Superparamagnetic Polymer Emulsion Particles from a Soap-Free Seeded Emulsion Polymerization and their Application for Lipase Immobilization

Superparamagnetic Polymer Emulsion Particlesfrom a Soap-Free Seeded Emulsion Polymerizationand their Application for Lipase Immobilization

Yanjun Cui & Xia Chen & Yanfeng Li & Xiao Liu &

Lin Lei & Yakui Zhang & Jiayu Qian

Received: 12 July 2013 /Accepted: 27 September 2013 /Published online: 10 October 2013# Springer Science+Business Media New York 2013

Abstract Using emulsion copolymer of styrene (St), glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) as seed latexes, the superparamagnetic polymer emulsionparticles were prepared by seeded emulsion copolymerization of butyl methacrylate (BMA),vinyl acetate (VAc) and ethylene glycol dimethacrylate in the presence of the seed latexes andsuperparamagnetic Fe3O4/SiOx nanoparticles (or Fe3O4-APTS nanoparticles) through a two-step process, without addition of any emulsifier. The magnetic emulsion particles named P(St-GMA-HEMA)/P(BMA-VAc) were characterized by transmission electron microscope andvibrating sample magnetometry. The results showed that the magnetic emulsion particles helda structure with a thinner shell (around 100 nm) and a bigger cavity (around 200 nm), andpossessed a certain level of magnetic response. The resulting magnetic emulsion particles wereemployed in the immobilization of lipase by two strategies to immobilized lipase onto theresulting magnetic composites directly (S-1) or using glutaraldehyde as a coupling agent (S-2),thus, experimental data showed that the thermal stability and reusability of immobilized lipasebased on S-2 were higher than that of S-1.

Keywords Polymer Emulsion Particles . Soap-Free Emulsion . MagneticMaterials .

Polymerization . Lipase Immobilization . Enzymatic Activity

Introduction

Nowadays, polymeric biomaterials are widely used in biochemistry, colloid science, andmedicine [1]. Particularly, nanosized superparamagnetic colloids with functional groupshave attracted many researchers for their extensive application, such as enzyme immobili-zation and separation and purification of biomolecules or cells from complex mixtures [2–5].

Appl Biochem Biotechnol (2014) 172:701–712DOI 10.1007/s12010-013-0563-x

Y. Cui : X. Chen :Y. Li (*) :X. Liu : L. Lei :Y. Zhang : J. QianState Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering,Lanzhou University, Lanzhou 730000, Chinae-mail: [email protected]

Y. CuiLanzhou Petrochemical Research Center of PetroChina, Lanzhou 730060, China

Superparamagnetic polymeric particles can be prepared via monomer polymerization in-cluding dispersion polymerization [6, 7], suspension polymerization [8, 9], conventionalemulsion polymerization [10, 11], seeded emulsion polymerization [12], etc. Among thesemethods, seeded emulsion polymerization has some obvious advantages over other poly-merization process, such as higher molecular weight and rate of polymerization, environ-mentally friendly [13], and controlled morphology [14]. However, until now, most latex wasprepared by traditional seeded emulsion polymerization in which emulsifier could poten-tially confer adverse effects on their biochemical applications.

Lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) is an important enzyme in biologicalsystems, where it catalyzes the hydrolysis of triacylglycerol to glycerol and fatty acids[15–18]. Their broad synthetic potential is largely due to the fact that lipases, in contrast tomost other enzymes, accept a wide range of substrates other than triglycerides such asaliphatic, alicyclic, bicyclic, and aromatic esters and even esters based on organometallicsandwich compounds, are quite stable in nonaqueous organic solvents, and thus, dependingon the solvent system used, can be applied to hydrolysis reactions or ester synthesis [19].Extensive research proved that lipases are very effective biocatalysts for the synthesis ofoptically pure compounds [15–17]. Immobilization of enzyme has been known to makeenzymes more suitable in reactions in organic solvents, operate at relatively higher temper-ature, as well as be easily separated from the reaction mixture and thus easy in retainingenzymes in bioreactors, enabling continuous operation of enzymatic processes [20, 21].

In this work, a facile approach to synthesis emulsifier-free superparamagnetic colloidswith narrow-size distributions was developed, as illustrated in Scheme 1. Firstly, P(St-co-GMA-co-HEMA) emulsion was synthesized by soap-free emulsion polymerization. Sec-ondly, Fe3O4/SiOx nanoparticles or Fe3O4-APTS nanoparticles were added into the emul-sion to prepare magnetic P(St-co-GMA-co-HEMA) latexes which were used as seeds toprepare unique superparamagnetic core-shell P(St-co-GMA-co-HEMA)/P(BMA-co-VAc)composite particles through seeded emulsion copolymerization without any emulsifier.Then, the resulting composites were used for lipase immobilization by two strategies (namedas S-1 and S-2) to immobilized lipase onto magnetic nanoparticles directly (S-1) or usingglutaraldehyde as a coupling agent (S-2).

Experimental

Materials

Glycidyl methacrylate (GMA) were obtained from Ciba Specialty Chemicals (China) Ltd.Guangzhou; Ethylene glycol dimethacrylate (EGDMA) were purchased from ALADDINReagent company (China); 2-hydroxyethyl methacrylate (HEMA), butyl methacrylate(BMA), styrene (St), and vinyl acetate (VAc) were obtained from Tianjin Chemical ReagentCompany (China). All the above reagents were purified by distillation under reduced pressureprior to use. Tetraethyl orthosilicate was purchased from SinopharmChemical Reagent Co., Ltd(China). γ-aminopropyltriethoxysilane (APTS) was purchased from Wuhan University Sili-cone NewMaterial Co., Ltd. (China). Lipase (from Candida rugosa, Type VII, 1,180 units/mgsolid) and BSAwere purchased from Sigma Chemical Co.; potassium persulphate (KPS) andferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), andother chemicals and solvents were used without further purification, obtained from TianjinChemical Reagent Company (China).

702 Appl Biochem Biotechnol (2014) 172:701–712

Preparation of Superparamagnetic Composites Based on Polymer Colloids

Preparation of Superparamagnetic Fe3O4/SiOx Nanoparticles and APTS-Modified Fe3O4

Nanoparticles

Magnetic nanoparticles Fe3O4 were prepared by chemical co-precipitating Fe2+ (FeCl2·4H2O)and Fe3+ (FeCl3·6H2O) ions in ammonia solution, as published previously [22]. To introducehighly reactive silanols on the surface of Fe3O4, Fe3O4/SiOx nanoparticles were prepared [23].APTS-modified magnetite nanoparticles were achieved by a reaction between APTS andhydroxyl groups on the surface of silica-coated Fe3O4 nanoparticles [23].

Preparation of Seed Latexes

The polymeric emulsions were prepared by soap-free emulsion polymerization carried out ina three-necked flask. A typical polymerization recipe is 62 g of H2O, 5 ml of St, 4.6 ml ofGMA, 0.5 ml of HEMA, 0.31 g of KPS, and 0.20 g of NH4HCO3 by batch emulsionpolymerization at 80 °C for 6 h. After that, 10 ml of the original polymeric emulsions wastransferred to another flask, and 0.2 g of Fe3O4/SiOx nanoparticles or 0.2 g APTS-modified

Scheme 1 Preparation approach of the superparamagnetic emulsion particles and their application for lipaseimmobilization

Appl Biochem Biotechnol (2014) 172:701–712 703

Fe3O4 nanoparticles were then added to the latex. After stirring at ambient temperature for120 min, two kinds of black seed latexes colloids were obtained, named as Fe3O4/SiOx seedand Fe3O4-APTS seed, respectively.

Preparation of Superparamagnetic Composite Polymer Colloids

Five milliliters of the two black seed latexes were diluted with 5 ml of H2O and werecharged into a flask. Two thirds of the KPS solution was introduced when the temperaturereached 80 °C, and without the addition of any emulsifier, the polymerization was conductedby adding into the system monomer mixture which contained 2.5 ml of BMA, 2.5 ml ofVAc, and 0.5 ml of EGDMA. The remaining KPS was introduced after 1.5 h. Thepolymerization was continued for an additional 3 h after completing the dropping ofmonomers. The resultant products were obtained by magnetic separation with permanentmagnet and thoroughly washed with ethanol and deionized water until neutral then was driedat room temperature under vacuum for 24 h.

Characterization of Superparamagnetic Polymer Colloids

The structure analyzed by IR spectra was recorded by a Fourier transform infrared spectro-photometer (FT-IR, Nicolet NEXUS 670, USA), and the sample and KBr were pressed toform a tablet. Crystal line properties of the magnetic nanoparticles were examined by the x-ray diffraction (XRD) (Rigaku D/MAX-2400 x-ray diffractometer with Ni-filtered Cu Kαradiation). The morphology of the latex nanoparticles was observed by a transmissionelectron microscopy (TEM, Hitachi H-7000 FA); the magnetization curves of samples weremeasured with a vibrating sample magnetometer (VSM, LDJ-9600, America LDJ Company)at room temperature.

Optimum Conditions of Enzymatic Activity

The effect of immobilizing time and lipase amount added on lipase immobilization weredetermined as the relative activity after incubation under a variety of the amount of lipaseadded (50 mg/g carrier–250 mg/g support).

Lipase Immobilization

The obtained superparamagnetic composite was used for immobilizing lipase. Two im-mobilization strategies (named S-1 and S-2) were adopted to immobilize lipase directlyonto the modified magnetic composites (S-1) or using glutaraldehyde as a coupling agent(S-2) [24]. For S-1, the resulting superparamagnetic nanoparticle was directly used forlipase immobilization without any activation. But for S-2, glutaraldehyde was added to theprecipitates of superparamagnetic nanoparticles before they were dispersed into a phos-phate buffer (0.1 M) under a gentle magnetic stirring for 4 h. Subsequently, the resultantnanoparticles were washed several times with deionized water by magnetic decantationbefore they were added into a phosphate buffer (0.1 M) containing lipase. Finally, whenthe two carriers were separately mixed with lipase, they were shaken for 5 h at roomtemperature to obtain immobilized lipase. The unbounded lipase was removed with amagnetization and the precipitates were washed carefully with phosphate buffer (0.1M, pH 7.0)for several times.


Determination of Lipase Activity and Immobilization Efficiency

The enzymatic activities of free and immobilized lipase were measured by the titration of thefatty acid which comes from the hydrolysis of olive oil [25]. One unit (U) of lipase activity isdefined as the amount of enzyme that hydrolyzes olive oil liberating 1.0-μmol fatty acid perminute under the assay conditions.

The activity yield remaining after immobilization was defined as follows:

Activityyield %ð Þ ¼ C=A� 100

Where A is the total activity of enzyme added in the initial immobilization solution, C isthe activity of the immobilized Candida rugosa lipase (CRL), respectively. The relativeactivity (percentage) was the ratio between the activity of every sample and the maximumactivity of sample. All experiments of activity measurement were carried out at least threetimes and the experimental error was less than 3 %.

The amount of protein in the lipase solution and in supernatant after immobilization wasdetermined by the Bradford method [26] and the amount of protein (p) bound on thesupports was calculated from the formula:

p ¼ Ci −C f

� �V

� �=W

Where p is the amount of bound lipase onto supports (milligrams per gram), Ci and Cf arethe concentrations of the lipase protein initial and final in the reaction medium (milligrams permilliliter), V is the volume of the reaction medium (milliliters) and W is the weight of thesupports (grams). All data used in this formula are the average of triplicate of experiments.

Thermal Stability and Reusability of Immobilized Lipase

Thermal stabilities of the free and immobilized lipase by the two strategies were studied bymeasuring the residual activities of the enzymes after incubation in phosphate buffer (0.1M, pH7.0) at 50 °C for 240 min with continuous shaking. A sample was removed after a 30-min timeinterval and assayed for enzymatic activity.

In addition, the reusability of the immobilized CRL was determined by hydrolysis ofolive oil by the recovered immobilized CRL with magnetic separation and compared withthe first running (activity defined as 100 %).

Kinetic Studies

Kinetics of the hydrolytic activity of free and lipase immobilized by two strategies were investi-gated using various initial concentrations (0.4–2.0 mg/ml) of olive oil as the substrate. The kineticconstants Vmax andKMwere calculated according to the equation and the Lineweaver–Burk plots.

Results and Discussion

Preparation and Characterization of Magnetic Nanoparticles

Scheme 1 shows the preparation routes of the magnetic emulsion particles. The magneticproperties of the composite particles were analyzed by VSM at room temperature. Saturation


magnetization Ms of magnetic nanoparticle was determined from the hysteresis loops aftersubtraction of paramagnetic component using linear interpolation, as shown in Fig. 1. Thesaturation magnetization of magnetic composites was found to be 1.82 emu/g (a, usingFe3O4/SiOx seed) and 4.21 emu/g (b, using Fe3O4-APTS seed). From the figure, we canconclude that magnetic composite using Fe3O4-APTS seed has better magnetic properties.With this saturation magnetization, the particles possessed a certain level of magneticresponse. In addition, there was no hysteresis in the magnetization with both remanenceand coercivity being zero, suggesting that these magnetic composites are superparamagnetic.With the large saturation magnetization, the magnetic composites can be separated from thereaction medium rapidly and easily in a magnetic field.

Figure 2a–d are the TEM images of (a) seed latex (b) Fe3O4/SiOx-seed latex (c) Fe3O4-APTS seed latex, and (d) magnetic emulsion composites using Fe3O4-APTS seed. The averagesize of the seed particles are around 200 nm with a narrow-size distribution. The addition ofmagnetic nanoparticles didn't change the morphology and size of the polymeric colloids.Figure 2b, c indicates that Fe3O4/SiOx and Fe3O4-APTS Fe3O4 nanoparticles were preferen-tially labeled onto the colloids. The amount of magnetic nanoparticles of 2c is increasingdramatically than that of 2b, which is in agreement with VSM results. Further, the magneticnanoparticles are less aggregation and dispersed evenly of 2b than that of 2c, owing to thecovalent binding of amino groups in APTS with epoxy group in seed latex. Because of thelarger saturation magnetization and higher magnetic nanoparticles content, 2c was chosen asseed for further studies.

A seeded swell emulsion polymerization was carried out against the seed colloids(Fig. 2a) using BMA and VAc as monomers and EGDMA as a cross-linking agent forsubmicrometer-sized particles. Figure 2d shows the structure of the composite spheres with athinner shell (around 100 nm) and bigger cavity (around 200 nm). Comparing Fig. 2d with2a, the diameter of the composite particles increased to about 300 nm after seeded emulsionpolymerization due to the coating of the crosslinked P(BMA-co-VAC) particles.

XRD was used to determine the crystal structure of the Fe3O4/SiOx-APTS support. TheXRD pattern is shown in Fig. 3. It is apparent that the diffraction pattern of our Fe3O4-APTSis close to the standard pattern for crystalline magnetite [27].

Fig. 1 Hysteresis loop of magnetic composites (a using Fe3O4/SiOx seed) and (b using Fe3O4-APTS seed)


The infrared (IR) spectra of the magnetic nanoparticles were used to ensure the success ofmodification, which is shown in Fig. 4a seed latex and 4b magnetic emulsion composites. Theabsorption bands of the stretching vibration of the epoxy groups from GMA should be at 1,251;906; and 845 cm−1 in Fig. 4a. But these peaks did not appear in 4b, which provided indirectproof of chemical reaction between amino groups in APTS and epoxy group in GMA. It showsmagnetic emulsion composites have the characteristic bands of Fe3O4 between around580 cm−1, which corresponds to the Fe–O bond of Fe3O4. From the IR spectra 4a and 4b, wecan see the absorption peaks of C–H stretching vibration at 2,800 cm−1, the absorption peaks ofC–O vibration at 1,100 cm−1 and stretching vibrations of C=O from GMA, BMA, and HEMA.A strong absorption peak around 3,400 cm−1 means the existence of hydroxy groups. All ofthese revealed that polymerization is successfully carried out.

Immobilization of Lipase

The effect of the lipase amount added on relative activity of the immobilized lipase is shownin Fig. 5. The relationship between relative activity and lipase amount added of S-1 and S-2obeys nearly the same rule. The amount of the lipase loading increased greatly with theinitial lipase amount and the relative activity reached a maximum value at an initial lipaseamount of 150 mg/g support of the two supports.

Fig. 2 TEM image of a seed latex, b Fe3O4/SiOx-seed latex, c Fe3O4-APTS seed latex, and d magneticemulsion particles

Fig. 3 XRD patterns of composite nanoparticles


Lipase was immobilized onto the support at the optimal conditions. Although the boundprotein of S-2 is more than that of S-1, the activity yield of S-1 is not that much. Thisobservation can be explained as one reason. For S-2, lipase was immobilized by multipointinteraction which not only increases the protein amount but also increases the steric hindrancepreventing the reorganization of the enzyme molecule to the proper conformation for thebinding to substrate.

Thermal Stability and Reusability

Figures 6 and 7 are comparisons of temperature effect and thermal stability of S1 and S2 inphosphate buffer (0.1 M, pH 7.0). The results of two immobilization strategies exhibited a

Fig. 4 FT-IR spectra of a seed latex; b magnetic emulsion composites

Fig. 5 Effect of lipase amount added on the relative activity of immobilized lipase (S-1 non-activated carrierand S-2 activated carrier). The error bar indicates standard deviations (n=3)


similar trend. From Fig. 6, it was observed that the S1 lost its 80 % activity at 80 °C. However,the immobilized lipase of S-2 retained its initial activity of nearly 40 % at 80 °C. Theimmobilized lipase of S-1 retained their initial activity of less than 60 % after 240 min andthat of S-2 retain about 60 %, as shown in Fig. 7. This could be explained by the formation ofcovalent bonds especially multipoint interaction between the enzyme and the carriers enhancingthe enzyme rigidity, protected it from unfolding and prevented the conformation transition ofthe enzyme at high temperature [28].

The reusability of immobilized lipase is very important for their application, especially inindustrial applications. To investigate the reusability, the immobilized lipase was washed withphosphate buffer (0.1 M, pH 7.0) after one catalysis run and reintroduced into a fresh olive oilsolution for another hydrolysis at 37 °C. Figure 8 shows the variation of activity of the lipaseimmobilizing by both strategies after multiple reusing by magnetic separation. It was observed

Fig. 6 Effect of temperature on the activity of immobilized CRL (S-1 non-activated carrier and S-2 activatedcarrier). The error bar indicates standard deviations (n=3)

Fig. 7 The thermal stability of the immobilized CRL (S-1 non-activated carrier and S-2 activated carrier). Theerror bar indicates standard deviations (n=3)


that the immobilized lipase of S-1 still retained 70% of its initial activity after the 10 reuses andthat of S-2 was nearly 80 %. This result confirmed that both strategies have a good durabilityand magnetic recovery. The decrease of activity was caused by the denaturation of the proteinand the leakage of protein from the carriers upon use.

Kinetic Studies

The KM value for the free lipase (0.80±0.02 mg/ml) was found to be higher than that of theimmobilized lipase (0.69±0.01 for S2 and 0.44±0.01 for S1), while the Vmax value for theformer preparation (19.05±0.49 U/mg protein) was found to be lower than that for the latterpreparation (43.48±0.68 U/mg protein for S2 and 29.41±0.67 U/mg protein for S1). The

Fig. 8 The reusability of the immobilized CRL (S-1 non-activated carrier and S-2 activated carrier). Theerror bar indicates standard deviations (n=3)

Fig. 9 The kinetic studies of the free and immobilized CRL (S-1 non-activated carrier and S-2 activated carrier)


decrease in KM of immobilized CRL indicated that the free enzyme has a better affinity for itssubstrate than that of immobilized enzyme, and S1 shows a better affinity for lipase than that ofS2 (Fig. 9).

Conclusions

Superparamagnetic P(St-co-GMA-co-HEMA)/P(BMA-co-VAc) composite colloids were suc-cessfully synthesized by soap-free seeded emulsion copolymerization. The TEM images showedthat the composite spheres had a thinner shell and bigger core structures. The VSM curvesshowed that the colloids were superparamagnetic with a saturation magnetization of 4.21 emu/g.The obvious advantages of these latexes were that the whole synthetic process involved noemulsifier and they possessed a certain level of magnetic response. These colloids were appliedfor lipase immobilization by two strategies (S-1 and S-2). Experimental data showed that thethermal stability and reusability of S-2 were better than that of S-1, but S1 shows a better affinityfor lipase than that of S2. Such approach is ideal for biochemical applications.

Acknowledgments The authors thank the financial supports from the National Natural Science Foundationof China (No.21374045, No.21074049), the National Natural Science Foundation for the scientific researchability training of undergraduate students majoring in chemistry by the two patterns based on the tutorialsystem and top students (J1103307) and the Opening Foundation of State Key Laboratory of Applied OrganicChemistry (SKLAOC-2009-35).

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Superparamagnetic Polymer Emulsion Particles from a Soap-Free Seeded Emulsion Polymerization and their Application for Lipase Immobilization