j chem technol biotechnol 2011; 86- 1386–1393 óxido de ferro na extração de proteinas de...

8/13/2019 J Chem Technol Biotechnol 2011; 86- 13861393 xido de ferro na extrao de proteinas de Moringa

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Research Article

Received: 29 October 2010 Revised: 10 May 2011 Accepted: 20 June 2011 Published online in Wiley Online Library: 6 September 2011

(wileyonlinelibrary.com) DOI 10.1002/jctb.2704

Application of magnetic iron oxide

nanoparticles prepared from microemulsionsfor protein purification

Chuka Okoli,a,b Magali Boutonnet,b Laurence Mariey,c Sven Jarsb

and Gunaratna Rajaraoa

Abstract

BACKGROUND: Magnetic nanoparticles are of immense interest for their applications in biotechnology. This paper reportsthe synthesis of magnetic iron oxide nanoparticles from two different water-in-oilmicroemulsion systems (ME-MIONs), theircharacterization and also their use in purification of coagulant protein.

RESULTS: ME-MIONs have demonstratedto be an efficientbinder in thepurification ofMoringaoleiferaprotein when comparedwiththe superparamagnetic ironoxide nanoparticles preparedfrom coprecipitationin aqueous media. The sizeand morphologyof the ME-MIONs were studied by transmission electron microscopy (TEM) while the structural characteristics were studied byX-ray diffraction (XRD). The microemulsion magnetic iron oxide nanoparticles (ME1-MION andME 2-MION) obtained have a sizerange 7 10 nm. The protein and ME-MIONs interaction was investigated by Fourier transform infrared spectroscopy (FT-IR);the presence of three peaks at2970, 2910 and 2870 cm1 respectively, confirms the binding of the protein. Thepurification andmolecular weight of the coagulant protein was 6.5 kDa as analyzed by SDS-PAGE.

CONCLUSION: The ME-MIONs have the advantage of being easily tailored in size, are highly efficient as well as magnetic, costeffective and versatile; they are, thus, very suitable for use in a novel purification technique for protein or biomolecules thatpossess similar characteristics to the Moringa oleifera coagulant protein.c 2011 Society of Chemical Industry

Keywords: microemulsion; magnetic nanoparticles; iron oxide; protein purification; magnetic separation;Moringa oleifera

INTRODUCTIONThe emergence of nanobiotechnology has contributedimmensely

in thedevelopmentof newtechniquesfor solving manyhealthand

environmental issues. Nanometer-sized magnetic nanoparticles

have received considerable attention because of their unique

physical and chemical properties owing to their extremely small

size and large specific surface area.1 3 Magnetic nanoparticles

have been used in many applications such as data storage

devices,4 6 and skin care products.7 In recent years, nanometer-

sized magneticnanoparticles have been used in both medical and

biological fields owing to their strong magnetic properties and

virtually no toxicity for applications including drug delivery, 811

biosensors,12,13 water purification systems14,15 and biomolecular

separation/purification.10,1619

Protein is the central dogma of cell function and study of

proteins is essential to a better understanding of the cell function.

Protein purification has been a fundamental requisite in advances

madein biotechnology.18,20 Pureprotein helps to resolve the basic

questions about the functional characteristics of protein while the

development of apt techniques is essential where more than one

stepis required for protein purification.1,16,18 Recombinantprotein

technologies are being developed in order to produce protein in

large quantities; however, many problems remain unresolved,

such as host contamination, solubility and structural integrity.20 In

biotechnology, affinity protein purification using antibody-based

separation or a matrix with specific tags for binding target protein

are commonly used methods.1618 The challenge is to use a

common matrix for purification of different proteins. Magnetic

nanoparticles can be used as a simple and quick method for the

efficientcaptureof selectedtargetbiomolecules in thepresence of

other suspended solids even fora small sample volume.21The use

of magnetic nanoparticles such as superparamagnetic iron oxide

nanoparticles for purification and immobilization of biomolecules

such as proteins/peptides, have the following advantages: (i) high

specific surface area can be obtained to bind large amounts

of protein samples; (ii) selective targeting of biomolecules with

Correspondence to: GunaratnaRajarao, Royal Institute of Technology (KTH),

Environmental Microbiology, Stockholm,106 91 Stockholm, Sweden.

E-mail: [email protected]

a Royal Institute of Technology (KTH), Environmental Microbiology, Stockholm,

106 91 Stockholm, Sweden

b Royal Institute of Technology (KTH), Chemical Technology, Stockholm, 100 44

Stockholm, Sweden

c Laboratoire Catalyse et Spectrochimie, ENSICAEN, Universite de Caen, CNRS,

140 50 Caen-Cedex, France

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Magnetic iron oxide nanoparticles for protein purification www.soci.org

desired surface characteristics and easy separation from the

reaction mixture by magnetic field; (iii) elimination of several

steps in the purification process such as centrifugation, filtration,

etc.1 Usingmagneticseparation offers a gentle andfast alternative;

targetsarecapturedonthesurfaceofthedesiredmagneticparticle

andarapidseparationisachievedfromthesamplebytheuseofan

external magnetic field. With most conventional or commercially

available protein purification/immobilization systems, the cost of

protein purification on a large scale is still a major challenge.

A promising approach is the use of magnetic iron oxide

nanoparticles prepared from microemulsion (ME-MIONs) for

the purification or immobilization of proteins. This technique

is based on microemulsion synthesis, which allows tailor-

made nanoparticles in order to achieve the desired size.22,23

Microemulsions are optically transparent, thermodynamically

stable solutions which consist of spherical aqueous nanodroplets,

so-called reverse micelles, stabilized by surfactant molecules. In

the aqueous core of the reverse micelle, iron metal precursor

can be solubilized and then precipitated to form iron oxide

nanoparticles.22 ME-MIONs are single domain magnetic dipoles

that show no preferred directional ordering in the absence of

an applied external magnetic field due to weak dipoledipoleinteractions. On application of a sufficiently high magnetic field

gradient, the nanoparticles exhibit a preferential ordering in the

direction of this external field.24

This paper reports a simple and fast method for protein

purification and/or immobilization using ME-MIONs obtained

from two different microemulsion compositions. The ME-MIONs

were synthesized and their characteristic properties studied. The

purification of coagulant protein is achieved by binding and

elution of theMoringa oleiferaprotein. The coagulation efficiency

of ME-MIONs purified proteins was examined and compared with

protein purified with superparamagnetic iron oxide nanoparticles

(SPION) prepared by co-precipitation in aqueous media (Okoli

etal., in press) and the results are discussed.

EXPERIMENTALMaterials

All chemicalswere of reagentgrade and wereused without further

purification. Ferric chloride hexahydrate (FeCl3.6H2O, >99%),

ferrous chloride tetrahydrate (FeCl2.4H2O,>99%), cetyl trimethyl

ammonium bromide (C19H42BrN), 1-butanol (C4H10O), n-octane

(C8H18) and hexanol (C6H13OH) were purchased from Sigma-

Aldrich, Sweden. Ammonia (NH3 , 32%), sodium chloride (NaCl),

ammonium acetate(CH3COONH4),andkaolinclay(Al2Si2O5(OH)4)

were purchased from Merck, Sweden. Ethanol (C2H5OH,>99.7%)

was obtained from Solveco, Sweden.Moringa oleiferaseeds were

obtained from Kenya.

Synthesis of magnetic iron oxide nanoparticles (ME-MIONs)by water-in-oil (w/o) microemulsion reaction method

The magnetic iron oxide nanoparticles of the present study were

prepared in a w/o microemulsion. In this work, twodifferent types

of microemulsion systems were investigated for the preparation

of ME-MIONs.

Each microemulsion system consists of a surfactant, cetyl

trimethyl ammoniumbromide (CTAB), a co-surfactant (1-butanol),

oil phase (n-octane or hexanol), iron precursors (FeCl3and FeCl2),

precipitating agent (32% NH3) and water (Milli-Q water). Mi-

croemulsion system 1 consists of CTAB/1-butanol/hexanol/water

and is termed microemulsion 1-magnetic iron oxide nanoparticles

(ME 1-MION) while microemulsion 2 consists of CTAB/1-butanol/n-

octane/water and is termed microemulsion 2-magnetic iron oxide

nanoparticles(ME 2-MION). The aqueous phasecontains either the

Fe salt precursors in a mole ratio of 2 : 1 [FeCl3: FeCl2] or aqueous

ammonia (NH3). It has been demonstrated that particles prepared

with this method exhibit a size range 650 nm with high surface

area to volume ratio and a superparamagnetic behavior.24

Microemulsion system 1 for synthesis of ME 1-MION

The ME 1-MION was synthesized by mixing two w/o microemul-

sion solutions; scheme 1 (a). Microemulsion 1 solution was

obtained by adding the iron precursor solutions (1.2 mol L1

FeCl3 + 0.6 mol L1 FeCl2) to the mixture of the surfactant,

co-surfactant and oil phase (molecular concentration of CTAB : 1-

butanol : hexanol : water= 1.5: 1 : 1.4: 1.6); for microemulsion 2, a

precipitating agent solution (NH3 , 32%) was added to themixture.

Microemulsion 2 was then added to microemulsion 1 dropwise in

order to obtain the formation of particles at 25 C upon vigorous

stirring for 2 h. The pH of the mixture was adjusted to 11 with 32%

ammonia solution. Formation of magnetic nanoparticles was in-

dicated by black coloration. The magnetic nanoparticles obtainedwere separatedusing an external magnetic field andthen washed

by several cycles of precipitation and resuspension in a mixture

of chloroform and methanol (1: 1), water and finally methanol to

remove all the surfactant and oil left in the system. The magnetic

nanoparticles were either dried at 70 C or suspended in Milli-Q

water and kept at 4 C until further use.

Microemulsion system 2 for synthesis of ME 2-MION

Microemulsion system 2 consists of a single-step mode of

preparation. In this experiment, only one type of microemulsion

solutionis needed forformation of thenanoparticles, scheme 1 (b).

For ME 2-MION preparation, the following conditions were used:

0.67 mol L1 FeCl3 and 0.42 mol L1 FeCl2 at mole ratio (2: 1)

in Milli-Q water; the molar concentration of CTAB : 1-butanol : n-

octane: water= 0.6 : 0.5 : 3 : 1.3. The addition of precursor solution

to the mixture of CTAB/1-butanol/n-octane will give rise to

the formation of a microemulsion. Formation of magnetic

nanoparticles was achieved by adding the precipitating agent

(NH3, 32%) dropwise to the microemulsion containing the

precursor upon vigorous stirring until pH 11 was achieved.

The reaction mixture was stirred for 2 h at 25 C. A dark brown

coloration indicates the formation of ME 2-MION in the reaction

mixture. The magnetic nanoparticles obtained were then washed

with a mixture of chloroformand methanol(1 : 1),water andfinally

methanolto removeallthe surfactant andoilleftin thesystem. The

magneticnanoparticles were either dried at 70 C or suspended in

Milli-Q water and kept at 4

C until further use.

Synthesis of superparamagnetic iron oxide nanoparticles(SPION)

SPIONs were preparedby co-precipitation in aqueousmedia (Okoli

etal., in press). Briefly, the iron source was prepared by dissolving

FeCl3 and FeCl2 in Milli-Q water at a molar ratio of 2 : 1 followed

by precipitation of nanoparticles with ammonia solution at 70 C

under vigorous mechanical stirring. The reaction was allowed to

proceed for 45 min under N2atmosphere in a closed system. The

black precipitated powder was collected by sedimentation with

the help of an external magnetic field and washed with Milli-Q

water. The magnetite obtained was re-suspended in water prior

to use.

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www.soci.org C Okolietal.

Scheme 1. Preparation of ME 1-MION (a) and ME 2-MION (b).

Characterization of magnetic iron oxide nanoparticles frommicroemulsion

The ME-MIONs were characterized by transmission electronmicroscopy (TEM; ZEISS, EM 906) with an accelerating voltage

of 200 kV, X-ray diffraction (XRD; Siemens D5000, using Cu

K radiation of wavelength; = 0.1540 nm). The investigated

ME-MION samples were either dried at 70 C or calcined at

450 C. Fourier transform infrared spectroscopy(FTIR; Nicolet 5700

spectrometer) was used to investigate the protein interaction on

the nanoparticle surfaces. The samples were analyzed as a disc

with an area of 2 cm2 and a mass of 15 mg.

Coagulant protein extraction and activity assay

Moringa oleifera coagulation protein (MOCP) extraction was

performed following the method described previously.25 The

Moringa oleiferaseeds were obtained from Kenya. Ethanol (95%)

was used to extract the oil from the crushed seeds and the active

components were extracted with Milli-Q water. Kaolin clay (1%)

wasaddedto 1 L oftap water, stirredfor half anhourandallowed to

settle for 24 h for complete hydration. Small-volume coagulation

activity assay on clay suspension was performed.26 A sample

volume of10 Lwasaddedtotheclaysuspensiontoafinalvolume

of 1 mL in cuvettesand mixed instantly.Absorbanceat 500 nm was

measured with Thermo Spectronic UV-visible spectrophotometer

at time 0 and after 60 min settling time. A decrease in absorbance

relative to control defines coagulation activity. The percentage

coagulation activity was calculated using the formula below;

whereT0is the initial absorbance andTf is the final absorbance.

Percentage activity (%) = T0 TfT0

100

Protein estimation and molecular weight determination

The protein content was estimated using the Bradford method

with bovine serum albumin (BSA, 1 mg mL1) asa standard.27This

method is based on binding of the dye Coomassie brilliant blue

to protein, and is measured at 595 nm. The protein profile and

the molecular weight of the purified protein and that of the crude

extract were determined and comparedusing 10% SDS-PAGEmini

gels.

Purification of coagulant protein with magnetic iron oxide

nanoparticlesIn this study, a batch system was employed in the purification

of the coagulant protein. ME 1-MION, ME 2-MION and SPION

suspensions were used as obtained without any heat treatment.

The magnetic iron oxide nanoparticles were washed three times

each with 10 mmol L1 ammonium acetate buffer, pH 6.7, to

equilibrate the particles and then suspended in water or buffer

with the following concentrations: ME 1-MION (36 mg mL1), ME

2-MION (44 mg mL1) and SPION (10 mg mL1). The volume of

each solution was adjusted such that the total weight of magnetic

particles was the same, 2.5 mg, in the resultant solution. The

immobilization of MOCP (2.6 mg mL1) on the particles was

performed in 400 L of 10 mmol L1 ammonium acetate buffer,

pH 6.7.After 60 minincubation at roomtemperature,the unbound

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protein was separated by applying an external magnetic field and

thepureproteinelutedwith0.6 and0.8 mol L1 NaClsolution. The

specific coagulation activity of the purified protein was calculated

by dividing the pure protein (% coagulation activity) by the total

protein content/mg/ml.

Adsorption kinetic studies

Studies of adsorption kinetics were carried out in a batch systemwith a fixed concentration of magnetic iron oxide nanoparticles

(0.5 mg) at varying protein concentrations (0.5 2.1 mg mL1).

The immobilization assay was performed in 1.5 mL of 10 mmol L1

ammonium acetatebuffer. After 1 h of immobilization,the samples

were separated and the supernatants collected to estimate the

protein content. Based on results for the controls, a change

in protein concentration was attributed to adsorption by the

magnetic nanoparticles. The amount of adsorbed protein was

estimated from the Langmuir equilibrium adsorption model:28

Ce

qe=

1

b(Xm)+

Ce

Xm(1)

where Ce is the amount of protein in solution at equilibrium

(mg L1), qe is the amount of protein adsorbed per weight

of adsorbent (mg g1), b is a constant related to the heat of

adsorption (mL g1) andXmis the maximum adsorption capacity

(mg g1).

RESULTS AND DISCUSSIONPreparation of magnetic iron oxide nanoparticles frommicroemulsion

The microemulsion system is one approach to obtaining magnetic

iron oxide nanoparticles with a uniform size distribution and

identical chemical and physical properties. In the present work,

magnetic iron oxide nanoparticles were obtained from twodifferent w/o microemulsion systems (Scheme 1).

The size and morphology of the ME-MIONs were determined

using TEM. ME-MIONs were examined as prepared. The influence

of w/o ratio in the microemulsion on the particle size was

investigated. Figure 1(a) and (b) presents TEM images of the

prepared nanoparticles. A part of the ME-MIONs agglomerated

dueto thelargesurfaceenergy of thenanoparticles.The prepared

ME-MIONs are almost spherical in shape and the size is in the

range of 710 nm. It is important to note that the oxidation of

iron oxide strongly depends on experimental parameters such

as temperature, reaction environment and surface conditions.

These parameters influence the composition of the iron oxide

nanoparticles; our findings suggest that the particles obtained

are either magnetite (Fe3O4) or maghemite (-Fe2O3) o r a

combination of both magnetite and maghemite as can be

seen from XRD results showing similar patterns in their crystal

structure.29,30 The ME-MION particle size obtained by TEM (Fig. 1)

is in agreement with the particle size values obtained from XRD

analysis (Fig. 2).

The XRD patterns of ME 1-MION and ME 2-MION are shown in

Fig. 2. It can be seen that the XRD patterns of both samples after

calcination at 450 C appear quite similar, but somewhat different

after drying at 70 C; this implies that both ME-MIONs have similar

spinel crystal structure at 450 C in contrastto thesamplesdried at

70 C. It is difficult to conclude whether the nanoparticles consist

of magnetite or maghemite. The broad diffraction peaks from the

nanoparticles imply that the original particle size is very small;

similar results have been reported by Li etal.31 From the XRD

analysis of the particles dried at 70 C, a particle size of 9.2 nm for

ME 1-MION and 7.8 nm for ME 2-MION was obtained.

Coagulant protein extraction and activity assay

Water was used to extract active coagulant protein extracted from

Moringa oleifera. In order to estimate the actual activity of the

coagulant protein, control samples are used and differences incontrol and coagulant protein are considered as real coagulation

activity.It has beenreported thatproteinactivitywith saltor buffer

extracts showcomparable activity but protein coagulation activity

from water extract tends to be higher. It is believed that protein

obtained by water extraction has a better binding environment

with the clay solution.25,32

Purification of coagulant protein with magnetic iron oxidenanoparticles

The coagulant protein was purified with ME 1-MION and ME

2-MION and their purification efficiency was compared with

SPION (15 nm size) prepared by a co-precipitation method. The

prepared magnetic particles exhibit similar size and magneticbehavior (Okoli etal., in press). Purification was achieved using

the same elution buffer and pH as described previously. In this

work, the active coagulantprotein waselutedfrom ME 1-MION, ME

2-MIONandSPIONwith0.6,0.8 and1 mol L1 NaCl concentrations,

respectively (Fig. 3). By increasing the salt concentration of the

elution buffer to 1 mol L1, it was envisaged that the remaining

protein molecules thatwere strongly bound to the particles would

be detached.

The protein purified with ME 1-MION and ME 2-MION showed

coagulation activity similar to that of SPION in the first elution

step. In subsequent elution steps (2nd and 3rd), the coagulation

activity of the ME-MIONs increased progressively compared with

the SPION. When 1 mol L1 NaCl was used, the activity of

Figure 1.TEM micrographs of ME 1-MION (a) and ME 2-MION (b).



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Figure 2. XRD patterns of ME 1-MION (a) and ME 2-MION (b) dried at 70 C and 450 C.

Figure 3. Results showing the coagulation activity of MOCP purified withdifferent nanoparticles after 60 min using different salt concentrations.

protein purified from SPION was reduced by 50% whereas the

purified proteins from ME 1-MION and ME 2-MION retained high

activity. This suggests that the binding with the latter magnetic

particles is stronger compared with that to SPION; however, the

probable binding mechanism is still under investigation (Okoli

etal., in press). The non-adsorbed protein separated from theparticles showed a significant low activity of less than 5%, which

confirms that more than 90% of the protein was attached to

the magnetic nanoparticles. The specific coagulation activity

confirms that purification with SPION showed an increase of

60% mg1 mL1 compared with the crude extract, whereas an

increase of 66% mg1 mL1 was observed after purification with

ME 1-MION and ME 2-MION (Table 1). On the other hand, the

preparation of microemulsion system by two different methods

presented similar particle size and protein binding. The presence

of surfactant molecules on ME-MIONs was critical in reducing

particle agglomeration while maintaining a small particle size

which possibly enhances a large specific surface area. We envisage

that the residual surfactant from microemulsion promotes better

Table 1. Total proteinconcentrationand specificcoagulation activity

Sample

Specific coagulationactivity

(%) mg1 mL1

Total proteincontent

(%) mg1 mL1

Crude extract 24 100

SPION 84 74

ME 1-MION 91 83

ME 2-MION 90 88

Figure 4. SDS-PAGE analysis of MOCP proteinsamples eluted with twosaltconcentrations. Lane 1 is a low molecular weight protein marker (Sigma),

lane 2 represents crude extract; lanes 3, 4 and 5 represent 0.6 mol L1elution with ME 1-MION, ME 2-MION and SPION, respectively. Lanes 6 and7 represent 0.8 mol L1 elution with ME 1-MION, ME 2-MION.

protein particle binding. The results presented in this work imply

that the functionality of the protein eluted from ME-MIONs is as

good as that of theSPION; however,the advantage of theformeris

that particle synthesis can be designedto achieve the desired size

with the possibility to improve their efficiency in adsorbing the

protein (Fig. 3). With the magnetic nanoparticles, it was possible

to selectively purify the active protein from a mixture of other

proteins; this was evident in the SDS-PAGE (Fig. 4) and MS/MS

peptide sequence analysis.



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Figure 5. FT-IR spectra of protein (a), ME 2-MION (b) and (c) ME 2-MION+ protein in air, in vacuum I and vacuum II at RT, 50 and 90

C.

Protein content/specific coagulation activity and molecularweight determination

The amounts of protein in crude extract and different fractions of

purified samples were estimated. The crude extract has a protein

content of 2.6 mg mL1. After purification withmagnetic particles,

the total recovery of the purified protein was 1.92 mg mL1,

2.15 mg mL1 and 2.30 mg mL1 for SPION, ME 1-MION and ME

2-MION, respectively. The highproteincontent in thecrude extractis as a resultof other proteinspresent in thesample;consequently,

a reduction in concentration of the purified protein shows that

the protein of interest has been eluted from the nanoparticles.

The total protein concentration and specific coagulation activity

measurementsin(%)mg1 mL1 areshown in Table 1. Theprotein

specific coagulation activity and total protein recovery increased

progressively after purification with the particles. The extracts

were further elucidated in 10% SDS gel electrophoresis.

SDS-PAGE was performed in order to verify the purification

efficiency and molecular weight of the eluted coagulant protein.

The molecularmass of thepurifiedprotein is approximately 6.5 kDa

as determined by SDS-PAGE using a low range molecular weight

marker from Sigma (Fig. 4).

Mass spectrometric analysis of the purified protein after SDS-

PAGE was carried out based on MS/MS peptide sequence analysis

(data not shown). The peptide sequences obtained confirmed that

the purified protein is identical or similar to the MOCP sequence

already published.25

Protein and ME-MIONs interaction study

FT-IR analysis was performed in order to understand theinteraction between the protein molecules and the magnetic

iron oxide nanoparticles prepared by microemulsion systems.

FT-IR studies of the following samples were carried out: protein

alone, ME 1-MION and ME 2-MION with and without protein

interaction. For each sample, several IR spectra were taken:

(i) spectrum in air; (ii) spectrum in primary vacuum (103 torr);

(iii) spectrum in secondary vacuum RT (room temperature)

(106 torr); (iv) spectrum in secondary vacuum at 50 C; and

(v) spectrum insecondaryvacuumat 90 C(abovethistemperature

the protein starts to denature). The IR spectra of the protein

show that the treatment in vacuum, at RT, 50 C or 90 C does

not modify the structure of the protein. Treatment in primary

vacuum modifies the spectrum, with a decrease of the NH peaks



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at 3297 and 1655 cm1. Peaks at 1655, 1538, 1238, 1023 cm1

correspond to NH vibrations while those at 1447 and 1405

may be attributes to CH (Fig. 5(a)).33 The ME 1-MION and

ME 2-MION present similar peaks, and it is observed that the

treatments in vacuum at RT, 50 C and 90 C affect the structure

of the iron oxide particles. After treatment in primary vacuum the

peaks at 3490 and 1634 cm1, corresponding to the adsorbed

water, decrease. Treatment in vacuum shows three peaks at

3690, 3660 and 3625 cm1, which can be distinguished from

the nanoparticles. These peaks correspond to the OH group.

The peaks at 900 and 800 cm1 correspond to the iron oxide

structure. FT-IR spectra of ME 2-MION areshown in Fig. 5(b). TheIR

spectra of ME 2-MION + protein were obtained after treatments

in vacuum, at RT, 50 C and 90 C. Similar to the ME-MIONs, the

treatmentshavenoeffectonthestructureoftheironoxide/protein

composite. After protein adsorption, the peaks at 3690, 3660

and 3625 cm1 disappeared in the spectrum; hence peaks at

2970, 2910, 2870 cm1 are characteristic of the adsorbed protein,

corresponding to CH vibrations (Fig. 5(c)), this suggests strong

binding between the protein and the solid support.33 In order to

verify if theproteinis denatured at temperatureshigherthan 90 C,

the sample was heated at temperatures of 120 and 150

C. After24 h at 150

C, new peaks at 2185 and 2091 cm1 appeared and

the structural peaks at 900 and 800 cm1 disappeared (Fig. 5(c)).

It was difficult to analyze the ME 1-MION + protein using the disc

method. An effort to deposit the powder onto a silicon sheet was

not successful.

Adsorption kinetic studies

In the present work, we compared the optimal protein adsorption

of theME-MIONs with that of SPION.The rate of protein adsorption

onto the nanoparticles was rapid in the beginning and then

decreased rapidly. The optimum equilibrium time can be up

to 120 min. For monolayer coverage, the Langmuir isotherm

model (Equation (1)) can be used to estimate the equilibriumadsorptionparameters.Theestimatedoptimaladsorptioncapacity

of the protein on ME 1-MION and ME 2-MION was, on average,

400 mg protein g1, which is similar to the value obtained for

SPION (450 mg g1). The maximum capacity of carboxyl methyl

cellulose (CMC), a commercial magnetic micro bead with similar

composition to the present nanoparticles, has been reported to

have a binding capacity 131 mg protein g1 beads.32 Similarly,

Ghebremichael etal. reported that the conventional protein

purification method using carboxymethyl (CM) Sepharose IEX

matrixhasa capacityof 21 mg g1 matrix forbindingcrudeprotein

(six times less than the CMC beads).25 In contrast, results from

our study show that with magnetic nanoparticles, the maximum

adsorption of the crude extract protein is three times higher than

that of CMC beads and twenty times higher than that of CM

Sepharose matrix. The size and specific surface structure of the

nanoparticles played a major role in their efficient binding with

the crude extract; hence, this makes the use of microemulsion

magnetic nanoparticles attractive for the purification of coagulant

protein.

CONCLUSIONSA novel approach to purify Moringa oleifera coagulant protein

using magnetic nanoparticles is reported. The method is based

on magnetic nanoparticles prepared from microemulsions (ME-

MIONs) that are able to capture proteins in a simple and

efficientway andwhich couldpromote large-scaleproduction.The

Moringa oleifera coagulant protein (MOCP) is captured onto the

magneticnanoparticles supposedly via an ionicchargeinteraction

mechanism. In order to study interactionof theME-MIONs/protein,

FT-IRanalysiswasusedtoinvestigatetheattachmentoftheprotein

onto the ME-MIONs. Consumption of the OH groups of iron oxide

(peak at 3660 cm1) was observedwhen theproteinwas added to

the particles. This indicates that the protein is indeed attached to

the surface of the oxide. In contrast to the conventional method,

purification of MOCP with ME-MIONs suggests a simple and cost

effective method for purifying coagulant proteins. The results

from kinetic studies show that the optimal adsorption capacity

of the protein on ME-MIONs is approximately three times higher

than that of commercial beads. It is proposed that this method be

extendedto thepurificationof proteinswith similarcharacteristics,

and possibly other biomolecules.

ACKNOWLEDGEMENTSThe authorswish to thank theSwedishResearch CouncilFormas for

financing this project. We would also like to extend our gratitude

to Dr Gustav Sundqvist, School of Biotechnology,KTH forthe massspectrometric analysis.

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J Chem Technol Biotechnol 2011; 86: 13861393 c 2011 Society of Chemical Industry wileyonlinelibrary com/jctb

j chem technol biotechnol 2011; 86- 1386–1393 óxido de ferro na extração de proteinas de...

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