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Journal of Molecular Catalysis B: Enzymatic 93 (2013) 1– 7

Contents lists available at SciVerse ScienceDirect

Journal of Molecular Catalysis B: Enzymatic

jo ur nal home p age: www.elsev ier .com/ locate /molcatb

lectrostatic immobilization of pectinase on negatively charged AOT-Fe3O4

anoparticles

tieh Bahrami, Parisa Hejazi ∗

iotechnology Research Laboratory, School of Chemical Engineering, Iran University of Science and Technology, P.O. Box 16846-13114, Tehran, Iran

a r t i c l e i n f o

rticle history:eceived 28 November 2012eceived in revised form 8 March 2013ccepted 11 March 2013vailable online xxx

eywords:nionic surfactantlectrostatic immobilizationagnetic nanoparticles

a b s t r a c t

Enzyme immobilization on magnetic nanoparticles (MNPs) has been a field of intense studies in biotech-nology during the past decade. The present study suggests MNPs negatively charged by docusate sodiumsalt (AOT) as a support for pectinase immobilization. AOT is a biocompatible anionic surfactant whichcan stabilize MNPs. Electrostatic adsorption can occur between enzyme with positive charge and oppo-sitely charged surface of MNPs (ca. 100 nm). The effect of three factors, i.e. initial enzyme concentration,aqueous pH and AOT concentration in different levels was investigated on pectinase immobilization.Maximum specific activity (1.98 U/mg enzyme) of immobilized pectinase and maximum enzyme load-ing of 610.5 mg enzyme/g support was attained through the experiments. Initial enzyme concentrationis significantly important on both loading and activity of immobilized enzyme, while pH and AOT con-

ectinase centration only affect the amount of immobilized enzyme. Immobilized enzyme on MNPs was recoveredeasily through magnetic separation. At near pH of immobilization, protein leakage in reusability of immo-bilized enzyme was low and activity loss was only 10–20% after six cycles. Since pH is associated withimmobilization by electrostatic adsorption, the medium pH was changed to improve the release of pro-tein from the support, as well. MNPs properties were investigated using Scanning Electron Microscopy(SEM), Fourier Transform Infrared (FT-IR) spectroscopy, and Dynamic Light Scattering (DLS) analysis.

. Introduction

Enzymes are biocatalysts playing key roles in different indus-ries. Pectinases as hydrolytic enzymes are used abundantly inarious industries such as fruit juice extraction, coffee and teaermentation, cotton scouring, water and wastewater treatment,nd bleaching of paper [1]. These industries need stable form ofnzymes to retain their activity by reusing several times, at higherHs, and temperatures [2]. Immobilization techniques such asovalent and ionic bonding, adsorption, entrapment, and encapsu-ation can successfully provide stable forms of enzymes [3]. Whilehe covalent binding method decreases an enzyme activity becausef direct contact of the enzyme with the support and its struc-ural changes, enzyme immobilization through simple adsorption

ethod preserves its activity.During the past decade, magnetic nanoparticles (MNPs) have

een a field of intense study because of their potential applicationsn pharmacy, biology, diagnostics, biotechnology, etc. [4,5]. The use

f MNPs offers many advantages because of their nano-scale size,hysical properties, biocompatibility and inoffensive toxicity pro-le. For example, metallic nanoparticles can easily penetrate the

∗ Corresponding author. Tel.: +98 21 77240496; fax: +98 21 77240495.E-mail address: phejazi@iust.ac.ir (P. Hejazi).

381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.molcatb.2013.03.010

© 2013 Elsevier B.V. All rights reserved.

human skin and have gained significant attention in targeted drugdelivery and early diagnosis of a skin disease [6].

Recently, immobilization of bioactive substances such as pro-teins on magnetic nanoparticles of iron oxide has been taken intoconsideration. Because of their magnetic properties, MNPs can beeasily separated from the reaction medium and reused in enzymeimmobilization systems. MNPs should be coated by organic mate-rials, surfactants or polymers to protect them from both oxidationand agglomeration processes [7]. The properties of coating mate-rial are important in enzyme immobilization since it serves as aninterface between enzyme and support. Following a proper surfacecoating, the biomolecules can link to the MNPs with electrostatic,ionic or covalent bindings. Previous studies have reported the effec-tiveness of adsorption coating surfactants such as sodium dodecylsulfate (SDS) and alkylbenzene sulfonate (LAS) in lipase immo-bilization on MNPs [8,9]. In these compounds, support improvedthe immobilization of lipase through hydrophobic surface, formedby, LAS coating [9]. Lei et al. [5] showed that combined magneticand chemical covalent immobilization of pectinase on compositesmembranes improves stability and activity of this enzyme. Theyattributed this improvement to physicochemical characteristics of

the nanoparticles.

Adsorption as a simple method involves either electrostaticor ionic interactions between a charged protein and oppositelycharged support. Some studies showed the efficacy of ionic

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A. Bahrami, P. Hejazi / Journal of Mo

xchange resins in electrostatic immobilization of pectinase, lipase,rotease, dextranase and xylanase [10]. Furthermore, adsorptionacilitates the release of the enzyme which is a highly efficientnd important property in targeting drug delivery in protein drugselease. Yu et al. [11] used bovine serum albumin (BSA) as a modelrug, delivered by composite microparticles consisted of chitosan,lginate and pectin. They showed that the release of BSA can befficiently sustained by changing pH. With regard to the aboveiscussion, positively charged protein can be immobilized on andeleased from negatively charged Fe3O4 nanoparticles through pHodification.Docusate sodium salt (AOT) is an anionic biocompatible sur-

actant widely used in bioseparation systems. It is mostly used ineverse micellar systems because of its low solubility, resulting innzyme recovery without contamination [12]. It also has antibacte-ial activity, which is an important trait in enzyme immobilizationor cell growth inhibition and contamination [13]. As a stabilizernd suspender, this surfactant covers Fe3O4 nanoparticles and pro-ides negative charge on its surface [6].

To the best of the authors’ knowledge, there has not yet been study on the Fe3O4 nanoparticles stabilized by AOT as a supportaterial for pectinase immobilization through adsorption. AOT as

n anionic surfactant covers the support surface and positivelyharged enzyme is bound to negatively charged MNPs throughlectrostatic interactions. In this study, Fe3O4 nanoparticles wereynthesized by a co-precipitation method and their properties werenvestigated using different analytical methods. They could eas-ly be recovered from the medium which can reduce downstreamrocessing costs, and as it is not toxic, they can be used in biologicalystems like enzyme immobilization processes and drug delivery.ectinase was used as a model enzyme in this study. The effects ofhree factors such as aqueous pH, initial enzyme concentration andOT concentration on pectinase immobilization onto MNPs weressessed. In addition, the catalytic ability, stability and reusabilityf immobilized pectinase for the hydrolysis of pectin were eval-ated. As this enzyme is considered as a model biomolecule inrug delivery, its release from the support by changing pH wasstimated.

. Materials and methods

.1. Materials

All chemicals had analytical grade and used without furtherurification. Crude pectinase from Aspergillus niger (EC 3.2.1.15),errous chloride (FeCl2·4H2O), ferric chloride (FeCl3), hydrazineydrate (N2H4·H2O), ethanol and aqueous ammonia (25% (v/v)queous solution) were used in the study (Merck, Germany). Bis2-ethylhexyl) sulfosucccinate sodium salt (AOT), pectin and d-alacturonic acid monohydrate used in the study were purchasedrom Sigma–Aldrich (Germany). The specific activity of the enzymeas higher than 1.0 U/mg enzyme of protein (40 ◦C, pH = 4.2).eionized water was used throughout the experiments.

.2. Magnetite nanoparticles synthesis

The Fe3O4 nanoparticles were synthesized using a co-recipitation method according to the method previouslyxplained by Hong et al. [4]. Deionized water (54.4 ml) and2H4·H2O (0.8 ml) were added into a capped 100 ml-vessel and

tirred for 30 min at 85 ◦C to eliminate the oxygen from the mix-ure. Aqueous solution of FeCl3·6H2O and FeCl2·4H2O (2:1 molaratio) were added dropwise to the vessel under continuous stirring.onsidering direct effect of temperature on morphology and size of

r Catalysis B: Enzymatic 93 (2013) 1– 7

nanoparticles, high temperature was chosen and it was increasedto 90 ◦C.

After the addition of the iron salts, NH4OH (12 ml) was quicklyinjected into the vessel under vigorous stirring. 4 ml of AOT-isooctane solution (0.05 and 0.1 M) was added to the suspension.The reaction was kept at 90 ◦C for 30 min. Nanoparticles werewashed with deionized water and anhydrous ethanol several timesand separated by magnetic decantation.

2.3. Pectinase immobilization

The amount of 25 mg of nanomagnetic support (synthesized inthe presence of AOT (0.05 M or 0.1 M)) were added to the enzymesolutions (0.25, 0.5, 0.75, 1, 1.5 and 2 mg/ml in the 0.2 M acetatebuffer at pHs 3.5, 4, 4.5 and 5) to immobilize the pectinase intotal volume of 11.5 ml. The mixture was then incubated for 4 hin an orbital shaker at room temperature. The amount of proteinadsorbed on the supports was determined by measuring the pro-tein concentration of initial pectinase solution and the remainingin the supernatant. The immobilized supports were washed withacetate buffer, separated by magnetic decantation, and stored at4 ◦C for further use.

2.4. Analysis methods

2.4.1. Analysis of MNPsFT-IR and DLS analyses of AOT coated MNPs were done to

investigate the properties. Also, SEM method was applied to deter-mine size of AOT-MNPs before and after enzyme immobilization.The FTIR spectra were recorded in FTIR spectrometer (8400F SHI-MADZU) with resolution 4 cm−1 in the range of 320–4500 cm−1 toconfirm the binding of AOT and pectinase to MNPs before and afterimmobilization. Samples were dried at the same condition at ambi-ent temperature. The mean size/size distribution was obtainedfrom particle size analyzer (DLS). Sample of Fe3O4 coated with AOTwere dispersed in deionized water with the help of sonicator andDLS analysis of sample were performed on a Nano ZS (red badge)ZEN 3600.

2.4.2. Bradford protein assayBradford method was used to estimate pectinase concentra-

tions [14], and pectinase without further purification was usedto draw the calibration curve. Range of enzyme concentrations of0.1–0.5 mg/ml were used for plotting the calibration curve. Theabsorbance of the samples was read at wavelength of 595 nm ina UVF 2800 LABOMED INC spectrophotometer.

2.4.3. Pectinase activity assayImmobilized and free pectinase activities were determined

using pectin as a substrate. One unit of activity was defined as theamount of enzyme required to hydrolyze 1.0 �mol of pectin per minat pH = 4 in 0.1 M acetate buffer and 40 ◦C. Pectin solution (pH = 4)was added to the enzyme stock (pH = 4) and the reaction time wasfor 30 min. The amount of formed reducing sugar was estimated bythe 3,5-dinitrosalicyclic acid (DNS) method proposed by Miller [15].d-Galacturonic acid monohydrate was used as the standard com-pound for creating the calibration curve in the pectinase activityassay.

2.5. Experimental design of pectinase immobilization

To evaluate pH, AOT concentration, and initial enzyme con-

centration effects on immobilization, four different pH levels, twoconcentrations of AOT, and six initial amounts of enzyme weretested by 48 experiments as shown in Table 1. All designed exper-iments were performed twice, and the corresponding average

A. Bahrami, P. Hejazi / Journal of Molecula

Tab

le

1D

esig

n

and

resu

lts

of

exp

erim

ents

of

pec

tin

ase

imm

obil

izat

ion

and

acti

vity

.

AO

T

con

c.

(M)

Init

ial e

nzy

me

con

c.

(mg/

ml)

pH

3.5

4

4.5

5

Imm

obil

ized

enzy

me

(mg

enzy

me/

g

sup

por

t)En

zym

e

acti

vity

(U/m

g

enzy

me)

Imm

obil

ized

enzy

me

(mg

enzy

me/

g

sup

por

t)En

zym

e

acti

vity

(U/m

g

enzy

me)

Imm

obil

ized

enzy

me

(mg

enzy

me/

g

sup

por

t)En

zym

e

acti

vity

(U/m

g

enzy

me)

Imm

obil

ized

enzy

me

(mg

enzy

me/

g su

pp

ort)

Enzy

me

acti

vity

(U/m

g

enzy

me)

0.05

0.25

63.8

1.24

59.1

1.32

55.3

1.55

52.3

1.37

0.5

177.

0

1.4

143.

7

1.71

119.

2

1.98

115.

9 1.

810.

7518

8.8

1.54

179.

4

1.38

163.

9

1.47

167.

7 1.

461

364.

3

1.26

364.

7

1.1

324.

9

1.13

322.

3 1

1.5

553.

5

0.73

540.

8

0.79

422.

8

0.8

465.

5

0.6

2

595.

7

0.56

604.

6

0.63

532.

3

0.63

348.

4

0.68

0.1

0.25

67.0

1.18

64.0

1.19

56.4

1.42

57.0

1.34

0.5

159.

6

1.56

161.

5

1.50

144.

8

1.55

137.

0

1.66

0.75

205.

4

1.54

223.

5

1.17

149.

3

1.5

160.

8

1.51

1

333.

7

1.5

349.

3

1.31

321.

2

1.1

356.

4

0.96

1.5

570.

9

0.7

558.

4

0.86

505.

7

0.75

504.

1

0.95

2

587.

6

0.7

610.

5

0.6

552.

9

0.68

442.

1

0.77

r Catalysis B: Enzymatic 93 (2013) 1– 7 3

amount and activity of immobilized enzyme are presented inTable 1. Data were evaluated to determine effective factors andinteractions by the Design Expert software (version 8) in generalfactorial design. To find the best situations for pectinase immobi-lization, three different goals were considered: the experiment inwhich higher amount of immobilized enzyme was achieved, theone that higher enzyme activity was measured, and the balancebetween both previous goals was investigated.

2.6. Stability experiments of immobilized enzyme

2.6.1. Determination of storage stability of free and immobilizedpectinase

The activity of the immobilized enzyme was measured after 30and 40 days of storage at 4 ◦C, and the remaining percentage ofimmobilized enzyme activity was calculated in each measurement.The measurement of activity was carried out in optimum conditionof enzyme activity (at pH = 4 and temperature of 40 ◦C).

2.6.2. Investigation of temperature and pH effects on free andimmobilized enzyme activity

The optimum temperature and pH for activity of this enzyme are40 ◦C and 4. Therefore, the stability of the enzyme was evaluated attwo higher temperatures and pHs. Activity of free and immobilizedenzyme were measured at 40, 50 and 60 ◦C under constant pH = 4;and also at pHs 4, 5, and 6 under constant temperature 40 ◦C, toevaluate their temperature and pH stability, respectively. Enzymeimmobilized in condition of 0.05 M AOT and pH = 4. For comparison,the amount of both free and immobilized enzyme was 0.125 mg.

2.7. Recycling process

The recovered particles were washed several times with bufferacetate. Then, the pectin solution at optimum pH of 4 was addedto the recovered particles, and hydrolysis was carried out underthe described conditions. The recycling process was repeated for6 times. This was performed only for some immobilized enzymesthat showed higher activities among immobilization experiments.

2.8. Experiments of immobilized pectinase release

Releasing the immobilized protein after immobilization throughchanging pH values was examined. According to pectinase iso-electric point (pI = 3.8), enzyme immobilized in acetate buffer atpH = 3.5, released with 0.1 M of phosphate buffer at pH = 6.7, andinversely enzyme immobilized at pH = 5, released with 0.2 M ofacetate buffer at pH = 3.5. To release the protein, enzyme immo-bilized on MNPs dispersed in the buffer during 4 h in the orbitalshaker for three times at the same conditions of immobilizationexperiments. The amount and activity of released protein in com-parison with the immobilized enzyme were measured.

3. Results and discussion

3.1. Characterization of MNPs

AOT was selected as an anionic surfactant to suspend and coatthe nanoparticles. AOT molecules in water form micelles which arecapable to entrap Fe3O4 molecules. Some of these micelles can formhydrogen bonds with oxygen atoms exist in structure of Fe3O4 [16].

3.1.1. FT-IR of nanoparticlesThe FT-IR spectrum of MNPs without pectinase is shown in Fig. 1.

A strong band exists at 594.03 and 592.11 cm−1, can be assigned tothe Fe O bond of the magnetite before and after immobilization,

4 A. Bahrami, P. Hejazi / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 1– 7

Ft

rngabe(aplteica

3

c1wItccin

ig. 1. FT-IR spectra of Fe3O4 nanoparticles before and after pectinase immobiliza-ion.

espectively [4]. In the IR absorption spectrum of MNPs after pecti-ase immobilization, peaks of 904 and 1026 cm−1 could be due toalacturonic acid and the presence of pectinase [17]. The two peakst 1419 and 1116 cm−1 (before) and peak of 1417 cm−1 (after) areecause of S O bond of sulfonation which could be due to pres-nce of AOT on the Fe3O4 nanoparticles [18]. The peaks at 1633before immobilization) and 1577 cm−1 (after immobilization) isssigned to the vibration of C O bond of AOT and the amide inrotein, respectively [19,20]. The peaks at 2939 (before immobi-

ization) and 2993 (after immobilization) cm−1 are attributed tohe vibration of CH [19]. The peak at 3413 cm−1 marks the pres-nce of amide groups (N H stretches) [21]. In spectrum, beforemmobilization, band of 3417 cm−1 could be due to OH of water. Itan be ascribed to the stretching vibrations of OH, which is alsollocated to OH−absorbed by Fe3O4 nanoparticles [19].

.1.2. DLS of nanoparticlesFig. 2 shows size distribution of AOT-coated Fe3O4 nanoparti-

les. About 41.8% of particles have the equal size or lower than05 nm. As the AOT molecules have the affinity to form micelles,hen they cover the surface of MNPs, they also aggregate in water.

t results in larger hydration sphere and contributes to larger par-icle size [22]. Although the effect of cluster size heterogeneity

ould not be removed completely, two following solutions wereonsidered to minimize them. Firstly, after synthesis of MNPs,mmobilization process was performed immediately; so there wasot enough time for them to settle or provide more aggregations.

Fig. 3. SEM images of Fe3O4 nanoparticles coated with AOT (left s

Fig. 2. DLS result for size distributions of AOT-coated MNPs.

Secondly, some experiments were chosen randomly and were car-ried out again up to 4 times. Decreasing or increasing trends ofimmobilization results were similar, and no dramatic differenceswere seen.

3.1.3. SEM of nanoparticlesFig. 3 shows the SEM images of AOT-Fe3O4 nanoparticles before

and after pectinase immobilization. It can be found from the twoimages that the mean size of the particles is near 100 nm, whichis in accordance with the result of DLS analysis. However, someaggregated particles can be observed in the images because ofaggregation during separation and drying.

3.2. Pectinase immobilization on Fe3O4 nanoparticles

Various initial pectinase concentrations (0.25–2 mg/ml acetatebuffer) at pH = 3.5–5 were tested on MNPs stabilized by AOT (0.05and 0.1 M) to determine the optimum enzyme loading and activ-ity. Results of the immobilization experiments in both amount ofprotein loading (mg enzyme/g support) and specific immobilizedenzyme activity (U/mg enzyme) are shown in Table 1.

3.2.1. Effect of initial enzyme concentrationAs shown in Fig. 4, increasing the initial enzyme concentra-

tion increases the amount of immobilized enzyme, whereas its

ide) before and (right side) after pectinase immobilization.

A. Bahrami, P. Hejazi / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 1– 7 5

0

100

200

300

400

500

600

700

0

0.5

1

1.5

2

0.25 0.5 0.75 1 1.25 1.5 1.75 2

Enzy

me

Imm

ob

iliz

ed (

mg/g

su

pp

ort

)

Enzy

me

Act

ivit

y (

U/m

g)

Initial Enzyme Co nc. (mg/ ml)

a

0

100

200

300

400

500

600

700

0

0.5

1

1.5

2

0.25 0.5 0.75 1 1.25 1.5 1.75 2

Enzy

me

Imm

obil

ized

(m

g/

g s

upport

)

Enzy

me

Act

ivit

y (

U/m

g)

Initial Enzyme Co nc. (mg/ ml)

Enzyme Activi ty(I mmobilized at pH= 3.5) Enzy me Activity ( Imm obilized at pH= 4)

Enzyme Activi ty ( Imm obilized at pH= 4.5) Enzy me Activity ( Imm obilized at pH= 5)

Immobilized Enzy me at p H=3 .5 Immobilized Enzy me at p H=4

Immobilized Enzy me at p H=4 .5 Immobilized Enzy me at p H=5

b

Fe

alWpasnc

cceselimI0ooeaeacba

st

0

0.5

1

1.5

2

2.5

100

120

140

160

180

200

3.5 4 4.5 5

Enzy

me

Act

ivit

y (

U/m

g)

Imm

obil

ized

Enzy

me

Am

ou

nt

(mg/

g s

up

po

rt)

pH

ig. 4. Effect of initial enzyme concentration on amount and activity of immobilizednzyme on nanoparticles coated with AOT (a) 0.05 M and (b) 0.1 M.

ctivity decreases. This can be because of spatial restrictions,imited active site accessibility, or denaturing of the protein [21].

u et al. [23] reported higher activity of cellulase immobilized onolyvinyl alcohol nanofibers at lower loadings because of substrateccessibility at the active site of the enzyme. Moreover, Lee et al. [8]howed that, in immobilization of lipase on hydrophobic magneticanoparticles, the excessive enzyme loading hinders the substrateonversion because of the protein–protein interaction.

The maximum amount and activity of immobilized enzymean be observed in Fig. 4. At condition of pH = 4, initial enzymeoncentration of 2 mg/ml and AOT concentration of 0.1 M, the high-st amount of immobilized enzyme of 610.5 mg enzyme/g supporthows the lowest enzyme activity of 0.6 U/mg enzyme. As it wasxpected, higher amount of enzyme immobilization occurred atower pHs where enzyme has more positive net charges. Consider-ng this experiment is important when immobilization amount is

ore important than its activity such as bioseparation experiments.n contrary, at condition of pH = 4.5, initial enzyme concentration of.5 mg/ml and 0.05 M AOT concentration, highest enzyme activityf 1.98 (U/mg protein) was attained while the immobilized enzymef 119.2 mg enzyme/g support was obtained (Fig. 4). The excessivenzyme loading inhibits the substrate conversion because of thebove reasons have been discussed. This can be the most importantxperiment when activity is of great significance such as biocat-lytic or drug delivery processes. Demir et al. [24] immobilizedommercial pectinase (Pectinex Ultra SP-L) on ion exchange resiny electrostatic adsorption. They reported the highest pectinase

ctivity was achieved at pH = 4.5.

Analysis of variance (ANOVA) for both responses using theoftware is reported in Table 2. Initial pectinase concentration ishe significant factor determining both the amount and activity

Fig. 5. Effect of aqueous pH on amount (solid line) and activity (dashed line) immo-bilized enzyme at 0.05 M (�,�) and 0.1 M (�,�) of AOT concentration (initial enzymeconcentration of 0.5 mg/ml).

of immobilized enzyme amount. As discussed above, increasingenzyme concentration decreases immobilized pectinase activityand vice versa. Specific enzyme activity response depends on theimmobilized enzyme amount, so only the initial enzyme amountaffects the enzyme activity while the other two factors have noeffects.

The best biocatalyst with balance between two responses is alsoestimated using the software and targets of two responses were setto maximum. The best experiment was chosen at pH = 3.5, initialenzyme concentration of 1000 mg enzyme/ml and AOT concentra-tion of 0.05 M. In this experiment, amount of immobilized enzymewas 338.8 mg enzyme/g support and its related specific activity was1.49 U/mg enzyme.

3.2.2. Effect of AOT concentrationAOT as an anionic surfactant provides negative charge on the

MNPs and the pH of the medium can affect the total net chargeof pectinase molecules according to its isoelectric point (pI = 3.8).In addition, the AOT amount on the support surface also affectsthe electrostatic attractions between enzyme molecules with netpositive charge (at pH < pI) and MNPs with negative charge.

Surfactant concentration did not affect the immobilization dra-matically, but as can be seen in Fig. 4, the level of immobilizationon support synthesized in the presence of 0.05 M AOT was slightlylower than the one with 0.1 M at high concentrations of enzyme.This can be attributed to the higher negative charge on MNPssurface that strengthen the electrostatic attraction. Enzyme immo-bilized on support covered with 0.05 M AOT faintly has shownhigher activity than the other one especially at low concentrationsof enzyme. Increasing surfactant amount can result in surroundingmore AOT molecule shells around the particles. Considering all lev-els of initial enzyme concentration, AOT concentration only affectsthe amount of immobilized enzyme (Table 2).

3.2.3. Effect of aqueous pHIncreasing the pH decreases the amount of immobilized enzyme

that can be assigned to poorer electrostatic affinities because ofincreasing pH over pectinase pI (3.8) and decreasing the amountof positive charges. The effect of aqueous pH on the amount ofimmobilized enzyme, for instance, at initial enzyme concentrationof 0.5 mg/ml is shown in Fig. 5.

Shamim et al. [25] investigated the impacts of adsorptionand desorption of Bovine Serum Albumin (BSA) on surface-

modified magnetic nanoparticles covered with thermosensitivepolymer (PNIPAM). At higher pH, a smaller amount of protein wasadsorbed because of the electrostatic repulsive force between pro-tein molecules and latex particles. The maximum amount of protein

6 A. Bahrami, P. Hejazi / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 1– 7

Table 2Results of ANOVA for amount and activity of immobilized enzyme.

Source Immobilized enzyme amount (mg enzyme/g support) Enzyme activity (U/mg)

F-value P-value F-value P-value

A – pH 31.43823 <0.0001* 1.442016 0.2700B – AOT conc. 7.112425 0.0176* 0.04057 0.8431C – initial enzyme conc. 871.8589 <0.0001* 119.1434 <0.0001*

AB 1.645627 0.2211 1.860407 0.17970 * *

0

wp

maea

ca(efirea

3

3

awictwcaers

3e

d(aaaTa

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catalyst at near pH of immobilization.However, the activity of immobilized enzyme at pH = 3.5

with AOT and enzyme concentrations of 0.05 M and 0.75 mg/mldecreases to 60%. In this case, the amount of immobilized enzyme

0

0.5

1

1.5

2

2.5

40 50 60

Enzy

me

Act

ivit

y (

U/m

g)

T (ºC)

a

0

0.5

1

1.5

2

2.5

4 5 6

Enzy

me

Act

ivit

y (

U/m

g)

pH

b

AC 7.692136

BC 1.398627

* Significant effect in 5% level (P-value < 0.05).

as adsorbed near the isoelectric point of BSA. Findings of theresent study were in agreement with their findings.

As can be seen in Fig. 5 and Table 2, pH of immobilizationedium did not significantly affect the enzyme activity, measured

t pH = 4 (optimum pH of pectinase activity). It can be found thatnzyme specific activity only depends on the immobilized enzymemount.

Aqueous pH can affect enzyme structure, stability and netharge, especially near the pI. Therefore, Interaction between pHnd initial enzyme concentration is important on both responsesTable 2). Furthermore, the main effect graphs of pH and initialnzyme concentration for both responses (not reported here) con-rm that interactions for the amount of immobilized enzyme occurespectively at higher enzyme concentration and higher pHs, whilenzyme activity interactions appear at lower enzyme concentrationround pI. They are all in accordance with the above discussion.

.3. Results of stability tests

.3.1. Storage stability of free and immobilized pectinaseElectrostatic immobilization offers a simple technique for sep-

rating and reusing enzymes over a longer period in comparisonith free enzymes. To evaluate storage stability of both free and

mmobilized enzyme, their activities were measured at the similaronditions after 30 and 40 days standing at constant tempera-ure 4 ◦C. Free pectinase was inactivated and lost its entire activityithin 30 days. Immobilization of pectinase on Fe3O4 nanoparti-

les reduced inactivation rate and about 65% of its activity retainedfter 30 days for all the immobilized enzymes at various pHs. Leit al. [5] immobilized pectinase on silica-coated (PEI/Fe3O4)n andeported that about 55% of its activity retain after 20 days at con-tant temperature 20 ◦C.

.3.2. Effect of temperature and pH on free and immobilizednzyme activity

Thermal and pH stability of free and immobilized enzyme wereetermined at pHs 4, 5, and 6 (T = 40 ◦C) and 40, 50, and 60 ◦CpH = 4). The reaction time was 30 min and after that enzymectivity was measured using Miller method. Fig. 6 shows enzymectivity decreases with temperature increasing and activity ofcidic pectinase (optimum pH = 4), reduces with pH increasing.hese reductions apparently are because of protein denaturationnd decreasing the reaction rate.

According to 50% reduction of both free and immobilizednzyme activity under pH and temperature increasing, it can beentioned that the system was unsuitable to achieve high tem-

erature and pH stability. The activity of immobilized enzyme wasimilar to its free counterpart at higher pHs and temperatures.

herefore, it can be concluded that enzyme structure did not changefter immobilization. Lei and Bi [26] immobilized pectinase ontohe amphiphilic block copolymers poly(styrene-b-acrylic acid), andeported the same optimum pH and temperature for both free and

.0002 2.820721 0.0266

.2803 2.125021 0.1185

immobilized enzyme (6 and 65 ◦C) indicating that they were notaffected by immobilization.

3.4. Reuse stability of the immobilized pectinase

Pectinase-immobilized Fe3O4 nanoparticles could be easilyrecovered from the reaction medium through magnetic decanta-tion and employed for several times. Since the stability andreusability are important characteristics in economic usage ofimmobilized enzymes in different industries, pectinase recyclingprocess was assessed. Fig. 7 shows the relative activity of enzymefor 6 cycles. In all experiments, the retained activity is about 85–95%of its first value. Difference between immobilization pHs (3.5, 4,4.5, and 5) and the pH during the activity measurements (pH = 4) islow; therefore, lower desorption occurs. It can be found, althoughthe immobilization technique is based on electrostatic bonding,enzyme leakage is low. Therefore, electrostatically immobilizedenzyme can be used for catalysis processes as an appropriate bio-

Free Enzy me Immobilized Enzy me

Fig. 6. Effects of (a) temperature and (b) aqueous pH on free (�) and immobilized(�) enzyme activity.

A. Bahrami, P. Hejazi / Journal of Molecula

50

60

70

80

90

100

0 1 2 3 4 5 6

Rel

ativ

e A

ctiv

ity%

Cycle Number

Immobilized at pH:3.5, 750 ppm, 0.05 M Immobilized at pH:4, 500 ppm, 0.05 M

Immobilize d at pH:4.5, 500 ppm, 0.0 5 M Immobilize d at pH:5, 50 0 ppm, 0.0 5 M

Immobilize d at pH: 3.5, 500 ppm, 0.1 M Immobilize d at pH:4, 50 0 ppm, 0.1 M

Immobilize d at pH: 4.5, 500 ppm, 0.1 M Immobilize d at pH: 5, 500 ppm, 0.1 M

ia

ip8fe

3

etceitdtsBaappai

r

Rdr3y

[[

[[

[[[[[

[

[[

[

Fig. 7. Variation of enzyme activity during cyclic uses at 40 ◦C and pH = 4.

s higher than the others, the immobilization pH is lower than pI,nd consequently enzyme leakage increases.

Demir et al. [24] immobilized commercial pectinase onto spher-cal anion exchange resin beads with electrostatic adsorption atH = 4.5, reported retained activity of immobilized pectinase was0% after 9 batches at pH = 4.5 and 35 ◦C. Since, pH is an importantactor in electrostatic immobilization; they kept pH constant for allxperiments near the optimum pH enzyme activity.

.5. Pectinase release

Covalent immobilization provides strong bonding betweennzyme and support, reducing enzyme leakage. In contrary, elec-rostatic adsorption and ionic interactions are based on poorerhemical affinities. This matter can be very useful in drug deliv-ry systems and also when support or enzyme is economicallymportant to release. Immobilization techniques are widely used inargeted drug-delivery systems where high desorption of proteinrugs can be achieved using pH changing [23]. Enzyme immobiliza-ion under acidic pHs and its release under basic pHs seems to be aimple way to achieve this purpose. Shamim et al. [25] immobilizedSA in acidic solution and released it in basic solution. As pectinasectivity decreases significantly through pH increasing (especiallyt pH more than 7), it is better to release the protein at acidicH. Enzyme immobilized at pH = 3.5, was released by increasingH to 6.7 (developing repulsion). Releasing yield was calculateds the proportion of the released enzyme amount to amount ofmmobilized enzyme:

eleasing yield (%) = mg released Enz.

mg immobilized Enz.

eleasing yield of the pectinase was 71.4%, whereas its activity

ecreased dramatically to 0.76 U/mg enzyme (about 50% activityeduction of its immobilized state). In other way, decreasing pH to.5 was applied for enzyme immobilized at pH = 5. The releasingield was 87.7%, and the activity decreased to 0.97 U/mg enzyme

[[[

[

r Catalysis B: Enzymatic 93 (2013) 1– 7 7

(about 50% of activity decline). Decreasing enzyme activity after therelease can be assigned to enzyme structural changes because of along period of shaking. Separated AOT molecules from MNPs’ sur-face may form micelles and entrap enzymes which inhibit substratediffusion and consequently results in reducing activity.

As mentioned previously, enzyme activity decreased to half ofits amount in immobilized state after release. The desired targetin drug delivery systems is delivering appropriate amount of drugto provide the medical treatment. However, protein drug shouldhave enough activity to react with the special target. So retainingspecific enzyme activity after release can be achieved by modifyingsupport’s surface and also by changing the way of release.

4. Conclusions

The present work proposed a simple protocol for enzyme immo-bilization. Pectinase was successfully immobilized on AOT-MNPsof Fe3O4 with a mean diameter particle size of about 100 nm.Nanoparticles’ surface covered with AOT results in electrostaticaffinity of enzyme and MNPs.

The initial pectinase and AOT concentrations and pH, weredetermined as effective factors on immobilized enzyme amountwhile only AOT concentration affects its activity. Electrostaticallyimmobilized enzyme on Fe3O4 nanoparticles can be used as bothbiocatalyst and drug carrier at near pH of immobilization (pH = 4).The same method for immobilization can be used to release theenzyme at acidic pH, which is important in protein drug deliveryand bioseparation processes. The long-term stability and retain-ing 85–95% of enzyme activity after 6 cycles are the advantagesof attaching pectinase to the surface of AOT-MNPs. Furthermore,evaluation of pH and thermal stability showed that immobilizationdid not affect the enzyme structure.

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