Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 83:97–104 (2008)

Immobilization of Pycnoporussanguineus laccase by metal affinityadsorption on magnetic chelator particlesFeng Wang,1,2 Chen Guo,1,2∗ Hui-Zhou Liu1,2 and Chun-Zhao Liu1,2∗1National Key Laboratory of Biochemical Engineering & Laboratory of Separation Science and Engineering, Institute of ProcessEngineering, Chinese Academy of Sciences, Beijing 100080, PR China2Graduate School of the Chinese Academy of Sciences, Beijing 100049, PR China

Abstract

BACKGROUND: Immobilized enzymes provide many advantages over free enzymes including repeated orcontinuous reuse, easy separation of the product from reaction media, easy recovery of the enzyme, andimprovement in enzyme stability. In order to improve catalytic activity of laccase and increase its industrialapplication, there is great interest in developing novel technologies on laccase immobilization.

RESULTS: Magnetic Cu2+-chelated particles, prepared by cerium-initiated graft polymerization of tentacle-typepolymer chains with iminodiacetic acid (IDA) as chelating ligand, were employed for Pycnoporus sanguineuslaccase immobilization. The particles showed an obvious high adsorption capacity of laccase (94.1 mg g−1 support)with an activity recovery of 68.0% after immobilization. The laccase exhibited improved stability in reactionconditions over a broad temperature range between 45 ◦C and 70 ◦C and an optimal pH value of 3.0 after beingadsorbed on the magnetic metal-chelated particles. The value of the Michaelis constant (Km) of the immobilizedlaccase (1.597 mmol L−1) was higher than that of the free one (0.761 mmol L−1), whereas the maximum velocity(Vmax) was lower for the adsorbed laccase. Storage stability and temperature endurance of the immobilized laccasewere found to increase greatly, and the immobilized laccase retained 87.8% of its initial activity after 10 successivebatch reactions.

CONCLUSION: The immobilized laccase not only can be operated magnetically, but also exhibits remarkablyimproved catalytic capacity and stability properties for various parameters, such as pH, temperature, reuse, andstorage time, which can provide economic advantages for large-scale biotechnological applications of laccase. 2007 Society of Chemical Industry

Keywords: laccase; immobilization; magnetic particles; metal chelating; Pycnoporus sanguineus

INTRODUCTIONImmobilized enzymes provide many advantages overfree enzymes, including repeated or continuous reuse,easy separation of the product from reaction media,easy recovery of the enzyme, and improvement inenzyme stability.1–3 A wide variety of methods havebeen employed in the immobilization of enzymes, suchas adsorption,4 entrapment,5 cross-link6 and covalentattachment.7 Among these immobilization techniques,adsorption is the most general, easiest to perform andoldest physical immobilization method.8 Simplicityand reversibility are the most important advantagesof this method. However, strong absorption betweenthe enzyme and support should be achievable inthe reversible immobilization methodology in orderto prevent enzyme desorption from immobilizationsupports. Noncovalent immobilization techniques,such as metal-chelated adsorption of enzyme ona metal-chelated adsorbent, can be a good option

because it saves time and labor, has simple operationand supports can be reused after desorption of theinactivated enzyme, thus reducing the final priceand generating fewer residues.8,9 Reversible metal-chelated immobilization has been used successfullywith a few enzymes, including catalase,8 α-amylase10

and invertase.11

Magnetic particles have many important applica-tions in the fields of cell labeling and separation,12

enzyme immobilization,13 magnetic resonance imag-ing (MRI) as a contrast agent,14 targeted drugdelivery,15 protein separations,16 etc.17 In recentlyyears, magnetic carrier technology has become veryattractive for the preparation of immobilized enzymes.The specific magnetic particles can be produced byimmobilization of an affinity ligand on the surfaceof prefabricated magnetic beads, which can be quicklyseparated from the reaction medium and controlled byapplying a magnetic field; then the catalytic efficiency

∗ Correspondence to: Chen Guo and Chun-Zhao Liu , National Key Laboratory of Biochemical Engineering & Laboratory of Separation Science and Engineering,Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, PR ChinaE-mail: cguo@home.ipe.ac.cn; czliu@home.ipe.ac.cn(Received 21 July 2007; revised version received 22 August 2007; accepted 23 August 2007)Published online 16 November 2007; DOI: 10.1002/jctb.1793

2007 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2007/$30.00

F. Wang et al.

and stability properties of the enzyme can be greatlyimproved.

Laccase (benzenediol:oxygen oxidoreductase, EC1.10.3.2) is a multi-copper oxidase produced by manyplants and numerous fungi,18 and it is able to oxidizea wide range of xenobiotic compounds such as syn-thetic dyes,19 chlorinated phenolics,20 and polycyclicaromatic hydrocarbons.21 The substrate specificity oflaccase in conjunction with appreciable stability prop-erties makes it a promising candidate in wastewatertreatment, biotransformation, biosensor and biofuelcell constructs.21–24 In order to improve laccasestability and increase its industrial application, lac-case has been recently reported to be successfullyimmobilized on different types of carriers, such asnanoparticles,21,25 chitosan,26–28 cellulose–polyaminecomposite,29 alginate,23,30 nylon membrane31 andshort-range ordered aluminum hydroxide.32 However,leakage and desorption were observed when alginateand nylon membranes were used as the laccase immo-bilization matrix,23,30,31 and the loss of enzyme activitywas still a major problem in laccase immobilization,which was attributed to many factors involving theenzyme itself, polymer matrix, reaction reagents andprocess conditions.25,26,28,32 Therefore, developmentof novel technologies to improve laccase immobiliza-tion is still an area of great interest.

In order to improve the catalytic efficiency ofimmobilized laccase, metal affinity magnetic parti-cles have been proposed as a suitable method forboth reversible and strong protein adsorption.8 Inthis way, protein immobilized on this flexible coat-ing matrix produces minimal distortion of proteinbecause the flexible polymer arm adapts itself tothe protein structure during the intense metal affin-ity adsorption. In addition, the high specific surfacearea of these particles for their small size inducesgood adsorption capacity. In this study, laccase wasimmobilized onto metal affinity magnetic particles viaadsorption. Nonporous micron-sized magnetic poly(vinyl acetate-divinylbenzene-g-glycidyl methacrylate-iminodiacetic acid) (PVA-DVB-g-GMA-IDA) parti-cles were prepared by a modified suspension polymer-ization method and cerium-initiated graft polymeriza-tion with a chelating group. Magnetic PVA-DVB-g-GMA-IDA-Cu2+ chelated particles were obtained byadding PVA-DVB-g-GMA-IDA beads to an aqueoussolution of Cu2+ ions. Then laccase was adsorbedonto the metal-chelating particles from the aqueousenzyme solution. Optimization of the immobilizationconditions was carried out, and the enzymatic proper-ties, reusability and storage stability of the immobilizedlaccase were investigated.

MATERIALS AND METHODSMaterialsGlycidyl methacrylate (GMA) and iminodiacetic acid(IDA) were purchased from Sigma - Aldrich (St Louis,MO, USA) and Acros Organics (Geel, Belgium),

respectively. All other materials were of analyticalgrade and were obtained from Beijing ChemicalReagents Company. Vinyl acetate (VAc) was distilledunder vacuum. Divinylbenzene (DVB) was used asa cross-linking agent. Benzoyl peroxide (BPO) wasused as an initiator. Poly (vinyl alcohol) (PVA-1788,degree of polymerization 1700, degree of hydrolysis88%) was used as a stabilizer. Pycnoporus sanguineuslaccase (molecular mass: 64 kDa) was provided bythe Institute of Microbiology, Chinese Academy ofSciences, Beijing, China. Its purity was tested byelectrophoresis with a single protein band and itsisoelectric pH is 3.0.

Preparation of immobilization particlesMagnetic PVA-DVB-g-GMA-IDA-Cu2+ particleswere synthesized according to a method previouslyreported.33 Oleic acid-coated magnetite nanoparti-cles were synthesized by a coprecipitation method.34

The magnetic PVAc-DVB particles were prepared bya modified suspension polymerization method andwere transformed into magnetic PVA-DVB particlesby ester exchange reaction. GMA-IDA monomer wasfirst prepared by reaction of GMA with IDA35 andthen GMA-IDA grafted magnetic PVA-DVB parti-cles by cerium-initiated graft polymerization. Finally,the magnetic PVA-DVB-g-GMA-IDA particles werecharged with copper ions, and then the fabricated mag-netic PVA-DVB-g-GMA-IDA-Cu2+ particles werewashed several times with water and 20 mmol L−1

sodium phosphate buffer (PBS, pH 8.0) to remove theexcess unbound Cu2+.

The fabricated magnetic polymer particles weredried in vacuum at 25 ◦C for 48 h, and then afragment of the dried particles mounted on a sampleholder was sputter-coated with gold for 2 min ina JEOL JEC-1200 sputter-coater (Tokyo, Japan).All samples were examined by a JEOL JSM-5600LV scanning electron microscopy (SEM) under highvacuum and at an accelerating voltage of 5.0 kV(7500×magnification). Immobilized Cu2+ capacitywas determined by suspending a known amount ofmagnetic particles into 0.5 mol L−1 EDTA to releasethe bound Cu2+. The Cu2+ concentration released inEDTA solution was detected by a Perkin Elmer (MA,USA) AAnalyst 100 atomic absorption spectrometer.

Immobilization of laccaseLaccase adsorption on the Cu2+-chelated magneticPVA-DVB-g-GMA-IDA particles was tested at vari-ous pH values, either in sodium acetate buffer (0.1 molL−1, pH 2.0–5.0) or in phosphate buffer (0.1 molL−1, pH 6.0–8.0). 10 mg of magnetic PVA-DVB-g-GMA-IDA-Cu2+ particles was added to 15 mL oflaccase solution (0.1 mg mL−1) prepared with the cor-responding buffer. The resulting suspensions weresubsequently incubated at 25 ◦C with shaking at150 rpm for a given time in order to reach adsorp-tion equilibrium. The laccase-adsorbed particles wereseparated from the enzyme solution magnetically and

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washed with the same buffer until no protein wasdetected in the supernatant. The elution solutionscontaining residual laccase were collected. The activi-ties of immobilized laccase were evaluated by the assayof the activity recovery, relative activity and residualactivity, respectively. The resulting immobilized lac-case were stored at 4 ◦C in fresh buffer until use.The amount of protein in the enzyme solution and inthe washing solution was determined by the Bradfordmethod,36 and the amount of protein bound on theparticles was calculated.37

To determine the adsorption capacities of the Cu2+-chelated magnetic PVA-DVB-g-GMA-IDA particles,the concentration of laccase in the medium was variedin the range 0.05–0.6 mg mL−1.

Assay of laccase activityThe activity array of the free and immobilizedlaccase was determined spectrophotometrically in areaction medium containing 0.1% (w/v) catecholas substrate in 100 mmol L−1 tartaric acid buffer(pH 3.0) at 25 ◦C in the absorbance at 450 nm.23

A suitable amount of laccase was added to thesubstrate solution and incubated on a rotary shaker at150 rpm for 20 min. During the process, the increasein the absorbance of supernatant was determined ina UNICO UV-2000 spectrophotometer (Shanghai,China). The molar absorption coefficient of catecholis 2211 L mol−1 cm−1.38 One unit of activity is definedas the amount of enzyme required to oxidize 1 µmol ofcatechol in 1 minute.

The activity recovery of the immobilized enzyme iscalculated from the formula:

R(%) =(

AiAf

)× 100%

where R is the activity recovery of the immobilizedenzyme (%), Ai the activity of the immobilizedenzyme (U) and Af is the activity of the same amountof free enzyme in solution as that immobilized onparticles (U).

Effect of pH and temperature on free andimmobilized laccase activityEffects of pH and temperature on free and immobilizedlaccase activity were determined as the relative activityafter incubation for 30 min (as described above) underdifferent levels of pH (2.0–5.0) and temperature(30–80 ◦C).

Determination of kinetic parameters andproperties of immobilized laccaseKinetic parameters (Km and Vmax) of the free laccaseand the immobilized laccase were determined bymeasuring initial rates of the reaction with catechol(0.1–10 mmol L−1) in tartaric acid buffer (0.1 molL−1, pH 3.0) at 25 ◦C. For this purpose, equivalentfree and immobilized laccase were added to catecholsolution of different concentrations between 0.1 and

10 mmol L−1 and initial activities were determined asdescribed above.

Thermal stability studies of the free and immo-bilized laccase were carried out by measuring theresidual activity of the enzyme exposed to differenttemperatures (25 ◦C, 50 ◦C) in tartaric acid buffer(0.1 mol L−1, pH 3.0), and the enzymatic activitiesof the free and immobilized laccase were determinedby the method described earlier. For storage stability,the activities of free and immobilized laccase in tar-taric acid buffer (0.1 mol L−1, pH 3.0) stored at 4 ◦Cwere measured in batch operating mode under theexperimental conditions given earlier.

The operating stability of immobilized laccase wasalso assessed. Several consecutive operating cycleswere performed by oxidizing catechol. At the endof each oxidation cycle, the immobilized laccasewas washed three times with PBS (pH 7.0) andthe procedure was repeated with a fresh aliquot ofsubstrate, as described by Davis and Burns.39

Desorption and reusability of magnetic particlesIn order to determine the reusability of magneticPVA-DVB-g-GMA-IDA-Cu2+ particles, the laccaseadsorption and desorption cycle was repeated 10times. Laccase desorption from these magnetic PVA-DVB-g-GMA-IDA-Cu2+ particles was carried outwith 50 mmol L−1 EDTA. The particles were washedseveral times with phosphate buffer (50 mmol L−1, pH7.0), and then were reused in enzyme immobilization.

All measurement experiments were carried out threetimes and the relative standard deviation was lessthan 2%.

RESULTS AND DISCUSSIONMagnetic PVA-DVB-g- GMA-IDA-Cu2+ particlesMagnetic metal-chelated affinity particles suitable forlaccase immobilization were prepared; the morphologyof the carrier particles is shown in Fig. 1(a). Theimmobilization matrix has a spherical form andmicron size, which offers an appropriate surfacearea to bind affinity ligands and can remain inaqueous suspension, thus permitting reaction kineticsof a ‘quasihomogeneous’ solution.33 According to theLewis-acid–Lewis-base concepts of Pearson,40 copperion, as a soft or borderline Lewis acid, exhibitsa preference for non-bonding lone pair electronsfrom nitrogen atoms in aromatic and aliphatic aminocontaining ligands. Thus, a strong binding can beestablished between amino acid side chain groups oflaccase (i.e. especially imidazole groups of the histidineresidues) and copper ions.41 The form of Cu2+ inthe magnetic Cu2+-chelated particles is depicted inFig. 2; the Cu2+ binding capacity was 5.07 mmol g−1

particles. From SEM images of magnetic particlesbefore (Fig. 1(a)) and after immobilization (Fig. 1(b)),the presence of immobilized enzymes on the matrixsurface is clearly observed.

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(a)

(b)

(c)

Figure 1. SEM micrographs of magnetic PVA-DVB-g-GMA-IDA-Cu2+

particles: (a) before immobilization; (b) after laccase immobilization;(c) after the 10th use.

Laccase immobilization parametersThe effect of pH on the immobilization of laccase ontomagnetic PVA-DVB-g-GMA-IDA-Cu2+ particles wasstudied in the pH value range 2.0–8.0, and results arepresented in Fig. 3. Proteins have no net charge at

CH2 CH

OH

CH2 N

CH2

CH2

C O

C O

O

O

CuC

O

O

OH2

OH2

OH2

Figure 2. Schematic diagram of the chelation of Cu2+ ions throughmagnetic particles.

2 50

20

40

60

80

100

Act

ivity

rec

over

y (%

)

pH

3 4 6 7 8

Figure 3. Effect of pH on laccase adsorption onto the magneticPVA-DVB-g-GMA-IDA-Cu2+ particles. All immobilizations werecarried out at 25 ◦C and 0.1 mg mL−1 of laccase concentration for1.5 h.

their isoelectric points, and therefore the maximumadsorption from aqueous solutions is usually observedat their isoelectric points.42 The isoelectric pH oflaccase used in this study was 3.0. Thus, the maximumadsorption of laccase was observed at pH 3.0. Thedecrease in enzyme adsorption capacity in more acidicand more alkaline regions was related to electrostaticrepulsion effects between the oppositely chargedgroups.10

Figure 4 indicates the changes in activity recoveryand amount of adsorption with reaction time. Theamount of laccase adsorption on the magnetic particlesincreased with prolonged reaction time and reachedequilibrium after 1.5 h. The highest activity recoverywas obtained for a reaction allowed to occur for1.5 h; activity recovery declined when reaction timewas prolonged further because of increasing thermaldeactivation.

The effect of the amount of enzyme added onactivity recovery of the immobilized laccase is shownin Fig. 5. The maximum activity recovery of theimmobilized laccase reached 68.0% when the addedenzyme concentration was 0.1 mg mL−1, wherethe adsorbed laccase reached 94.1 mg g−1 magneticparticles. The activity recovery decreased graduallywhen the laccase concentration was more than 0.1 mgmL−1. This is because a larger amount of enzymeis immobilized to form an intermolecular space,

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0 20

10

20

30

40

50

60

70

Activity recoveryAbsorbed enzyme

Abs

orbe

d en

zym

e (m

g/g)

Time (h)

0

20

40

60

80

100

120A

ctiv

ity r

ecov

ery

(%)

1 3 4

Figure 4. Changes of activity recovery and amount of adsorptionwith reaction time. All reactions were carried out at pH 3.0 (0.1 molL−1 tartaric acid buffer), 25 ◦C and 0.1 mg mL−1 laccaseconcentration. (°) activity recovery, (�) adsorbed enzyme.

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.70

10

20

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40

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60

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80

0

20

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120

Activity recoveryAbsorbed enzymeRelative activity

Enzyme concentration (mg/ml)

Act

ivity

rec

over

y (%

)

0

50

100

150

200

250

Rel

ativ

e ac

tivity

(%

)

Abs

orbe

d en

zym

e (m

g/g)

Figure 5. Effect of amount of enzyme added on laccaseimmobilization onto magnetic supports. All immobilizations werecarried out at pH 3.0 (0.1 mol L−1 tartaric acid buffer) and 25 ◦C for1.5 h. (°) activity recovery, (�) adsorbed enzyme, (�) relative activity.

inhibiting the immobilized enzyme, which will restraindispersion of the substrate and product.43

The magnetic particles reported here have a goodadsorption capacity for laccase, which is higher thanthat previously reported for magnetic microbeads:17–44.4 mg g−1,44 10 mg g−1,25 and 12.9 mg g−1.45

Laccase immobilizations on non-magnetic particleswere studied by other researchers and the adsorptioncapacity of these particles was low, e.g. 7.1 mg g−1 ona nylon membrance,31 6.18 mg g−1 on aluminumhydroxide,32 and 1 mg g−1 on controlled porosityglass.46 Compared with other particles, the magneticparticles prepared in this study have a nonporousstructure, which has the advantage of higher resistanceto fouling and better mass transfer.33 Althoughsome new immobilization matrices have smaller size(nanoparticles), their adsorption capacities are muchlower than that of the magnetic metal-chelated affinityparticles.25,45 The high adsorption capacity of themagnetic metal-chelated affinity particles is attributed

2.0 2.5 3.0 3.5 4.0 4.5 5.040

50

60

70

80

90

100

Immobilized laccaseFree laccase

pH

Rel

ativ

e ac

tivity

(%

)

Figure 6. Effect of pH on the activity of free and immobilized laccase.(°) free laccase, (�) immobilized laccase.

20 30 40 50 60 70 8040

50

60

70

80

90

100

110

Rel

ativ

e ac

tivity

(%

)

Temperature (°C)

Immobilized laccaseFree laccase

Figure 7. Effect of temperature on the activity of free andimmobilized laccase. (°) free laccase, (�) immobilized laccase.

to the tentacle-type polymer chains grafted on themagnetic particle. Each of the chains possesses severalaffinity ligands, and the steric hindrance of enzymeprotein to the ligands is reduced. In comparisonwith laccase immobilization studies reported by otherresearchers,26,28,44 the activity recovery of the laccaseimmobilized onto these magnetic PVA-DVB-g-GMA-IDA-Cu2+ particles reached a high level due to thenonporous structure and the flexible grafted tentaclechains reaching out of the solution on the magneticpolymer particles, which is beneficial for substratecombining with enzyme and the product diffusing tothe reaction medium.

Conditions affecting on immobilized enzymeactivityThe effects of pH on the enzyme activity of free andimmobilized laccase for catechol were determined inthe pH range 2.0–5.0, and the results are illustrated inFig. 6. The maximum relative activity of the free andimmobilized laccase was observed at pH 3.0. Therewas no change in optimum laccase before and afterimmobilization, which was similar to the result forα-amylase immobilization on poly-(EGDMA-VIM)-Cu2+ particles.10 As shown in Fig. 7, the immobilizedlaccase on the magnetic particles also had the same

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00

20

40

60

80

100R

elat

ive

activ

ity (

%)

Time (h)

Immobilized enzyme, 25°CFree enzyme, 25°CImmobilized enzyme, 50°CFree enzyme, 50°C

1 2 3 4

Figure 8. Thermal stability of free and immobilized laccase. (°) freelaccase, 25 ◦C; (ž) free laccase, 50 ◦C; (�) immobilized laccase,25 ◦C; (�) immobilized laccase, 50 ◦C.

optimum temperature as the free enzyme (55 ◦C).Compared with the free laccase, the immobilizedpreparation gave a significantly broader profile, therelative activity being maintained at over 85% withinthe temperature range 45–70 ◦C. The increasedstability of immobilized laccase was due to therestricted conformational mobility of the moleculesfollowing immobilization.47

It is well known that enzymes in solution are notstable, and that their activities decrease graduallyduring use. Thermal stability tests were carried outwith the free and immobilized laccase in tartaric acidbuffer at various temperatures, and the results areshown in Fig. 8. Immobilization enhanced heat anddenaturation resistance of laccase remarkably. Afterincubation at 25 ◦C or 50 ◦C for 4 h, the immobilizedlaccase retained a higher activity than the free laccase.The improvement in the thermal stability of laccasewas probably due to a reduction in molecular mobilityby multi-point binding between the enzyme and themetal-chelated particles.48

Storage stability is an important advantage ofimmobilized enzymes over free enzymes because freeenzymes can lose their activities fairly quickly. Theimmobilized laccase on magnetic PVA-DVB-g-GMA-IDA-Cu2+ particles retained its full activity for a 1-month storage period at 4 ◦C, whereas the free laccaselost 60% of its initial activity over the same period(data not shown). No enzyme release from themagnetic particles was observed during this storageperiod. This result indicates that the stability of

0 2 5 7 10 1180

85

90

95

100

Absorbed enzymeActivity recovery

Regeneration times

Abs

orbe

d en

zym

e (m

g/g)

60

62

64

66

68

70

72

74

76

78

Act

ivity

rec

over

y (%

)

1 3 4 6 8 9

Figure 9. Reuse of magnetic PVA-DVB-g-GMA-DA-Cu2+ particles.Laccase immobilization was carried out at 0.1 mg mL−1 of laccase in0.1 mol L−1 acetate buffer (pH 3.0) at 25 ◦C. (°) activity recovery; (�)adsorbed enzyme.

immobilized laccase is greatly improved over thatof the free enzyme. Of the immobilization methods,fixation of enzyme molecules on a surface often givesrise to the highest stabilization effect on enzymeactivities because the active conformation of theimmobilized enzyme is stabilized by multipoint bondformation between the substrate and the enzymemolecules.7

Compared with free enzyme, immobilized enzymecan be easily separated from the product solutionand reused.2 It was observed that the immobilizedlaccase maintained 87.8% of its original activityafter the 10th reuse (data not shown). This resultindicates that immobilized laccase on magneticparticles has high operational stability, which canbe attributed to the protection against heat anddenaturing agents as a result of multi-point attachmentof the laccase on the magnetic Cu2+-chelated particles.High operational stability has also been observed inthe immobilization of urease and invertase by metalaffinity adsorption.11,49

Desorption of laccase from the magnetic Cu2+-chelated PVA-DVB-g-GMA-IDA particles was car-ried out in a batch system. It was then repeatedlyused for the adsorption of laccase. No remark-able change was observed in the adsorption capacityand activity recovery of laccase during 10 successiveadsorption–desorption cycles (Fig. 9). The magneticenzyme-immobilized matrix kept its spherical shapeafter the 10th reuse, and the structure of the magneticparticles was not damaged or collapsed (Fig. 1(c)).

Table 1. Kinetic constants of free and immobilized laccase

Form of enzymeKm

(mmol L−1)Vmax [µmol mg−1

protein min−1]ECa

(mg g−1 support) Vmax/Kmb R (%)c

Laccase activity(U g−1 particles)

Free 0.761 1.128 – 1.48 – –Immobilized 1.597 0.767 94.1 0.48 68.0 72.2

a Enzyme content per gram particles.b Catalytic efficiency was defined as the ratio Vmax/Km.c R (%) is the activity recovery of the immobilized enzyme.

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These results show that the magnetic PVA-DVB-g-GMA-IDA-Cu2+ particles can be repeatedly used inenzyme immobilization, without detectable losses intheir initial adsorption capacity and recovered activity.

Kinetic parametersKinetic constants, the Michaelis constant (Km) andthe maximal initial rate of the reaction (Vmax) for thefree and immobilized laccase were determined usingcatechol as a substrate, and the results are shown inTable 1. The Km value of the immobilized laccasewas 2.1 times higher than that of the free laccase.The Vmax value of the free laccase (1.128 µmol mg−1

protein min−1) was found to be higher than thatof the immobilized laccase (0.767 µmol mg−1 proteinmin−1). The change in affinity of the laccase for itssubstrate is probably caused by structural changesin the enzyme introduced by the immobilizationprocedure and by lower accessibility of the substrateto the active site of the immobilized enzyme.8,37

CONCLUSIONSMagnetic PVA-DVB-g-GMA-IDA particles were pre-pared from magnetic PVA-DVB particles and GMA-IDA monomer via cerium-initiated graft polymer-ization. The Cu2+-chelated magnetic particles wereused for the reversible immobilization of laccase viametal-affinity adsorption. These particles not onlycan be operated magnetically but also exhibit a highadsorption capacity of laccase. The immobilized lac-case exhibited remarkably improved catalytic capacityand stability properties with variables such as pH,temperature, reuse, and storage time. Together withthese results, the reusable magnetic beads can provideeconomic advantages for large-scale biotechnologicalapplications of laccase.

ACKNOWLEDGEMENTSThis work is financially supported by the NationalHigh Technology Research and Development Pro-gram of China (863 Program) (No.20060102Z2049).

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