A novel approach for efficient immobilization and stabilization of papain on magnetic gold nanocomposites

Colloids and Surfaces B: Biointerfaces 101 (2013) 280–289

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A novel approach for efficient immobilization and stabilization of papain on magnetic gold nanocomposites Banalata Sahoo a , Sumanta Kumar Sahu b , Dipsikha Bhattacharya a , Dibakar Dhara a,∗ , Panchanan Pramanik a,∗ a b

Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India Department of Applied Chemistry, Indian School of Mines, Dhanbad 826004, India

a r t i c l e

i n f o

Article history: Received 9 January 2012 Received in revised form 2 July 2012 Accepted 6 July 2012 Available online xxx Keywords: Enzyme immobilization Magnetic gold nancomposites MPTS, Nanobiocatalyst Papain

a b s t r a c t In the present study, a facile functionalization of magnetic nanoparticles has been described for the immobilization of enzyme that offers many advantages for reuse and excellent efficiencies. The magnetic gold nanocomposites have been fabricated for the successful immobilization of an industrially important enzyme “papain”. For immobilization of papain on magnetic gold nanocomposites, magnetic nanoparticles were modified with 3-(mercaptopropyl) trimethoxy silane (MPTS). Further, the citrate stabilized gold nanoparticles were chemisorbed on these thiol coated magnetic nanoparticles to fabricate the desired magnetic gold nanocomposites. Papain containing net positive charge (isoelectric point of papain = 8.75) in PBS buffer (pH 7.4) has immobilized on the surface of the negatively charged magnetic gold nanocomposites through the ionic or electrostatic interaction. The Michaelis–Menten kinetic constant (Km ) and maximum reaction velocity (Vmax ) for free papain were 0.236 × 105 g ml−1 and 4.08 g ml−1 /s respectively whereas for immobilized papain, Km and Vmax values were 0.308 × 105 g ml−1 and 5.4 g ml−1 /s respectively. The loading amount of papain on magnetic gold nanocomposites was 54 mg/g support and the activity recovery of the immobilized papain reached to 47 (±5)% compared to native papain. The main advantage of this papain nanobiocatalyst is the easy isolation of enzyme from the reaction medium. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The development of cost-effective and efficient strategies for enzyme immobilization is a promising field of biotechnology. Immobilization of enzymes and cells on the fabricated support with retention of their catalytic properties is one of the most important research areas of biological field [1]. To date, several reports have been published on the enzyme immobilization using various solid supports such as silica nanoparticle [2], porous support (sepabeads) [3], polyelectrolyte capsules [4], polymer film [5], and mesoporous molecular sieves [6] etc. with satisfactory enzymatic efficacy. Number of well-developed techniques such as physical adsorption, entrapment, ion exchange, and covalent bonding have been adopted to immobilize enzymes on the solid supports [7]. Immobilization is an important tool to improve enzyme properties. As for example, the stabilization of multimeric enzymes is achieved using cross-linked enzyme aggregates (CLEAs) technology, while multipoint covalent attachment improves the rigidification of the enzyme. Moreover, enzyme immobilized on non-porous

∗ Corresponding author. Tel.: +91 9775664551; fax: +91 3222 255303. E-mail addresses: [email protected] (B. Sahoo), [email protected] (D. Dhara), [email protected] (P. Pramanik). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.07.003

nanoparticles have kinetic advantages compared to other porous supports, because some stabilization effects of the enzyme immobilization on porous solids may be decreased or even fully lost [8]. Over the past two decades, magnetic nanoparticles have been widely used as promising nanomaterials in biomedical fields such as separation of biomolecules [9], magnetic resonance imaging contrast agent [10], drug delivery [11], protein separation [12], and biodetection [13]. Moreover, these functionalized magnetic nanoparticles have also aroused enormous interests as a unique support material for enzyme immobilization and protein binding over the previously used solid carriers [14–16]. The efficacy of MNPs for enzyme immobilization has presumed because of their tailored surface chemistry, high surface area to volume ratio, and special magnetic behavior [16]. For the fabrication of enzyme immobilized magnetic nanoparticles (as solid supports) with improved efficacy, it is necessary to engineer their surface with desired functionalities. A variety of suitable coating materials have already established as surface modifying agents to render functionalized magnetic nanoparticle with higher hydrophilicity and biocompatibility. Among the various coating materials used for surface modification of magnetic nanoparticles, gold nanoparticles have received increasing attention due to their exceptional optical and electronic properties. It is a well-known fact that gold nanoparticles are highly biocompatible materials, providing a facile environment

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for the enzyme binding on their surface [17]. However, the goldenzyme nanocomposites without magnetic nanoparticles have faced severe troubles during its recovery as well as recycling because of their problematic way of isolation. Thus, there is considerable interest in the development of stable, reusable, and highly active magnetic gold nanocomposites for enzyme tagging. The excellent properties of gold nanoparticles help to stabilize the surface of these composite nanoparticles more efficiently in corrosive biological conditions and have readily functionalized through specific biomolecules exploiting the well-developed Au S chemistry [18,19]. Varieties of magnetic gold nanocomposites have proposed for the immobilization of enzymes and antibodies [20–23]. Jeong et al. have reported the enhanced reusability of hexa-arginine-tagged esterase immobilized on gold-coated magnetic nanoparticles [24]. This bifunctional magnetic gold material with superior magnetic properties and potential surface chemistry performs easy binding of enzyme and its recovery. The bifunctional magnetic gold nanomaterial for the immobilization of biomolecule is a great achievement of nanotechnology. Among the various enzymes, we have selected papain as our enzyme of choice, which consists of a single peptide chain containing 211 amino acids. It is one of the most industrial proteases and widely used in food, pharmaceutical, leather, cosmetic, and textile industries [25]. The application of papain includes protein structural studies, peptide mapping, preparation of Fab fragment from IgG, solubilization of integral membrane protein, and production of glycopeptides from purified proteoglycans. It catalyzes the hydrolysis of varieties of peptide, amide, and ester linkages [26]. A number of methods have ascribed to immobilize papain on different supports [27–29]. With the aim to design a simple and efficient method for papain immobilization using magnetic gold nanocomposites, we have generated highly hydrophilic thiol functionalized magnetic nanoparticles using MPTS as a silane-coupling agent. The gold nanoparticles are chemically adsorbed on the MPTS functionalized magnetic nanoparticles through conventional thiol chemistry. Immobilization of papain on magnetic gold nanocomposites is speculated via both electrostatic and multipoint non-covalent interaction. This immobilization protocol is time saving, provides stable immobilization for papain and this bioconjugation protocol forms a new class of nanobiocatalyst. The enzyme specific activity at broad pH ranges, thermal stability, enzyme loading efficiency and kinetic behavior of immobilized papain are compared with free papain in solution. The immobilized papain shows higher catalytic activity with a broader temperature ranges compared to free papain. The papain nanobiocatalyst can be easily isolated from reaction mixture by simple separation with the help of a magnet and demonstrate significant catalytic activity over six times successive reuse.

2. Materials and methods 2.1. Materials FeSO4 ·7H2 O and FeCl3 (anhydrous) were obtained from Merck, Germany. HAuCl4 ·3H2 O, 3-mercaptopropyl trimethoxy silane (MPTS) were obtained from Aldrich Chemicals, USA. Papain (Carica papaya, Enz No-3.4.22.2, activity 3.3 units/mg) was obtained from Sigma–Aldrich. Casein, trisodium citrate dihydrate, ethylenediamine tetra acetic acid (EDTA), l-cysteine hydrochloride, sodium carbonate (Na2 CO3 ), trichloroacetic acid (CCl3 ·COOH), ethanol, NaOH, NH3 solution (28%), and Folin Ciocalteau’s reagents were procured from Merck (Germany). All the chemicals were used without further treatment. The water used in this work was milli-Q ultrapure water.

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2.2. Synthesis and surface modification of magnetic nanoparticles Fe3 O4 magnetic nanoparticles were prepared by simple coprecipitation method as previously reported by our group with little modification [30]. In a 100 ml three-necked flask equipped with a mechanical stirrer, 0.324 g of FeCl3 and 0.278 g of FeSO4 ·7H2 O were taken in 40 ml of deoxygenated millipore water under argon flow. The pH of the reaction mixture was maintained 10.0 by adding 28% ammonia solution drop wisely in a controlled manner and the reaction mixture was heated at 80 ◦ C for 30 min. The particles were separated magnetically and washed five times with milli-Q water followed by acetone and dried under vacuum. For surface modification, 0.1 g of magnetic nanoparticles were dispersed in 80 ml ethanol in an ultrasonicator for 30 min. Then ammonia solution was added to maintain pH 9–10. The reaction mixture was stirred at 30 ◦ C. To the above solution, 100 ␮l MPTS was added dropwise under vigorous stirring condition and the solution was stirred for 6 h at 30 ◦ C. The surface functionalized magnetite nanoparticles were magnetically separated, washed with ethanol and finally dried. 2.3. Preparation of gold nanoparticles Gold nanoparticles were prepared according to the reported procedure with little modification [31]. In brief, a 5 mmol/l solution of hydrogen tetrachloroaurate trihydrate (HAuCl4 ) (Sigma–Aldrich) was dissolved in ultrapure water and brought to a boil with vigorous stirring. To this solution 1 ml of 1% trisodium citrate was added. After addition of trisodium citrate, the heating was turned off. Upon addition of citrate solution, a color change was observed from colorless to purple to ruby red within 10 min. The colloidal solution was stirred for an additional 30 min and then cooled at room temperature. 2.4. Synthesis of magnetic gold nancomposites For the surface modification by gold nanoparticle on magnetite, 20 mg of MPTS coated magnetic nanoparticle was added to the above as synthesized gold nanoparticle and dispersed for 30 min. After dispersion, the solution was stirred for 5 h till gold solution become colorless. The nano-gold coated magnetic nanoparticle were separated magnetically and washed with milli-Q water followed by acetone and dried in air. 2.5. Conjugation of papain on magnetic gold nanocomposites 10 mg of above magnetic gold nanocomposites were treated with 8 mg of papain containing 8 ml of phosphate buffer (0.01 M, pH 7.4) and the resulting solution was stirred for 24 h at 4 ◦ C. After 24 h, enzyme tagged magnetic nanoparticles were washed five to six times with PBS (pH 7.4). The supernatant after immobilization was collected to determine the amount of enzyme bound to the magnetic gold nanocomposites surface. The loss in absorbance at 280 nm in the supernatant was used to quantify the amount of bound enzyme on magnetic gold nanocomposites. The papainnanogold-magnetic conjugate was stored at 4 ◦ C in 0.01 M PBS prior to further experimentation. 2.6. Measurements of biocatalytic activity of immobilized papain The specific activity of free papain and papain– nanobioconjugate was determined by reaction with 2% casein at 37 ◦ C in 0.01 M PBS [25]. Before the catalytic activity studies, the activation of the papain was carried out according to the following procedure. 10 mg of conjugated papain (54 ␮g/mg of particle) in 10 mmol/l phosphate buffer was mixed with 50 mmol/l

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l-cysteine hydrochloride and 3 mmol/l ethylenediamine tetra acetic acid (EDTA) and incubated for 30 min at room temperature. The activated papain (0.5 ml) was added to the test tube containing 0.5 ml of casein (2%) solution. The reaction was carried out at a certain temperature for 15 min and stopped by the addition of 2 ml of 10% trichloroacetic acid (TCA). The liquid solution was separated from the conjugated papain via a permanent magnet, and 1 ml of 0.5 mol/l Na2 CO3 was added to 0.5 ml of such solution and kept for 10 min. After that, 0.1 ml of Folin Ciocalteau’s reagent was added. The tubes were incubated for 30 min in the dark for color development. The color was measured against the reagent blank at 660 nm in a spectrophotometer. The activity was expressed as the amount of enzyme required to release 1 ␮g of tyrosine per minute per milliliter. In the case of native papain, the activity measurement was performed following the above procedures and conditions similar to conjugated papain, except that, 100 ␮l of 1 mg/ml of native papain solution in 10 mM PBS (pH 7.4) was used and magnetic separation was unnecessary.

4. Results and discussion

2.7. Reusability studies of papain magnetic gold nanoconjugate

4.1. Characterization of magnetic gold nanocomposites

In order to determine the reusability behavior of immobilized papain, the immobilized papain nanobiocatalyst was stored at 4 ◦ C in PBS buffer for a period of one month. The biocatalytic activity of immobilized papain was checked as above procedure in six repeated cycles in every five days interval. For every repetitive cycle, the papain biconjugate material was washed with PBS buffer 5–6 times and stored in the same buffer for further use.

The schematic presentation for the immobilization of papain on magnetic gold nanocomposites is shown in Scheme 1. The immobilization of papain on nanocomposites includes following steps: magnetic Fe3 O4 nanoparticles are prepared by the co-precipitation technique from ferrous and ferric solution with 1:2 stoichiometric ratios under alkaline condition. The high density of hydroxyl groups on magnetite surface allows further modification of magnetite by MPTS (a silane coupling agent). Colloidal gold solution is then chemisorbed on MPTS modified magnetic nanoparticles to form a new composite material having the properties of both magnetic and gold nanoparticles. Finally, magnetic gold nanocomposite interacts with enzyme molecules through both electrostatic interaction and multipoint non-covalent interaction. Control experiment was performed to check whether the immobilization occurs on free iron oxide and MPTS modified magnetic nanoparticles. We have observed that the papain immobilization is not taking place on free iron oxide nanoparticles and MPTS modified magnetic nanoparticles. On the other hand, there is successful papain immobilization is observed on magnetic gold nanocomposites. The immobilization efficiency (Ie ) is defined as the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in stock solution. Based on the preface of experiments, 0.8 mg papain is used per mg of magnetic gold nanocomposites so that the enzyme is in excess for incubation. Fig. 1a displays the XRD patterns of Fe3 O4 , MPTS–Fe3 O4 , and magnetic gold nanocomposites. The position and relative intensity of all peaks match well with standard XRD pattern of Fe3 O4 (JCPDS card no-82–1533) indicating that each sample is a Fe3 O4 crystal. The broadening of XRD peaks indicates nanocrystalline nature of each sample. The XRD pattern of the Fe3 O4 and MPTS–Fe3 O4 show peaks at 35.33◦ , 41.55◦ , 50.53◦ , 63.7◦ , 67.64◦ , and 74.36◦ which are marked respectively by their indices (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0). Three additional peaks at 44◦ , 52◦ , and 78◦ , which represent the Bragg reflections from (2 0 0), (2 2 0) and (3 1 1) planes of Au nanoparticle, showing clearly the existence of Au nanoparticles in the magnetic gold nanocomposites. Thermal analysis is performed to confirm the coating formation on the surface of the magnetite. Fig. 1b shows comparative weight loss for uncoated iron oxide and Fe3 O4 –MPTS. In case of free Fe3 O4 , the weight loss around 5% is due to the physically adsorbed water molecules on magnetite surface whereas in case of MPTS–Fe3 O4 two stage weight losses are observed. The weight loss within 100–200 ◦ C is due to physically adsorbed moisture and MPTS whereas the major weight loss above 300 ◦ C attributes to the decomposition followed by removal of chemically bonded MPTS.

2.8. Determination of kinetic parameters for both immobilized and free papain The kinetic parameters of Michaelis–Menten equation (Km and Vmax ) for both free and immobilized papain were determined by measuring the initial rates of the reaction and varying casein concentration from 0.25% to 2.0% at 30 ◦ C in phosphate buffer pH 7.4. The Michaelis–Menten constant (Km ) and maximum reaction velocity (Vmax ) were calculated from Lineweaver–Burk plot. The reciprocal of substrate concentration (1/S) was plotted against the reciprocal of reaction rate (1/V) according to the following equation: 1 = V

 K  m Vmax

×

1 1 + Vmax [S]

where [S] is the concentration of substrate, V and Vmax represented the initial and maximum rate of reactions, respectively. Km is the Michaelis–Menten constant (the substrate concentration when the rate is half of Vmax ). 3. Characterizations The phase formation and crystallographic state of uncoated as well as functionalized magnetic nanoparticles were determined by Phillips PW 1710 X-ray diffractometer (XRD) with ˚ Presence of surface funcNi-filtered Co–K␣ radiation ( = 1.79 A). tional groups was investigated by fourier transform infrared (FTIR) spectroscopy. The samples were prepared in KBr medium in the range 400–4000 cm−1 with a model Thermo Nicolet Nexux FTIR (model 870). The surface composition of MPTS coated nanoparticles were obtained by analyzing X-ray photoelectron spectroscopy (XPS) data using Al K˛ excitation source in an ESCA–2000 Multilab apparatus (VG microtech). Thermal analysis was done with a thermal analyzer (Pyris Diamond TG/DTA) with a heating rate 8 ◦ C/min with in temperature range 50–1000 ◦ C. The size and morphology of the nanoparticles were observed using a Phillips CM 200 transmission electron microscope (TEM) with an acceleration

voltage 200 kV. The nanoparticles were thoroughly dispersed in water by ultra-sonication and placing a drop of solution on the carbon coated copper grid. The hydrodynamic size of the particle was measured by dynamic light scattering (DLS) technique, using a Brookhaven 90 Plus particle size analyzer. The laser light of wavelength ( = 660 nm) was scattered with an angle  = 90◦ at 27 ◦ C placing the dispersion in a polystyrene cuvette. Magnetic measurements were performed using vibration sample magnetometry (VSM) analysis. Zeta potential was measured by making aqueous dispersion of particles (0.05 mg/ml) at 30 ◦ C in Nano-Zetasizer (Malvern Instruments). The binding of colloidal gold to the thiolfunctionalized nanoparticle, papain binding to the magnetic gold nanocomposites and papain activity were monitored by UV–Vis spectroscopy on a Shimadzu UV-1700 spectrophotometer.

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Scheme 1. The schematic presentation for the immobilization of papain on magnetic gold nanocomposites.

In order to verify the coating formation on the magnetite surface through the silanation reaction, an FTIR spectrum of the MPTS–treated magnetite sample is analyzed. Fig. 2a displays FTIR spectra of Fe3 O4 , MPTS, and Fe3 O4 –MPTS nanoparticles. Pure magnetite nanoparticles demonstrate peaks at 582 cm−1 corresponds to Fe O stretching and 3400 cm−1 corresponding to broad OH groups on magnetite surface. The presence of siloxane groups Si O Si is confirmed by the band at 1040 cm−1 indicating the attachment of mercaptosilane molecules onto magnetic nanoparticles. The observed C H stretching bands 2917 cm−1 and 2842 cm−1 in the coated magnetic nanoparticles reveal the presence of MPTS on the surface of the magnetic nanoparticles. A band at 1112 cm−1 is observed which corresponds to Fe O Si bond vibration. All the vibrational bands of MPTS functionalized nanoparticles match well with the original peaks of MPTS. Again, to further confirm the surface modification on the nanoparticles XPS is determined. XPS is highly useful analysis for the presence of S Au bonding on the nancomposite surfaces. The usual spectrum of magnetic gold nancomposites is presented in Fig. 2b. The presence of sulfur (S2p) corresponding to the binding energy 162.5 eV is shown in figure. The existence of Au on MPTS modified Fe3 O4 nanoparticles are confirmed from the high-resolution XPS spectrum. The peaks at binding energies 84.8, 88.5 eV are ascribed to Au0 (4f 7/2 ) and Au0 (4f 5/2 ) electrons respectively. The average hydrodynamic diameters (HDs) of MPTS–Fe3 O4 and magnetic gold nanocomposites are studied in phosphate buffer saline at pH 7.4 and the data is shown in Fig. 3. The HD sizes of MPTS–Fe3 O4 nanoparticles are obtained in the range of 135–155 nm (shown in Fig. 3a) at physiological pH 7.4, whereas the HD size of magnetic gold nanocomposites is found in the range 55–85 nm (shown in Fig. 3b). This decrease in HD size of aqueous

dispersion of magnetic gold nanocomposites might be attributed from higher surface negatively charged citrate, which prevents the aggregation of particles. Zeta potential measurements are convenient to study the surface modification of magnetic-gold nanocomposite. Fig. 3c depicts the zeta-potential curve of aqueous dispersion of magnetic gold nanocomposites. The prepared Fe3 O4 nanoparticles have zeta potential value of −7.0 mV. Upon modification with the gold nanoparticle on magnetite surface, the zeta potential decreases to −45.0 mV indicating surface modification by the citrate stabilized gold nanoparticles. The zeta potential value of magnetic gold nanocomposites give high negative potential value varying pH ranges from 3.0 to 11.0. The surface charge potential of magnetic gold nanocomposites in water with various pH values could be explained by negatively charged citrate ions on nanocomposites surface. These variations in zeta potential support successful surface modification of the gold nanoparticles on magnetic nanoparticles. The photograph of aqueous dispersion of magnetic nanoparticles, colloidal gold solution and aqueous dispersion of magnetic gold nanocomposites are presented in Fig. 3d. From figure it is clearly observed that the aqueous dispersion of all the compounds show different colors. Fig. 4 displays HRTEM image of Fe3 O4 –MPTS nanoparticles, which are prepared in aqueous medium. The TEM image illustrates that the particles are spherical in shape with some agglomeration, which is obvious because of the magnetic nature of the particles. In the magnetic gold nanocomposites, the presence of gold nanoparticles with magnetite nanoparticles is observed in the highresolution TEM image. Same type of TEM image has been observed for Fe3 O4 /Au core/shell nanoparticles prepared by Xie et al. [32]. The selected-area electron diffraction (SAED) pattern confirms the

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Fig. 6a displays UV–Vis spectra of the as-prepared colloidal gold solution and the decant gold solution after stirring with MPTS modified magnetic nanoparticles. The surface plasmon resonance of the as-prepared colloidal gold solution can be clearly observed at 530 nm. After shaking the colloidal gold solution with mercaptosilane coated magnetite nanoparticles, the decant solution after magnetic separation is collected. It is observed that there is loss in intensity of the surface plasmon resonance due to decrease in the concentration of gold nanoparticles in the decant solution. This indicates binding of colloidal gold particles to the mercaptosilane coated magnetite nanoparticles through thiol groups on magnetite surface. 4.2. Studies of immobilization of papain on magnetic gold nanocomposites Fig. 6b displays UV–Vis peak of papain stock solution (1 mg/ml) in 0.01 M PBS buffer, pH 7.4. It illustrates UV absorbance value 2.048 at 280 nm. The decant papain solution after immobilization and magnetic separation, is collected which exhibit absorbance value at 1.033 at 280 nm. The decrease in the UV absorbance value of papain solution confirms the conjugation of papain to the magnetic gold nanocomposites. This mode of enzyme immobilization is very simple, low cost and time saving. 4.3. Studies of papain activity assay

Fig. 1. (a) X-ray diffraction patterns (XRD) for pure Fe3 O4 nanoparticles, MPTS functionalized Fe3 O4 nanoparticles and magnetic gold nanocomposites (* marks assigned the presence of gold in the composite) and (b) weight loss analysis from TGA curves of as prepared Fe3 O4 and MPTS functionalized Fe3 O4 nanoparticles.

polycrystalline nature of the embedded magnetite particles and magnetic gold nanocomposites. The individual planes have identified from the SAED pattern. The EDX result indicates that the elemental compositions of the nanocomposites are Fe, Si, S, C, O, and Au. Here, all the results from XRD, XPS and TEM authenticate a conclusion that Au nanoparticles have immobilized to the surface of Fe3 O4 nanoparticles. Importantly, these intact Au nanoparticles are quite stable and enable us rapidly and efficiently for immobilization of enzyme therefore, it would greatly facilitate their applications in biotechnology. The magnetic measurements of both free Fe3 O4 and coated Fe3 O4 nanoparticle have obtained at 300 K. The saturation magnetization values for the uncoated, MPTS coated Fe3 O4 , magnetic gold nanocomposites are presented in Fig. 5, and the values are found to be 65 emu/g, 57 emu/g and 43 emu/g respectively. All the particles show superparamagnetic behavior and the particles do not show any coercivity. The decrease in magnetization value suggests that the successful coating on the original Fe3 O4 nanoparticles. The high saturation magnetizations of magnetic gold nanocomposite enable us for easy separation of enzyme from reaction mixture with a help of simple magnet.

The stability of enzyme with the retention of the biocatalytic activity of enzyme is the most important criteria of enzyme immobilization. The biocatalytic activity of free papain and immobilized papain are investigated under identical assay condition. It is also possible that binding of papain to the magnetic gold nanocomposites occurs via electrostatic interactions between the negatively charged surface of the nanocomposites and the positively charged enzyme. There are many literatures reported the binding of protein and enzyme through electrostatic interaction with different support materials [33–35]. The isoelectric point of papain (pI) is 8.75. From zeta potential measurement, magnetic gold nanocomposites contain highly surface negative charges due to citrate ions on gold surface (zeta potential is −45.5 mV). The immobilization of papain is carried in PBS buffer (pH 7.4). At this pH, papain is positively charged and magnetic gold nanocomposites contain highly negative charges on its surface. As a result, the binding of papain on the magnetic gold material takes place through electrostatic interaction. Moreover, there may be non-covalent interactions occur between nitrogen and sulfur atoms of enzyme with magnetic gold nanocomposites. Moreover, to clarify the mode of immobilization of papain on magnetic gold nanocomposites the effect of NaCl concentration on the immobilization rate was performed. We have observed that the immobilization rate decreases in the presence of moderate concentration of NaCl. Therefore, the ionic exchange effect plays an important role for papain immobilization. 4.4. Effect of pH on papain activity Fig. 7A displays plot of the biocatalytic activity of free papain molecules in solution and papain bound to the magnetic gold nanocomposites for reactions carried out after preincubating the enzyme/bioconjugate as a function of solution pH in the range of 3–11. From figure, the optimum activity for free papain is pH 6.0 whereas the optimum activity of immobilized papain is shifted toward pH 9.0. Li et al. have reported the optimum medium pH values for free and immobilized papain are 6.0 and 7.0 respectively [36]. The variation of the activity of immobilized papain with pH is due the following reasons. The surface charge of papain varies with the pH. Hence, it is possible that the immobilized

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Fig. 2. (a) FTIR spectra of Fe3 O4 nanoparticles, MPTS and Fe3 O4 –MPTS coated nanoparticles and (b) X-ray photoelectron spectroscopy (XPS) of magnetic gold nanocomposites.

enzyme would detach from the magnetic gold nanocomposites, subsequently leading to lower activity compared to free papain and the shift of the optimum activity. The shift of optimum pH after enzyme immobilization is also related to the multipoint noncovalent interaction of papain and support, which preserves the activity [37]. Though free papain shows higher catalytic activity than

immobilized papain, the catalytic performance of immobilized papain is well retained in a wider pH range between 5.0 and 11.0. Apart from ionic interaction, the stabilization of papain on magnetic gold nanocomposites may occur through multipoint non-covalent interaction. We have incubated the immobilized enzyme at pH 3, 4, 9, 11 and we have observed that there is less amount of

Fig. 3. DLS distribution of: (a) MPTS coated Fe3 O4 ; (b) magnetic gold nanocomposites; (c) zeta potential curve for magnetic gold nanocomposites at various pH (Error bars represents ± standard deviations, n = 3) and (d) color comparison of Fe3 O4 nanoparticles (black color), colloidal gold nanoparticle solutions (wine red color), and magnetic gold nanocomposites (brown color). (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)

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Fig. 4. TEM micrographs, SAED pattern and EDX analysis of: (a) Fe3 O4 –MPTS nanoparticles and (b) magnetic gold nanocomposites.

enzyme, which is desorped. Due to the desorption of papain in the above mentioned pH, from nanocomposites we have observed less activity compared to free papain, which we have discussed in our previous manuscript. 4.5. Hydrolysis activity of immobilized papain at different temperatures The effect of temperature on the activity of free and conjugated papain are investigated in phosphate buffer (0.01 M, pH 7.4) in the temperature range 30–90 ◦ C. The optimal activity for free papain and immobilized papain are obtained at 50 ◦ C and 60 ◦ C respectively as shown in Fig. 7B. The optimum temperature for papain immobilized on carboxymethyl chitosan coated magnetite nanoparticle was 75 ◦ C whereas in case of native papain the highest enzyme activity was observed at 55 ◦ C [24]. This shift toward higher temperature for immobilized papain is due to multipoint ionic interaction between support and bound enzyme. Compared with free papain the enzymatic activity of immobilized papain is higher for a broader temperature profile. The increase in activity of immobilized papain with temperature is due to the restriction in conformational change of papain upon heating due to rigid binding of papain on the magnetic gold material. The enzymatic activity of free papain decreases, which may due to conformational change, which deteriorates the activity.

the hydrolysis rates of casein versus the concentration of product formed at room temperature, which is shown in Fig. 7C. From figure, it is depicted that casein is catalytically hydrolysed by immobilized papain. On adding excess substrate concentration and keeping it for prolonged period does not show any more hydrolysis that means the active site of the enzyme becomes fully saturated with substrate. The maximum specific activity of the immobilized papain is 47% with respect to free papain when the free papain concentration is 100 ␮l (1.0 mg/ml) and on 10 mg (54 ␮g/mg particle) of immobilized papain on magnetic gold nanocomposite. The units of papain on magnetic gold nanocomposites surface is 0.18 units/mg particle.

4.6. Specific activity of immobilized papain The “specific activity” is expressed as the ␮mol of product formed per unit time per mg of enzyme. Specific activity is the activity per unit mass of enzyme. The specific activity of conjugated papain is predicted through the determination of casein hydrolysis rate. The enzymatic activity of papain immobilized on magnetic gold nanocomposites is studied by plotting the graph between

Fig. 5. VSM curve for: (a) free Fe3 O4 nanoparticles; (b) MPTS–Fe3 O4 nanoparticles and (c) magnetic gold nanocomposites.

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papain 0.236 × 105 g ml−1 and 4.08 g ml−1 /s respectively whereas for immobilized papain, Km and Vmax values are 0.308 × 105 g ml−1 and 5.4 g ml−1 /s respectively. Higher value of Vmax of immobilized papain denotes higher rate of product formation compared to free papain. The apparent Km value of the immobilized papain is higher than that of free papain. The higher value of Km upon immobilization is due to decrease in affinity of papain for casein probably caused by diffusional limitation of substrate to the active site of enzyme. Shaw et al. reported that the immobilized papain had higher apparent Km value at all temperature tested with either casein and benzoyl l-arginine ethyl ester (BAEE) as substrate and the effect was more pronounced for casein as substrate [38]. The effects may be due to steric hinderance of carrier, which caused the difusional limitation of substrate around the microenvironment of immobilized enzymes [38]. Bhattacharyya et al. have reported that Km has increased 10 times for immobilized papain compared to free papain, which indicated that affinity of the enzyme toward its substrate was reduced after immobilization of papain on alginate bead [39]. 4.8. Papain loading efficiency “Papain loading efficiency” is defined as follows: Weight of enzyme on nanoparticles × 100 Weight of enzyme taken for immobilization

Fig. 6. UV–Vis data of: (a) free gold nanoparticle and decant gold solution after binding with MPTS–Fe3 O4 nanoparticle; (b) free papain and decant papain after immobilization.

The immobilization yield is defined as the percentage of the ratio of total activity of immobilized enzyme (Ci ) to the total activity of initial free enzyme enzyme taken. Immobilization yield =

Ci × 100 Cs

We have observed 76% of immobilization yield. 4.7. Kinetic studies of immobilized papain The kinetic study of casein hydrolysis by immobilized papain are studied to determine the kinetic parameters like Vmax and Km , which are obtained from Lineweaver–Burk plot shown in Fig. 7D. For the determination of kinetic parameters, we have used 10 mg of immobilized papain (54 ␮g of protein/mg particle) and 100 ␮g of free enzyme for our studies. The kinetic parameter Vmax indicates the maximum velocity of product formation and Km represents the affinity of the enzyme toward its substrate. The Michaelis–Menten constant (Km ) and maximum reaction rate (Vmax ) for both free and immobilized papain are determined by using casein as substrate. The kinetics constants Km and Vmax are calculated free

The papain loading efficiency on magnetic gold nanocomposites is carried in PBS buffer (pH 7.4) keeping magnetic gold nanocomposites concentration constant and varying papain concentration, which is shown in Fig. 7E. The maximum enzyme loading of 54 ␮g/mg particle is obtained when papain concentration is 1.0 mg/ml and particle concentration is 10 mg/ml. For papain loading efficiency, the enzyme taken for immobilization is less, and for loading efficiency calculation we have used total enzyme bound on 10 mg nanoparticles. Therefore, we have obtained maximum papain loading efficiency more than 70%. The enzyme loading efficiency on magnetic gold support is carried out at two different reaction times. After 24 h the maximum loading of 50.2 ␮g/mg particle is obtained whereas after 48 h the maximum amount of protein loaded on nanocomposite surface is 54 ␮g/mg particle. 4.9. Effect of batch operation on hydrolytic activity of immobilized papain Reusability of immobilized enzyme without much loss of catalytic activity is the main purpose of enzyme immobilization. The most significant benefit of magnetic gold support material for enzyme immobilization is their enduring stability. For reusability studies, the immobilized papain is stored at 4 ◦ C in PBS buffer. After checking its activity, the immobilized papain can be easily separated from product by magnet and again washed with PBS buffer five to six times and stored in the same condition for subsequent catalytic activity. The catalytic activity of immobilized papain is checked in six successive repeated cycles over a period of one month as shown in Fig. 7F. Our hybrid enzyme-nanocomposites retain 70% of its initial catalytic activity after five times successive reuse. The immobilized enzymes are reused many times, different from the free enzyme, which is used only once. This long-term stability illustrates the advantage of attaching enzymes chemically to the magnetic gold nanocomposites. Hydrolysis activity of the immobilized enzyme over a wider range of pH and temperature, described above, gives economically a great advantage over the free enzyme.

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Fig. 7. (A) Effect of pH on relative activity of native papain and immobilized papain; (B) effect of temperature on relative activity of native papain and immobilized papain; (C) variation of reaction rate with substrate concentration for immobilized papain; (D) Lineweaver–Burk plot for the maximum hydrolysis rate (Vmax ) of free papain and immobilized papain at the room temperature; (E) plot of papain loading efficiency vs. papain concentration after 24 h after 48 h and papain loading amount (␮g/mg particle) and (F) reusability of the immobilized papain (error bars represents ±standard deviations, n = 3).

5. Conclusion The catalytic efficacy of papain by immobilizing onto magnetic gold nanocomposite, with concomitant enhancement in enzyme immobilization efficiency, activity, and storage stability opens up new avenues in biotechnological applications. This immobilization strategy furnished good reusability, so the immobilized papain can be successively reused six times without significant loss of its catalytic performance. This process of enzyme immobilization is simple, robust and applicable to other enzymes for the potential applications in industry. This magnetic material may be a promising material for immobilization of other industrially important enzymes. Acknowledgements The authors gratefully acknowledge CSIR, New Delhi and DBT, Govt. of India for providing financial support for this work and

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