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Journal of Magnetism and Magnetic Materials 320 (2008) 2350– 2355

Contents lists available at ScienceDirect

Journal of Magnetism and Magnetic Materials

0304-88

doi:10.1

� Corr

E-m

journal homepage: www.elsevier.com/locate/jmmm

Preparation and application of polymer-grafted magnetic nanoparticles forlipase immobilization

Yang Yong a, Yongxiao Bai b, Yanfeng Li a,�, Lei Lin a, Yanjun Cui a, Chungu Xia c

a State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering & Environmental Technology,

Lanzhou University, Lanzhou 730000, Chinab Institute of Materials Science and Engineering, Lanzhou University, Lanzhou 730000, Chinac State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China

a r t i c l e i n f o

Article history:

Received 22 January 2008

Received in revised form

11 April 2008Available online 2 May 2008

Keywords:

Fe3O4 nanoparticle

Polymerization

Functionalized

Immobilized lipase

53/$ - see front matter & 2008 Elsevier B.V. A

016/j.jmmm.2008.04.158

esponding author. Tel.: +86 9318912528; fax

ail address: liyf@lzu.edu.cn (Y.F. Li).

a b s t r a c t

Functionalized superparamagnetic particles were prepared by graft polymerization of glycidyl

methacrylate and methacryloxyethyl trimethyl ammonium chloride onto the surface of modified-

Fe3O4 nanoparticles. The resultant particles were characterized by X-ray powder diffraction,

transmission electron microscopy, Fourier transform infrared spectroscopy, and vibrating sample

magnetometry. The results indicate that the polymer chains had been effectively grafted onto the

surface of Fe3O4 nanoparticles. The functionalized particles remained dispersive and superparamag-

netic. Lipase was immobilized on the magnetic particles under mild conditions by electrostatic

adsorption and covalent binding with the activity recovery up to 70.4%. The immobilized lipase had

better thermal stability compared to free lipase.

& 2008 Elsevier B.V. All rights reserved.

1. Introduction

In recent years, superparamagnetic nanoparticles of ironoxides have shown great potential applications in many biologicalfields, including bioseparation [1,2], tumor hyperthermia [3],magnetic resonance imaging (MRI) diagnostic contrast agents[4], magnetically guided site-specific drug delivery agents [5], andbiomolecules immobilization [6,7]. The application for biomole-cules immobilization mainly based on the solid-phase magneticfeature which is able to achieve a rapidly easy separation andrecovery from the reaction medium in an external magnetic field.In order to increase the loading amount of the biomolecules onthe magnetic particles and improve the stability of immobilizedbiomolecules, the preparation of surface functionalized magneticparticles with water soluble, biocompatible and reactive groups ismuch desired. Several magnetic particles have been functiona-lized by reacting with organic micromolecules on the surface [8],or incorporating magnetic nanoparticles during the synthesis ofthe supporting polymers by suspension polymerization or disper-sion polymerization [9,10]. Now polymers grafting of magneticnanoparticles is now one of the most attractive methods to realizesuch functionalities, as the polymer chains offer flexibility anddiversity to control the chemical composition and functionalgroups on the surface of nanoparticles [11].

ll rights reserved.

: +86 9318912113.

Enzymes have been demonstrated to be efficient biocata-lysts in many biomass conversion processes, and are becominga viable alternative for many applications [12]. The enzymaticconversions usually operate under mild conditions and pro-duce less by-product due to the biocatalysts’ high specificity.However, the industrial applications of biocatalysts have notyet reached a significant level for the high costs of the enzymesand the inability in their separation, recycling, and reusing.These deficiencies have attracted researchers to improve theirfunctionality, stabilize catalytic properties and reusability byusing different immobilization matrices (i.e., membranes orbeads) or by various immobilization methods, such as covalentbinding [13], entrapment [14], or adsorption [15]. It has beendescribed that combining with two methods would greatlyincrease the enzyme loading amount and improve the stabilityof the enzyme [16].

In this paper, magnetic Fe3O4 nanoparticles were pre-pared by the chemical co-precipitation of Fe3+ and Fe2+

ions. Then, the nanoparticles were modified directly by vinyl-triethoxysilicane (VTES) to introduce reactive groups onto theparticles surface, and glycidyl methacrylate (GMA) and metha-cryloxyethyl trimethyl ammonium chloride (MATAC) were graftedonto modified nanoparticles by surface-initiated radical polymer-ization. The functionalized magnetic particles were used forimmobilizing lipase by electrostatic adsorption and covalentbinding. The properties of the immobilized lipase, suchas activity recovery, thermal stability and reusability wereinvestigated.

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2. Experimental

2.1. Materials

GMA and MATAC were obtained from Ciba Specialty Chemicals(China) Ltd. Guangzhou; VTES was purchased from WuhanUniversity Silicone New Material Co. Ltd. (China); Lipase (fromCandida rugosa, Type VII, 1180 units/mg solid) was purchased fromSigma Chemical Co.; Azobisisobutyronitrile (AIBN), ferric chloridehexahydrate (FeCl3 �6H2O), ferrous chloride tetrahydrate(FeCl2 �4H2O), ammonium hydroxide (25 wt%) and other chemi-cals were of analytical grade, obtained from Tianjing ChemicalReagent Company (China).

2.2. Synthesis of Fe3O4 nanoparticles

The preparation of Fe3O4 nanoparticles was followed by achemical co-precipitation of Fe2+ and Fe3+ ions described previously[17]. With some modifications, fifty milliliters of 1.0 M FeCl2 and1.75 M FeCl3 solutions were prepared with deionized water in twobeakers, and then transferred to a 250 ml three-necked flasktogether, stirred under nitrogen. When the solution was heated to60 1C, NH3 �H2O (25 wt%) was added dropwise until pH ¼ 10–11.After base was added, the solution immediately became darkbrown, which indicates iron oxide has been formed in the system.The solution was heated at 80 1C for 1 h. The precipitates wereisolated from the solvent by magnetic decantation and repeatedlywashed with deionized water until neutral, then were dried atroom temperature under vacuum for 12 h.

2.3. Preparation of VTES-modified Fe3O4 nanoparticles

VTES-modified magnetite nanoparticles were achieved by thereaction between VTES and the hydroxyl groups on the surface ofmagnetite. Typically, 1.0 g of Fe3O4 nanoparticles were dispersedin 60 ml of ethanol by sonication for about 1 h, then 12 ml ofNH3 �H2O was added and sonicated to homogenize for 10 min.Under continuous mechanical stirring, 6.0 ml of VTES was addedto the reaction mixture. The reaction was allowed to proceed at50 1C for 8 h under continuous stirring. After that, the resultantproducts were obtained by magnetic separation with permanentmagnet and were thoroughly washed with ethanol and deionizedwater until neutral, then were dried at room temperature undervacuum for 12 h.

2.4. Synthesis of poly(GMA-MATAC)-grafted Fe3O4 nanoparticles

The graft polymerization was conducted under various reactionconditions. In a typical protocol, 0.5 g of VTES-modified Fe3O4

nanoparticles, 0.069 g of AIBN, 44 ml of ethanol and 10 ml ofdeionized water were put in a flask and vibrated with ultrasonic for30 min under nitrogen to be dispersed uniformly. Then the flaskwas placed in a water bath at 70 1C, mechanically stirred at 400 rpmunder nitrogen, and a mixture of GMA (4 ml, 0.03 mol), MATAC(5.6 ml, 0.03 mol), ethanol (7 ml) and deionized water (7 ml) wasadded dropwise into the flask in 1 h. The graft polymerization wasundergone at 70 1C for 6 h. After that, the products were collectedby magnetic separation and washed with ethanol and distilledwater several times, then were extracted in ethanol for 48 h, anddried at room temperature under vacuum for 12 h.

2.5. Immobilization of lipase

Due to the epoxy groups and positive electrical charges on thesurface of poly(GMA-MATAC)-grafted Fe3O4 nanoparticles, lipase

immobilization was carried out by mixing the enzyme solutionwith the particles directly. The particles (1.0 g) were added into40 ml of phosphate buffer (0.1 M, pH 6.5) containing lipase (0.2 g).The mixture was placed in a shaking incubator at 30 1C and150 rpm, and continuously shaken for 6 h to finish the immobi-lization of lipase. The immobilized lipase was recovered bymagnetic separation, and washed with phosphate buffer (0.1 M,pH 6.5) three times to remove unbound lipase. The receivedimmobilized lipase was held at 4 1C prior to use.

The enzymatic activities of free and immobilized lipase weremeasured by titrating the fatty acid, which comes from thehydrolysis of the olive oil [18]. One unit of lipase activity (U) isdefined as the amount of enzyme that hydrolyzes olive oilliberating 1.0 mmol fatty acid per minute under the assayconditions.

The activity recovery (%) was the ratio between the activity ofimmobilized lipase and the activity of free lipase.

2.6. Thermal stability and reusability

Thermal stabilities of the free and immobilized lipase werestudied by measuring the relative activities of the lipase afterincubation in phosphate buffer (0.1 M, pH 7.0) for 30 min in therange 25–80 1C with continuous shaking.

In addition, reusability of the immobilized lipase was deter-mined by hydrolysis of olive oil by recovered immobilized lipasewith magnetic separation and compared with that in the first run(activity defined as 100%).

The relative activity (%) was the ratio between the activity ofevery sample and the maximum activity of the sample (activitydefined as 100%).

2.7. Characterization

Power X-ray diffraction (XRD, Rigaku D/MAX-2400 X-raydiffractometer with Ni-filtered Cu Ka radiation) was used toinvestigate the crystal structure of the magnetic nanoparticles.The size and shape of the nanoparticles were determined bytransmission electron microscope (TEM, Hitachi H-600, Japan),the sample was dispersed in ethanol and spread a small drop ontoa 400 mesh copper grid. The IR spectra were recorded by a Fouriertransform infrared spectrophotometer (FT-IR, Nicolet NEXUS 670,USA), and the sample and KBr were pressed to form a tablet. Themagnetization curves of samples were measured with a vibratingsample magnetometry (VSM, LAKESHORE-7304, USA) at roomtemperature.

3. Results and discussion

3.1. Preparation of poly(GMA-MATAC)-grafted Fe3O4 nanoparticles

The processes for synthesis of poly(GMA-MATAC)-graftedFe3O4 nanoparticles and the immobilization of lipase onto themare shown in Scheme 1. The Fe3O4 nanoparticles were synthesizedby a chemical co-precipitation of Fe2+ and Fe3+ ions under alkalinecondition. The concentration ratio of Fe2+/Fe3+ was selected to be1:1.75 rather than the stoichiometric ratio of 1:2, because Fe2+ isprone to be oxidized and become Fe3+ in solution. The Fe3O4

nanoparticles prepared by the co-precipitation method have anumber of hydroxyl groups on the surface from contacting withthe aqueous phase. VTES-modified Fe3O4 nanoparticles wereachieved by the reaction between VTES and the hydroxyl groupson the surface of magnetite. Two reactions were involved in theprocess. First, the VTES was hydrolyzed to be highly reactive

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Scheme 1. Surface modification and graft polymerization of magnetic nanoparticles, and immobilization of lipase.

Fig. 1. XRD patterns of (a) pure Fe3O4 nanoparticles, (b) poly(GMA-MATAC)-

grafted Fe3O4 nanoparticles.

Y. Yong et al. / Journal of Magnetism and Magnetic Materials 320 (2008) 2350–23552352

silanols species in the solution phase under alkaline condition.Then, their condensation with surface free -OH groups ofmagnetite to render stable Fe–O–Si bonds takes place. Oligomer-ization of the silanols in solution also occurs as a competingreaction with their covalent binding to the surface. Surface-grafted polymerization by GMA and MATAC also involves tworeactions, which take place simultaneously. On the surface ofVTES-modified Fe3O4 nanoparticles, the graft polymerizationoccurs, while the random polymerization takes place in thesolution. In order to decrease the random polymerization,the following strategies were adopted. On the one hand, afterAIBN was dissolved in the modified nanoparticles suspendedsolution, the solution was placed overnight to make thenanoparticles absorb AIBN onto the surface furthest. On theother side, an optimal concentration of initiator was selected,and the monomers were added dropwise in the reaction. Theungraft oligomers would be separated by magnetic decantationafter reaction.

3.2. Characterization of Fe3O4 and poly(GMA-MATAC)-grafted Fe3O4

nanoparticles

Fig. 1 shows the XRD patterns of pure Fe3O4 and poly(GMA-MATAC)-grafted Fe3O4 nanoparticles. It is apparent that thediffraction pattern of our Fe3O4 nanoparticles is close tothe standard pattern for crystalline magnetite (Fig. 1(a)). Thecharacteristic diffraction peaks marked, respectively, by theirindices (2 2 0), (311), (4 0 0), (4 2 2), (511), and (4 4 0) could bewell indexed to the inverse cubic spinel structure of Fe3O4 (JCPDScard no. 85-1436), were also observed from poly(GMA-MATAC)-grafted Fe3O4 nanoparticles (Fig. 1(b)). This reveals that modifiedand grafted polymerized, on the surface of Fe3O4 nanoparticles,did not lead to their crystal phase change. The average crystallitesize D was about 15 nm, obtained from Sherrer equation D ¼ Kl/(b cos y), where K is constant, l is X-ray wavelength, and b is thepeak width of half-maximum.

The TEM micrographs of pure Fe3O4 nanoparticles (Fig. 2(a))and Fe3O4 nanoparticles grafted by poly(GMA-MATAC) (Fig. 2(b))are shown. Observing the photograph (a), nanoparticles wereaggregated seriously, which was due to the nano-size of the Fe3O4,and they were about 2075 nm, according to the result of XRD.After graft polymerization, the size of particles was changed to be200–400 nm, and the dispersion of particles was improved greatly(Fig. 2(b)), which can be explained by the electrostatic repulsion

force and steric hindrance between the polymer chains on thesurface of Fe3O4 nanoparticles.

To evaluate the effect of graft polymerization, the homopoly-mers and unreacted monomers were extracted in ethanol to beseparated from the grafted nanoparticles. FT-IR spectroscopy wasused to show the structure of Fe3O4 (Fig. 3(a)), VTES-modifiedFe3O4 (Fig. 3(b)) and poly(GMA-MATAC)-grafted Fe3O4 (Fig. 3(c)).From the IR spectra presented in Fig. 3, the absorption peaks at568 cm�1 belonged to the stretching vibration mode of Fe–Obonds in Fe3O4. Comparing with the IR spectrum (a), the IRspectrum (b) of VTES-modified Fe3O4 possessed absorption peakspresented at 1603 and 1278 cm�1 should be attached to thestretching vibrations of CQC and the bending vibration of Si–Cbonds, peak at 1411 cm�1 due to the bending vibration of QCH2

group, additional peaks centered at 1116, 1041, 962 and 759 cm�1

were most probably due to the symmetric and asymmetricstretching vibration of framework and terminal Si–O– groups.All of these revealed the existence of VTES. It indicated that thereactive groups had been introduced onto the surface ofmagnetite. The reaction mechanism was given in Scheme 1. InFig. 3(c), the absorption peaks of CQC and QCH2 groupsdisappeared, and additional peaks at 1724, 1486, 1447 and1387 cm�1 due to the stretching vibrations of CQO, the bendingvibration of –CH2–, –CH– and –CH3, absorption peaks at 1147, 906and 847 cm�1 belonged to the stretching vibration of the epoxygroups from GMA. However, the identification of peak attributable

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Fig. 2. TEM micrographs of (a) pure Fe3O4 nanoparticles, (b) poly(GMA-MATAC)-

grafted Fe3O4 nanoparticles.

Fig. 3. FT-IR spectra of (a) pure Fe3O4 nanoparticles, (b) Fe3O4 nanoparticles

modified by VTES, (c) poly(GMA-MATAC)-grafted Fe3O4 nanoparticles.

Fig. 4. The magnetic behavior of magnetic nanoparticles.

Y. Yong et al. / Journal of Magnetism and Magnetic Materials 320 (2008) 2350–2355 2353

to the stretching vibrations of C–N (normally at about 1100 cm�1)was problematic due to overlapping other peaks, but the elementanalysis method demonstrated the presence of N element of theMATAC in poly(GMA-MATAC)-grafted Fe3O4 nanoparticles. Over-all, these FT-IR spectra provided supportive evidence that the–CHQCH2 group initiated polymerization of GMA and MATACpolymer chains were successfully grafted onto the Fe3O4 nano-particles surface.

The magnetic properties of the magnetic nanoparticles wereanalyzed by VSM at room temperature. Fig. 4 shows thehysteresis loops of the samples. The saturation magnetiza-tion is found to be 45.5 and 27.3 emu/g for VTES-modifiedFe3O4 and poly(GMA-MATAC)-grafted Fe3O4, respectively, lessthan the pure Fe3O4 nanoparticles (58.9 emu/g). This differ-ence suggests that a large amount of silane and polymers coatedon the surface of Fe3O4 nanoparticles. With the large satura-tion magnetization, the poly(GMA-MATAC)-grafted Fe3O4

could be separated from the reaction medium rapidly andeasily in a magnetic field. In addition, there is no hysteresisin the magnetization with both remanence and coercivitybeing zero, suggesting that these magnetic nanoparticles aresuperparamagnetic. When the external magnetic field isremoved, the magnetic nanoparticles could be well dispersedby gentle shaking. These magnetic properties are criticalin the applications of the biomedical and bioengineeringfields.

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Fig. 6. Reuse of the immobilized lipase for hydrolyzing olive oil.

Y. Yong et al. / Journal of Magnetism and Magnetic Materials 320 (2008) 2350–23552354

3.3. Immobilization of lipase

Poly(GMA-MATAC)-grafted Fe3O4 nanoparticles were preparedand used for immobilizing lipase under mild conditions (Scheme1). Through optimal graft polymerization, GMA and MATAC weregrafted on the surface of VTES modified-Fe3O4 nanoparticles,which induced large amount of positive electrical charges andreactive epoxy groups on the surface. The activity recovery ofthese particles immobilized lipase was assayed by hydrolysis oliveoil, and the maximum activity recovery was up to 70.4%. Theenzyme loading amount on the particles was determined by theBradford method [19]. It was 105.2 mg protein/g particles, betterthan other literatures [20,21], which adopted a single immobiliza-tion method. This was probably explained by the reactionbetween the functional groups on the surface of particles andlipase. When these particles were added to the lipase solutionwith a definite pH value, lipase molecule would be absorbedaround the particles by electrostatic adsorption first, and then beimmobilized onto the particles via a covalent reaction betweenepoxy groups of the particles and the amino groups (or thiol,hydroxyl groups) of the lipase. It has been described that theprevious adsorption of enzyme on the epoxy support is necessaryto achieve a significant covalent immobilization of the enzymedue to the extremely low reactivity of the epoxy supports withsoluble enzyme [16].

3.4. Thermal stability and reusability of immobilized lipase

Thermal stabilities of the free and immobilized lipase weredetermined by using olive oil as substrate at pH 7.0 in thetemperature range 25–80 1C, as shown in Fig. 5. It was found thatthe optimum temperature for the free enzyme was approximately35–40 1C, while it shifted to 35–45 1C for the immobilized enzyme,which is wider than that of the free lipase. The increase inoptimum temperature was caused by changing physical andchemical properties of the enzyme. The covalent bonds formationvia epoxy groups and amino groups reduced the conformationalflexibility and might result in higher activation energy for themolecule to reorganize the proper conformation for binding to thesubstrate [22]. The immobilized lipase was not inactivated attemperature above 40 1C. As the temperature was raising, therelative activity of the free enzyme decreased greater than for theimmobilized lipase. The immobilized lipase held approximately

Fig. 5. Thermal stabilities of the free and the immobilized lipases. Incubated in

phosphate buffer (0.1 M, pH 7.0) for 30 min in the range 25–80 1C.

35% at 80 1C, but the free enzyme remained less than 10%. Thiswas either due to the creation of conformational limitation on theenzyme movement as a result of covalent bonds formationbetween the enzyme and the carriers or a low restriction in thediffusion of the substrate at high temperature. Thus, theimmobilized lipases showed their catalytic activities at a higherreaction temperature.

The reusability of immobilized enzyme is very important fortheir applications, especially in industrial applications. To in-vestigate the reusability, the immobilized lipase was washed withphosphate buffer (0.1 M, pH 7.0) after one catalysis run andreintroduced into a fresh olive oil solution for another hydrolysisat 37 1C. Fig. 6 shows the variation of activity of the immobilizedlipase after multiple reusing by magnetic separation. It wasobserved that the immobilized lipase still retained 70% of itsinitial activity after five reuses. This result confirmed that theimmobilized lipase on magnetic particles has good durability andmagnetic recovery. The decrease in activity was caused by thedenaturation of the protein and the leakage of protein from thecarriers upon use.

4. Conclusions

Functionalized superparamagnetic particles were prepared viatwo steps. First, magnetite (Fe3O4) nanoparticles were modifiedby Vinyltriethoxysilicane, and introduced reactive groups onto thenanoparticles’ surface. Second, GMA and MATAC were graftedonto the surface of modified-Fe3O4 nanoparticles by surface-initiated radical polymerization. The results indicate that thepolymer chains had been effectively grafted onto the surface ofFe3O4 nanoparticles. The functionalized particles remained dis-persive and superparamagnetic. The saturation magnetizationwas found to be 27.3 emu/g. These particles were employed inimmobilizing lipase under mild conditions, and could significantlyimprove the stability of lipase.

Acknowledgments

The authors are grateful to the financial support from the StateKey Laboratory for Oxo Synthesis and Selective Oxidation OpenFoundation (OSSO0602).

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References

[1] A.K. Gupta, M. Gupta, Biomaterials 26 (2005) 3995.[2] J. Krizzova, A. Spanova, B. Rittich, D. Horak, J. Chromatogr. A 1064 (2005) 247.[3] A. Ito, M. Shinkai, H. Honda, T. Kobayashi, J. Biosci. Bioeng. 100 (2005) 1.[4] S. Mornet, S. Vasseur, F. Grasset, G. Goglio, A. Demourgues, J. Portier, E. Pollert,

E. Duguet., Prog. Solid State Chem. 34 (2006) 237.[5] T. Neuberger, B. Schopf, H. Hofmann, M. Hofmann, B. von Rechenberg, J. Magn.

Magn. Mater. 293 (2005) 483.[6] A. del Campo, T. Sen, J.-P. Lellouche, I.J. Bruce, J. Magn. Magn. Mater. 293

(2005) 33.[7] Z.M. Saiyed, S. Sharma, R. Godawat, S.D. Telang, C.N. Ramchand, J. Biotechnol.

131 (2007) 240.[8] A. Dyal, K. Loos, M. Noto, S.W. Chang, C. Spagnoli, K.V.P.M. Shafi, A. Ulman,

M. Cowman, R.A. Gross, J. Am. Chem. Soc. 125 (2003) 1684.[9] T.-H. Chung, H.-C. Pan, W.-C. Lee, J. Magn. Magn. Mater. 311 (2007) 36.

[10] A. Pich, S. Bhattacharya, A. Ghosh, H.-J.P. Adler, Polymer 46 (2005) 4596.

[11] Q.-L. Fan, K.-G. Neoh, E.-T. Kang, B. Shuter, S.-C. Wang, Biomaterials 28 (2007)5426.

[12] P.D. Marıa, J.V. Sinisterra, S.W. Tsai, A.R. Alcantara, Biotechnol. Adv. 24 (2006)493.

[13] J.C. Tiller, R. Rieseler, P. Berlin, D. Klemm, Biomacromolecules 3 (2002) 1021.[14] Y.J. Wang, F. Caruso, Chem. Mater. 17 (2005) 953.[15] S. Koutsopoulos, J. Oost, W. Norde, Langmuir 20 (2004) 6401.[16] B.C.C. Pessela, C. Mateo, A.V. Carrascosa, A. Vian, J.L. Garcıa, G. Rivas,

C. Alfonso, J.M. Guisan, R.F. Lafuente, Biomacromolecules 4 (2003) 107.[17] R. Massart, V. Cabuil, J. Chim. Phys. Phys. Chim. Biol. 84 (1987) 967.[18] N. Watanabe, O. Yasude, M. Yasuji, Y. Koichi, Agric. Biol. Chem. 41 (1977) 1353.[19] M.M. Bradford, Anal. Biochem. 72 (1976) 248.[20] A. Dyal, K. Loos, M. Noto, S.W. Chang, C. Spagnoli, J. Am. Chem. Soc. 125 (2003)

1684.[21] S.S. Kanwar, R.K. Kaushal, A. Aggarwal, S. Chauhan, S.S. Chimni, G.S. Chauhan,

J. Appl. Polym. Sci. 105 (2007) 1437.[22] M.Y. Arıca, G. Bayramoglu, N. Bıc-ak, Process Biochem. 39 (2004) 2007.