability to form strong linkages with amino, hydroxyl, and thiols group under mild condition [3, 4]. e modifications of epoxy ring with amine groups endow PGMA with excellent affinity to a variety of proteins, which makes it applicable in many areas and easily available for immobilization of the enzyme [5]. On the other hand, poly(n-butyl acrylate) (PnBA) provides hydrophobic property of copolymer. As the microspheres are hydrophobic, laccase immobilization will be confined to the surface of the spheres thus allowing the enzymatic reaction of enzyme and analyte to occur at the surface [6]. PGMA based materials are used as sup- ports in various forms, such as powder, membrane, and gel with different geometrical configurations for immobilization of several enzymes [7]. Recently, PGMA supports were investigated for enzyme immobilization using physical and chemical immobilization techniques. It is because smaller diameter of the microspheres provides more active sites for enzymes reaction thus improving immobilization efficiency. Immobilization is always a successful strategy for stabi- lizing enzymes or improving their performance [8]. Bezerra et al. (2015) gave guidelines on the selection of suitable solid supports and immobilization conditions [9]. Dur´ an et al. (2002) comprehensively reviewed the different immobiliza- tions of laccase, including a variety of supports available, immobilization techniques, and potential industrial applica- tions [10]. Several approaches have been reported for the immobilization of laccase such as entrapment into polymer Hindawi International Journal of Polymer Science Volume 2017, Article ID 5657271, 8 pages https://doi.org/10.1155/2017/5657271

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Research ArticleEffects of Temperature and pH on Immobilized Laccase Activityin Conjugated Methacrylate-Acrylate Microspheres

Siti Zulaikha Mazlan1 and Sharina Abu Hanifah1,2,3

1School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia,43600 Bangi, Selangor, Malaysia2Polymer Research Center, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia3Center for Water Research and Analysis, Faculty of Science and Technology, Universiti Kebangsaan Malaysia,43600 Bangi, Selangor, Malaysia

Correspondence should be addressed to Sharina Abu Hanifah; [email protected]

Received 12 July 2017; Revised 7 September 2017; Accepted 17 September 2017; Published 18 October 2017

Academic Editor: Matthias Schnabelrauch

Copyright © 2017 Siti Zulaikha Mazlan and Sharina Abu Hanifah. This is an open access article distributed under the CreativeCommons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.

Immobilization of laccase on the functionalized methacrylate-acrylate copolymer microspheres was studied. Poly(glycidylmethacrylate-co-n-butyl acrylate) microspheres consisting of epoxy groups were synthesized using facile emulsion photocuringtechnique. The epoxy groups in poly(GMA-co-nBA) microspheres were then converted to amino groups. Laccase immobilizationis based on covalent binding via amino groups on the enzyme surface and aldehyde group on the microspheres. The FTIRspectra showed peak at 1646 cm−1 assigned to the conformation of the polymerization that referred to GMA and nBA monomers,respectively. After modification of the polymer, intensity of FTIR peaks assigned to the epoxy ring at 844 cm−1 and 904 cm−1 wasdecreased. The results obtained from FTIR exhibit a good agreement with the epoxy content method. The activity of laccase-immobilizedmicrospheres increased upon increasing the epoxy content. Furthermore, poly(GMA-co-nBA)microspheres revealeduniform size below 2 𝜇mthat contributes to large surface area of themicrospheres to be used as amatrix, thus increasing the enzymecapacity and enzymatic reaction. Immobilized enzyme also shifted to higher pH and temperature compared to free enzyme.

1. Introduction

The selection of a suitable carrier material is a key fac-tor in enzyme covalent immobilization [1]. Poly(glycidylmethacrylate) (PGMA) containing epoxy group has beenwidely applied due to its attractive properties and discoveredas an ideal support for enzyme immobilization [2]. It hasthe ability to form strong linkages with amino, hydroxyl, andthiols group undermild condition [3, 4].Themodifications ofepoxy ring with amine groups endow PGMA with excellentaffinity to a variety of proteins, which makes it applicablein many areas and easily available for immobilization ofthe enzyme [5]. On the other hand, poly(n-butyl acrylate)(PnBA) provides hydrophobic property of copolymer. Asthe microspheres are hydrophobic, laccase immobilizationwill be confined to the surface of the spheres thus allowingthe enzymatic reaction of enzyme and analyte to occur at

the surface [6]. PGMA based materials are used as sup-ports in various forms, such as powder, membrane, and gelwith different geometrical configurations for immobilizationof several enzymes [7]. Recently, PGMA supports wereinvestigated for enzyme immobilization using physical andchemical immobilization techniques. It is because smallerdiameter of the microspheres provides more active sites forenzymes reaction thus improving immobilization efficiency.

Immobilization is always a successful strategy for stabi-lizing enzymes or improving their performance [8]. Bezerraet al. (2015) gave guidelines on the selection of suitable solidsupports and immobilization conditions [9]. Duran et al.(2002) comprehensively reviewed the different immobiliza-tions of laccase, including a variety of supports available,immobilization techniques, and potential industrial applica-tions [10]. Several approaches have been reported for theimmobilization of laccase such as entrapment into polymer

HindawiInternational Journal of Polymer ScienceVolume 2017, Article ID 5657271, 8 pageshttps://doi.org/10.1155/2017/5657271

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2 International Journal of Polymer Science

support [11, 12], absorption method [13], or covalent linkages[9, 14]. Among various immobilization methods, covalentattachment is the most frequently used because it providesstrong and stable enzyme attachment and, in some cases,reduces enzyme deactivation rates. It was reported that theimmobilized enzyme by covalent linkage is usually morestable in the reaction system [15]. Immobilization via covalentbinding prevents enzyme leaching and improves enzymestabilization [5]. As biomolecules, their structures, functions,and biological activities have to be maintained. Besidesthat, they must be tightly bound to the surface to preventleaching [16]. Physical properties of polymeric support suchas surface area, porosity, and functional group density caneasily be tailored in accordance with their specific needs.Among them, polymericmicrospheres and nanospheres haveattracted much attention due to their large-scale applicationsincluding ion exchange, bioseparation, biosensor, and biore-actor [6].

In this study, we have successfully synthesizedpoly(glycidyl methacrylate-co-n-butyl acrylate) referredto as poly(GMA-co-nBA) microspheres by a rapid methodof emulsion photopolymerization. The prepared polymermicrospheres were used for laccase immobilization viacovalent linkage. Among the great variety of enzymes, laccasehas gained much attention for being capable of catalyzinga wide range of popular reactions such as delignification,waste detoxification and decontamination, decolourizationof dyes, degradation of polycyclic aromatic hydrocarbons,and bioremediation and as bioreceptor in biosensorsapplication [17, 18]. Thus, laccase was chosen as an enzymeto understand the interaction of epoxy-immobilized laccaseconjugated methacrylate-acrylate copolymer microspheres.The efficiency of laccase immobilization was evaluated inthe aspects of size distribution of polymer microspheres,pH effect, and thermal property. This article is an extensionversion from the previous work reported by Mazlan andHanifah [17]. To the best of our knowledge, poly(GMA-co-nBA) microspheres have never been used as laccaseimmobilization support in order to improve the stability ofimmobilized laccase.

2. Experimental

2.1. Material. The following chemicals were obtained fromcommercial sources: glycidyl methacrylate, GMA (Sigma-Aldrich), n-butyl acrylate, nBA (Merck), ethylene glycoldimethacrylate, EGDMA (Sigma-Aldrich), sodium dodecylsulphate, SDS (Systerm), 2,2-dimethoxy-2-phenylacetophe-none, DMPP (Sigma-Aldrich), glutaric dialdehyde (Sigma-Aldrich), Bradford reagent (Sigma-Aldrich), and bovineserum albumin, BSA (Sigma-Aldrich). Deionized water wasused for making aqueous solution during the experiments.

2.2. Synthesis of Poly(GMA-co-nBA)Microspheres. Poly(GMA-co-nBA) microspheres were prepared via photopolymeriza-tion in the form of emulsion. Two compositions of GMAand nBA monomers were prepared in sample bottles toproduce GN91 and GN82, respectively. For GN91 copolymer,

a mixture of 90% v/v of GMA monomer, 10% v/v of nBAmonomer, 0.09 g DMPP, 400 𝜇L EGDMA, 0.1 g SDS, and10mL deionized water was prepared in a sample bottle. Thesame amount of DMPP, EGDMA, SDS, and deionized waterwas used for the preparation ofGN82, except that it contained80% v/v GMA monomer and 20% v/v nBA monomer. Themixture turned into milky white solution after five-minutesonication. The solution was photocured for five minutesunder continuous purging with nitrogen gas in an ultravioletexposure unit (R.S. Ltd.) of 15-watt light intensity at a wave-length of 350 nm. Poly(GMA-co-nBA) microspheres wereisolated by centrifugation (4,000 rpm, KUBOTA) for 8minand then thoroughly washed a few times with methanol.Finally poly(GMA-co-nBA)microspheres were dried at roomtemperature and kept at 4∘C when not in use [18].

2.3. Surface Modification of Poly(GMA-co-nBA)Microspheres.The epoxy groups of the poly(GMA-co-nBA) microsphereswere aminated with 0.5M ammonia at 65∘C in a beakercontaining 25 g of microspheres and magnetically stirred for5 h. After reaction, the aminated poly(GMA-co-nBA) micro-spheres were washed with deionized water. The aminatedpoly(GMA-co-nBA) microspheres (5 g) were equilibrated inphosphate buffer (10mL, 50mM, pH 7.0) for 18 h and trans-ferred to the medium containing glutaric dialdehyde (20mL,0.01% v/v).The activation reaction was carried out at 25∘C for12 h, while the medium was continuously stirred. After thereaction period, excess glutaric dialdehyde was removed bywashing sequentially with distilled water, acetic acid solution(100mM, 100mL), and phosphate buffer (100mM, pH 7.0).The product of modified poly(GMA-co-nBA) microsphereswas dried in a vacuum oven at 40∘C and stored at roomtemperature [19].

2.4. Immobilization of Laccase onto Poly(GMA-co-nBA)Microspheres. Immobilization of laccase on the modifiedpoly(GMA-co-nBA) microspheres was carried out at roomtemperature. An amount of 1ml from 20mg/ml laccasesolution was added to 100mg beads in the presence of 5mgsodium borohydride (NaBH

4) and stirred for 1 hour. After 1

hour, physically bound enzyme was removed by rinsing thesupports with phosphate buffer (50mM, pH7.0). It was storedat 4∘C in the phosphate buffer until use [10].

2.5. Microspheres Characterization. The composition ofcopolymer microspheres was analyzed by Attenuated TotalReflection-Fourier Transform Infrared (ATR FT-IR) (modelFTIR-8400S, Shimadzu, Japan) equipped with a MCT-Adetector at a resolution of 4 cm−1. The morphology andsize of copolymer microspheres were observed by scanningelectron microscopy (SEM, LEO 1450VP) at resolutionvoltage of 20 kV. Dry copolymer microspheres were placedon a piece of glass slide and then deposited within a thin layerof gold to reduce the charge effect from primary electronbeam, which may cause scanning error [20]. Size anddistribution of copolymer microspheres were determinedbased on a random selection of 300 microspheres from ascanning electron micrograph [6]. The amount of available

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International Journal of Polymer Science 3

surface functional epoxy groups content of the copolymermicrospheres was determined by pyridine–HCl method asdescribed previously [18].

2.6. Laccase Optimization. Bradford protein assay was con-ducted to determine laccase concentration and to ascertainthe optimum amount of glutaric dialdehyde required in thepreparation of poly(GMA-co-nBA) microspheres [21]. Forthis purpose, 1.4 g of poly(GMA-co-nBA) microspheres foreach glutaric dialdehyde (GA) was used (GA = 0.001; 0.010;0.020; 0.030%) and placed on a screen-printed electrode(SPE) and dried at 4∘C. After 24 hrs, 2 𝜇L laccase solution(0.05mg 𝜇L−1) was dropped onto the surface of poly(GMA-co-nBA) microspheres deposited on SPE and left at 4∘Cfor 24 hrs. Finally the SPE with immobilized laccase on thespheres was immersed in 3mL of 0.05M phosphate buffersolution at pH 5 for 30min. In order to determine the amountof laccase present in the solution of microspheres, a mixtureof 100 𝜇L phosphate buffer for washing, 100𝜇L NaOH, and800 𝜇LBradford reagent weremixed and incubated for 6min.The mixture was then measured using a spectrophotometer(Cary 50) at 595 nm. For the calibration of Bradfordmicroas-say, a series of BSA standard solutions were prepared (0, 10,20, 30, 40, and 50 𝜇gmL−1) in 0.05M phosphate buffer at pH5 [6].

Laccase activitywas determined by the oxidation ofABTSmethod [22]. The assay mixture contained 5.1mM ABTS,1mM sodium phosphate (pH 5), and a suitable amount ofenzyme. Oxidation of ABTS was monitored by determiningthe increase at 414 nm. Absorbance was read at 414 nm in aspectrophotometer (Perkin Elmer Lambda 900UL/VIS/NIR)against a suitable blank. One unit was defined as the amountof the laccase that oxidized 1 𝜇mol of ABTS substrate permin. The effect of pH on the activity of free and immobilizedlaccase was carried out over pH range 2.0–7.0 and at 30∘C.Concentration of laccase solution used was 2.0mM andwas prepared in 0.05M sodium phosphate in the pH range2.0–7.0. The effect of temperature on laccase activity wasstudied in the range 20–50∘C with a laccase concentrationof 2.0mM in 50mM phosphate buffer pH 5. Results for pHand temperature are presented in a normalised form with thehighest value of each set being assigned the value of 100%activity [22, 23].

3. Results and Discussion

3.1. Structural Analysis of Poly(GMA-co-nBA) Microspheres.In the present study, poly(GMA-co-nBA) microspheres wereprepared from GMA and nBA via emulsion photopolymer-ization. The copolymer was then modified at the epoxy ringto allow covalent binding of laccase with the copolymer.The proposed mechanism of the poly(GMA-co-nBA) micro-spheres modification is presented in Figure 1.

The theoretical epoxy group in copolymer microsphereswas dependent on GMA content in the monomer form. Thetheoretical epoxy was defined as the proportion of epoxy intotal polymer by assuming that all of the monomers wereconverted to the polymer. It was found that the theoreticalvalue of poly(GMA-co-nBA) microspheres was 5.33mmol/g

and based on the determination of epoxy content by titrationwas 3.44mmol/g. Percent conversion of epoxy groups wasapproximately 65% and this value was equivalent to otherfinding [24]. Some parts of the epoxy groups were hiddenin the inner copolymer as they were not chemically reactiveunder the titration conditions [10].

The activation of aminated poly(GMA-co-nBA) micro-spheres was achieved by the reaction with glutaric dialdehydeunder mild condition. Laccase was then covalently immobi-lized via amino groups to activate poly(GMA-co-nBA)micro-spheres. In this coupling reaction, alkylated functionalizationof poly(GMA-co-nBA) microspheres may occur after thealdehyde functionalization step. Furthermore, the glutaricdialdehyde can readily react with an amino group in mildcondition; thus, aldehyde group content should be closed tothe content of the amino group on the microspheres [25].

FTIR spectra were used to ensure the presence of epoxygroups in poly(GMA-co-nBA) microspheres and the con-jugated poly(GMA-co-nBA) microspheres as presented inFigure 2. Both FTIR spectra of polymer microspheres gavecharacteristic peaks at 1722 cm−1 and 2956 cm−1 due to C=Oof ester group and methyl vibration, respectively, as shown inFigure 2. The most important adsorption bands at 1156 cm−1and 910 cm−1 represent epoxy group. The transmittance wassignificantly decreased after ammonia was introduced intopoly(GMA-co-nBA) microspheres.

During synthesis of polymer microspheres, glutaricdialdehyde was chosen for its reactive aldehyde groups tobind with amino-bearing laccase [26]. Long chain alkylsulphate surfactant, which is amphiphilic, was used to sta-bilize the emulsion system and to prevent monomers fromforming larger droplets by allowing small droplets to remainstable during emulsion phase [27, 28]. Photopolymerizationcaused droplets that containGMA and nBAmonomers in thepresence of photoinitiator to be converted into poly(GMA-co-nBA)microspheres at room temperature.Thismethod canbe simply terminated by removing the light source [6].

Surface morphology of methacrylate-acrylate micro-spheres was investigated using SEM and the micrographsare presented in Figure 3. The micrographs revealed that thesynthesized copolymers were of spherical shape. However,when nBA composition was increased from 10% to 20%,the microspheres tend to be more aggregated as exhibitedby the micrograph (Figure 3(d)). This may be explainedby the fact that a higher quantity of nBA leads to themerging of the monomer into larger droplets thus resultingin an increase in the size of the microspheres formed afterphotopolymerization. The increase of sticky properties iscontributed by nBA [29].

Figure 4 demonstrates narrow size distribution with thehighest percentage of microspheres diameter in the range of0.1 𝜇m to 2.0 𝜇m for GN91 copolymer compared to the rangeof 1.0 to 4.0 𝜇m for GN82. Methacrylate-acrylate micro-spheres in this study, particularly GN91, produced smallerparticles size in comparison to previous findings [29, 30]. Asa result, the total surface area will be increased and improveenzyme binding onto the polymer microspheres as reportedby other studies [24, 30].

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4 International Journal of Polymer Science

C

C

O

CHO

O

HCC

O

OC C

C

O

CHO

OC C

C

O

Om n

+

DMPP

GMA nBA poly (GMA-co-nBA) microspheres

CHO CH

OH

CH

OHCH

C

HON

OHC CHOCHO

(1) Synthesis of poly (GMA-co-nBA) microspheres

(2) Amination

(3) Addition

CHC

OHN

CHO

(4) Immobilization

CHC

OHNH

CNH

H

UV

#(2

#(2

#(2

#(2

#(2

#(2

#(2

#(2#(2

#(2

#(2

#(3#(3

(2#

(2#

(3# (3#

(2#

(2

(2 (2

(2

(2 (2

.(3

.(2

#(2

.(2

5 B, 65∘C

12 B, RT

1 B, RT.;"(4, Laccase

Figure 1: Proposed schematic reaction of poly(GMA-co-nBA) microspheres for laccase immobilization by covalent binding.

800120016002000

(A)

(B)

Epoxy group

20

40

60

80

100

120

% tr

ansm

ittan

ce

Wavenumber (=G−1)

Figure 2: FTIR spectra of poly(GMA-co-nBA) microspheres (A)and poly(GMA-co-nBA) microspheres (B) after conjugation. FTIRspectra showed the decreasing peak intensity of epoxy group beforeand after polymer conjugation.

3.2. Effect of Glutaric Dialdehyde as Cross-Linking Agent.The amount of immobilized laccase on the GN91 copolymermicrospheres with varying compositions of glutaric dialde-hyde as an active functional group was determined usingBradford assay. Figure 5 shows that the amount of laccaseimmobilized on GN91 copolymer microspheres increasedwhen the concentration of glutaric dialdehyde increased from0.001 to 0.030% but decreased after 0.010%. It may be dueto an excessive cross-linking between the immobilizationmaterials where the bulk of the immobilized enzyme blockedfurther access by the free enzyme [25]. This limitation ofbinding capacity is attributed to the stearic effect between freeand immobilized enzyme as shown in Figure 6.

Poor strength of cross-linked microspheres may occurwhen low concentration of glutaric dialdehyde was used.When glutaric dialdehyde concentration was increased, theamount of free aldehyde groups on the microspheres’ surfacewill be increased thus enhancing the laccase loading capacity.However as the concentration of glutaric dialdehyde was

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International Journal of Polymer Science 5

(a) (b)

(c) (d)

Figure 3: SEMmicrographs of polyGMA (a), polynBA (b), GN91 copolymer (c), and GN82 copolymer (d) at 2.00 Kx magnification and sizedistribution of poly(GMA-co-nBA) microspheres in various compositions.

0

10

20

30

40

50

60

70

Dist

ribut

ion

of m

icro

sphe

res (

%)

0–0.

09

5.1–

6.0

2.1–

3.0

3.1–

4.0

6.1–

7.0

0.1–

1.0

1.1–

2.0

4.1–

5.0

Range of size (G)

GMA polymernBA polymer

GN91 copolymerGN82 copolymer

Figure 4: Size distribution for each of conjugated polymers.

more than 10%, the excessive cross-linking within the immo-bilization matrix may be blocked and change the enzymeconformation, hence resulting in the declination in theactivity of the immobilized enzyme [27].

3.3. Temperature and pH Effects. The effect of temperatureon the free and immobilized laccase activities is shown

38.39

67.62

27.4515.00

0

10

20

30

40

50

60

70

Imm

obili

zed

lacc

ase (

%)

0.001 0.010 0.020 0.0300.000Concentration of glutaric dialdehyde (%)

Figure 5: Effect of glutaric dialdehyde concentrations towardenzyme binding capacity.

in Figure 7. The temperature profile of the immobilizedlaccase improved the stability of the optimum temperaturevalue in comparison to the free laccase. It means thatthe immobilization method preserved the enzyme activity.The temperature profile of immobilized laccase was alsobroader than free laccase. The optimum temperature forfree laccase appeared at 40∘C and 50∘C for immobilizedlaccase. It shows that immobilized laccase could withstandhigher temperature conditions compared to free laccase. The

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6 International Journal of Polymer Science

Stearic effect

Bead

Free enzyme

Immobilized enzyme

Figure 6: Schematic representation of stearic effect (red arrow)between free and immobilized enzyme.

Free laccaseImmobilized laccase

30 40 50 60 70 80 9020Temperature (∘C)

0

20

40

60

80

100

Relat

ive l

acca

se ac

tivity

(%)

Figure 7: Effect of temperature on enzyme activity in comparisonof immobilized laccase with free laccase.

shifting in temperature is caused by changing the physical andchemical properties of immobilized enzyme. The covalentbond formation via amino groups of immobilized laccasemight also reduce conformational flexibility and result inhigher activation energy for the molecule to reorganiseproper conformation of substrate binding to substrate.

The pH effect on the activity of free and immobilizedlaccase was also examined in the pH range 2.0–7.0 at 30∘Cand the result is presented in Figure 8. The optimum pHfor free laccase was found at pH 4.0 which was similarto that reported previously [22]. On the other hand, theoptimum pH for immobilized laccase was shifted to pH5.0. The microenvironment of the immobilized enzyme andbulk solution usually has unequal partitioning of H+ andOH− concentrations due to electrostatic interactions withthe matrix, which often leads to the displacements in thepH activity profile [25, 27]. Furthermore, pH profiles of theimmobilized laccase are broader than free enzyme, whichmeans that the immobilization method preserved enzymeactivity in a wider pH range [31, 32]. These results couldprobably be attributed to the stabilization of laccase resulting

Free laccaseImmobilized laccase

0

20

40

60

80

100

Rela

tive l

akas

e act

ivity

(%)

3 4 5 6 72pH

Figure 8: Effect of pH on enzyme activity between immobilized andfree laccase.

from itsmultipoint attachments on the surface of poly(GMA-co-nBA) microspheres.

4. Conclusion

The conjugated copolymer microspheres of glycidyl metha-crylate (GMA) with n-butyl acrylate (nBA) were successfullysynthesized by using emulsion photopolymerization. Themicrographs revealed that the synthesized copolymers wereof spherical shape. Uniform size distribution of microspheresoffers higher surface area thus enhancing laccase bindingcapacity. The immobilization procedure for laccase that usedcovalent binding has improved its temperature and pHstability.

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper.

Acknowledgments

This work was financially supported in part by Ministry ofHigher Education Malaysia (Research Grant FRGS/1/2016/TK07/UKM/02/2) and Universiti Kebangsaan Malaysia(Research Grant GUP-2016-061). Special thanks are due alsoto the Centre for Research and InstrumentationManagement(CRIM), Universiti Kebangsaan Malaysia.

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