Article pubs.acs.org/Langmuir

Enhancement of Enzymatic Activity by Magnetic Spherical Polyelectrolyte Brushes: A Potential Recycling Strategy for Enzymes Yisheng Xu,†,‡ Siyi Wang,†,§ Haoya Han,† Kaimin Chen,†,∥ Li Qin,† Jun Xu,† Jie Wang,† Li Li,† and Xuhong Guo*,† †

State Key Laboratory of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, China Zhejiang Provincial Key Laboratory for Chemical & Biochemical Processing Technology of Farm Products, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China § Testing Center, Shanghai Research Institute of Chemical Industry, Shanghai 200062, China ∥ College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, China ‡

S Supporting Information *

ABSTRACT: Interactions between amyloglucosidase and magnetic spherical polyelectrolyte brushes (MSPB) were studied by turbidimetric titration, which reveals reversible and tunable behaviors of pH-dependent enzyme−SPB binding. Quantitative thermodyanmic parameters including binding affinity and stoichiometry between enzyme and SPBs were further measured by isothermal titration calorimetry (ITC). A large amount of enzyme can be loaded in MSPB without loss of MSPB stability. We demonstrated that the enzymatic activity of amyloglucosidase bound in MSPB could be greatly enhanced (catalytic reaction rate, kbound = 1.36kfree) compared to free enzyme acitivity in solution. This is tremendous improvement from other carrier systems that usually lead to a significant decrease of enzymatic activity. Both the high enzyme loading capacity and the enhancement of the catalytic activity probably arise from the Coulombic interactions between the enzyme and MSPB. These findings provide a practical strategy for enhancement of enzyme activity and enzyme recycling by MSPB.

1. INTRODUCTION Immobilization of enzymes on solid substrates such as latex nanoparticles, silica-based nanoparticles, and flat substrates and even hydrogels is an important process for applicable biotechnology.1−3 So far, a variety of systems with versatile features have been designed for enzyme support,4,5 drug delivery,6−8 and biosensor.9 However, immobilization of enzymes on these supports often induces partial or complete loss of catalytic activity.10−18 Surface-initiated partial unfolding of proteins usually accounts for the decrease of the activity of enzymes.13 In some extreme cases, strong interactions between substrates and proteins or peptides can even promote the amyloidgenesis. Previously, Ballauff et al. reported an interesting way to immobilize enzymes onto nanoparticles modified with densely grafted polyelectrolytes. Spherical polyelectrolyte brushes (SPBs) are appropriate substrates for protein immobilization © 2014 American Chemical Society

because they barely affect the biological functions of enzymes.19−22 The secondary structure of immobilized proteins is almost preserved on SPBs as well as their enzymatic activities. In addition, SPBs also provide a large surface area for adequate binding of proteins. The involvement of nonspecific interactions, e.g., electrostatics, van der Waals, and hydrophobic interactions,21,23 enables the immobilization to be reversible by controlling the solution conditions such as ionic strength and pH.20,24,25 More recently, they reported microgels as ideal nanoparticle carriers for enzyme immobilization.26,27 The activity of β-D-glucosidase adsorbed on a suitable core−shell microgel was enhanced significantly.22 Received: June 13, 2014 Revised: August 30, 2014 Published: September 2, 2014 11156

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Chemical Reagent Co., Ltd., were distilled under reduced pressure and stored in a refrigerator at 4 °C before use. Oleic acid (OA), n-octane, acetone, ammonium hydroxide (25 wt %), and hydrochloride acid (36 wt %) were bought from Lingfeng Chemical Reagent Co., Ltd. Sodium chloride (NaCl), potassium persulfate (KPS), sodium dodecyl sulfate (SDS), and 2-(N-morpholino)ethanesulfonic acid (MES) were purchased from J & K Chemical and used as received. The 2hydroxy-4′-hydroxyethoxy-2-methylpropiophenone (IRGACURE 2959) was bought from Ciba Chemicals Inc. Pyridine, 3,5dinitrosalicylic acid, and methacryloyl chloride purchased from TCI were used without further purification. Hexadecane (99%) was purchased from Acros Organics and methacryloyl chloride from Technical Choices, Inc. The 2,2′-azobis(isobutyronitrile) (AIBN) (from Shanghai No.4 Reagent & H.V. Chemical Company) was recrystallized in methanol before use. Millipore Milli-Q water was used in all experiments. 2.2. Synthesis and Characterizations of MSPB. There are four steps for the synthesis of magnetic spherical polyelectrolyte brushes as shown in Figure 1: First of all, oleic acid (OA), FeCl3·7H2O, and ammonium hydroxide were added together and coprecipitated to form magnetic nanoparticles (MNPs). The detailed information was included in our previous work.27−29 In the second step, magnetic polystyrene lattices were synthesized by miniemulsion polymerization. A certain amount of MNP was mixed with 0.10 g of hexadecane, 0.075 g of AIBN, and 2.50 g of styrene. The mixture was ultrasonicated for 15 min in an ice bath and then added into 200 mL of SDS solution (0.1 wt %) followed by miniemulsification for 60 min. In the third step, at the end of the miniemulsion polymerization, 4.5 g of acetone solution with 0.5 g of photoinitiator (2-[p-(2-hydroxy-2methylpropiophenone)]ethylene glycol methacrylate) (HMEM) synthesized in our laboratory was added slowly in 30 min. Magnetic polystyrene lattices with a thin layer of HMEM were thus obtained after further 2.5 h reaction. The lattices were purified in Milli-Q water by dialysis to get rid of unreacted monomer and surfactant. In the final step, magnetic polystyrene lattices were added into a UV reactor (range of wavelengths: 200−600 nm; power: 150 W) and diluted to 0.5 wt % with water. Calculated amount of acrylic acid (AA) was added. Photoemulsion polymerization was performed with UV radiation at room temperature with vigorous stirring for 2.5 h. The obtained MSPB emulsion was purified by dialysis against water until the conductance of the eluent became constant. The synthesis of photoinitiator HMEM was reported in our previous publication.31 First, 30.0 g of 2-hydroxy-4′-hydroxyethoxy-2methylpropiophenone dissolved in 150 mL of acetone was mixed with 10 mL of pyridine by mechanical stirring in an ice bath under dark condition. Second, 13.6 g of methacryloyl chloride dissolved in 50 mL of acetone was added. The addition rate was controlled at each drop of solution at intervals of 6 s. After the addition of methacryloyl chloride, the reaction system was stirred for 30 min in an ice bath. Third, the Schotten−Baumann reaction carried out for 12 h in an oil bath at 30 °C after the remove of ice bath. The yellow crude product was purified by silica gel column after filtration and evaporation to remove solvent under dark conditions. Finally, 8 g of acetone solution was mixed with 1 g of photoinitiator HMEM and stored at 4 °C. Dynamic light scattering (DLS) was conducted with a particle sizing system of NICOMP 380 ZLS at a fixed scattering angle of 90° at 25 °C. The core hydrodynamic radius of MSPB was 60 ± 2 nm. The thickness of the brush layer was also found to be 30 ± 5 nm by subtracting the core radius from the hydrodynamic radius of MSPB. Xray diffraction (XRD) was performed on a Bruker D8 Advance X-ray diffractometer with a scan rate of 6°/min. A thermal gravimetric analysis (TGA) instrument WRT-2P was employed to determine the magnetite content in particles. The high-resolution transmission electron microscopy (HRTEM) was obtained using a JEOL-2100F electron microscope operating at 200 kV. Samples were magnetically separated before TEM. 2.3. Turbidimetric Titration. Turbidity, reported as 100 − %T, was measured with a Brinkmann PC 950 colorimeter (420 nm filter), equipped with a 2 cm path length optical probe. All solutions were filtered through 0.45 μm filters before the measurements. The pH

Reuse of enzymes is another issue needs to be addressed in order to reduce the processing cost. It is more efficient to reuse them after enzymatic reactions. However, difficiulty was found to recyle these enzymes out from the complex enzymatic reaction medium. One strategy is to attach enzyme onto appropriate supporting materials which makes the isolation of enzyme from the reaction medium possible as long as the enzyme-supporting material complex can be isolated from the medium. Reverse of the complex then gives the purified and active enzymes. For example, incorporation of a unique magetic core can easily achieve the magnetic-steering movement, magnetic-response recyclability, and magnetic separation and thus redispersion of enzymes.28,29 MSPB seems to be a good candidate for enzyme immobilization, drug delivery, protein separation, high-performance diagnostic assay, and nano-sized bioreactors. In this work, we desgined and prepared magnetic spherical polyelectrolyte brushes (MSPB) as nano-size bioreactors for immobilization of amyloglucosidase for amylolysis. MSPB used here consist of a polystyrene core embeded with iron oxide nanoparticles (Fe3O4-NPs), onto which poly(acrylic acid) (PAA) chains are chemically tethered with a high grafting density (Figure 1). Because of mutual electrostatic repulsion of

Figure 1. Synthetic scheme of the magnetic spherical polyelectrolyte brushes and the adsorption of enzymes. 2-[p-(2-Hydroxy-2methylpropiophenone)]ethylene glycol methacrylate (HMEM) is a homemade photoinitiator, and acrylic acid (AA) is an anionic monomer.

neighboring PAA chains, these colloidal particles show high stability and present high surface area for potential protein adsorption. Such iron oxide NPs embedded PS core demonstrates strong superparamagnetic force, enabling simple recovery of the protein−NP complexes29,30 Both qualitative and quantitative analysis were applied to study the enzyme-NP phase behaviors and enzyme immobilization thermodynamics. We show enzyme molecules significantly adsorb into MSPB through nonspecific electrostatic interactions, but the activity is well retained. These studies provide helpful insights for industry oriented enzyme immobilization and followed recycling under controlled conditions.

2. EXPERIMENTAL SECTION 2.1. Materials. Amyloglucosidase (from aspergillus niger, isoelectric point 3.6), standard sodium hydroxide (NaOH) solution (0.1 M), potassium sodium tartrate tetrahydrate, and sodium hydroxide were purchased from Sigma-Aldrich. Ferric chloride hexahydrate (FeCl3·6H2O), ferrous sulfate heptahydrate (FeSO4·7H2O), and anhydrous ethyl alcohol were purchased from Sinopharm Chemical Reagent Co., Ltd. Acrylic acid and styrene purchased from Sinopharm 11157

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Figure 2. (a) Schematic representation of amylolysis catalyzed by amyloglucosidase. Amyloglucosidase cleaves starch into D-glucose in aqueous solution. (b) Schematic representation of color reaction between 3,5-dinitrosalicylic acid and D-glucose. (c) Product of color reaction is 3-amino-5nitrosalicylic acid, which subsequently can be monitored by UV/vis spectroscopy. The color of product varies with amylolysis time, which is shown in (c).

Figure 3. (a) TEM images of MSPB and iron oxide NPs (inset). (b) Size of magnetic polystyrene core and MSPB by DLS: (△) size of magnetic polystyrene core; (○) size of MSPB. (c) XRD patterns for synthesized iron oxide NPs (black line) and MSPB (blue line). (d) TGA patterns for synthesized iron oxide NPs (black curve) and MSPB (blue curve). (e) Separation and redispersion of MSPB guided by magnetic field: (1) magnetic separation by a NbFeB magnet; (2) MSPB aggregated on the vial wall at the magnet side after removal of magnetic field immediately. dependence of solution turbidity was obtained by observing the change of turbidity upon addition of 0.1 M HCl (or 0.1 M NaOH) by a 2.0 mL microburet to an enzyme−SPB mixture at a certain ionic strength. Generally, MSPB and enzyme concentration were 0.004 and 0.020 g/L, respectively. Nitrogen was purged in all titrations. SPB-free blanks and enzyme-free blanks were subtracted to eliminate the effect of free enzyme scattering. An Orion pH meter (Ross Ultra

Combination pH, 8156BNUWP, Orion) was employed in all experiments. 2.4. Isothermal Titration Calorimetry. Isothermal titration calorimetry (ITC, Microcal ITC200, GE Healthcare) was used to examine the thermodynamics of enzyme binding to MSPB. Enzymes and MSPB solutions were prepared in 5 mM MES buffer at different pH values. All solutions were filtered by 0.22 μm Millipore filters. After 11158

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instrument stabilization at 25 °C, 20 successive injections of protein solutions (40 μL of 0.41 mM total protein solution) were used to titrate 200 μL of 3.2 × 10−6 mM MSPB solution with an interval of 3 min between injections. The stirring rate was set at 1000 rpm during the experiments. Before data analysis, heats of protein dilutions as background signals were corrected. Microcal Origin software was used to analyze ITC data. All parameters are model-dependent. A one-site independent binding model was employed to fit the binding isotherms. 2.5. Adsorption Experiments. NaCl was added to adjust the ionic strength of all MES buffer solutions of MSPB. The mixture of enzyme and MSPB was stirred for 24 h at 4 °C for equilibration. After equilibrium, the MSPB−enzyme solution was purified by ultrafiltration to remove the unbound enzyme. The amount of unbound protein in the supernatant was determined by UV−vis at 278 nm (UV-2550, UV−vis spectrophotometer, Shimadzu). The amount of adsorbed protein was calculated by subtracting the amount of unbound protein from the total amount of proteins. Consequently, MSPB with immobilized enzymes was used as nano-bioreactors to catalyze hydrolysis in aqueous solution. Product glucose can be removed from the system by ultrafiltration, and MSPB with bound enzymes can be recycled for next enzymatic catalysis. 2.6. Determination of Enzymatic Activity. The activities of native and immobilized amyloglucosidase were determined by following the hydrolysis of starch and color reaction at 540 nm with a Shimadzu UV−vis-2550 spectrophotometer. Kinetic studies were performed at various temperatures (20−60 °C) using the concentration range of enzyme of 150−510 mg/g MSPB in MES buffer at pH 5. According to previous studies,32 the presence of glucose can be assayed by the reaction of D-glucose molecules with 3,5-dinitrosalicylic acid. Hereby, glucose, released from a starch substrate by amyloglucosidase (as Figure 2a), reduces 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid which shows a characteristic absorbance at 540 nm, causing the color changes from orange yellowish to red (see Figure 2b,c). In order to determine the amount of released glucose molecules after using the MSPB−enzyme system on a starch substrate, a calibration curve was created at different glucose concentrations. The solution consists of 3, 5-dinitrosalicylic acid, potassium sodium tartrate tetrahydrate, and sodium hydroxide dissolved in DI water. According to previous studies,32 the presence of glucose can be assayed by the reaction of glucose molecules with 3,5-dinitrosalicylic acid. Therefore, given amounts of the MSPB and enzyme solutions were mixed with a starch substrate (1% w/v) and incubated in 20 mL glass tubes for various reaction times (3 min−24 h) at corresponding temperatures (20−60 °C). After reaction, 3,5-dinitrosalicylic acid was added. The reaction was performed at 100 °C for 15 min. After catalysis, the solutions were diluted with DI water and measured by UV−vis at λ = 540 nm. For all measurements, background signals of blank solutions of MSPB, color reagent, and starch standard were subtracted in the UV−vis assay.

phase of Fe3O4 (JCPDS Card No. 19-629).33,34 The additional peak at 20° is from the PS shell of MSPB (blue spectrum in Figure 3c). Oleic acid attached on MNP surface via coordination interaction between carboxylate group in oleic acid and iron atom.35 The amount of oleic acid was determined to be ca. 25 wt % (Figure 3d, black curve). Successful grow of polymer brushes was clearly observed from the TGA results (Figure 3d, blue curve). The magnetite content in MSPB was ca. 24 wt % as determined by TGA. As shown in Figure 3e, MSPB can be aggregated and separated by external magnetic field, which can also be observed by colorimetry (Brinkmann, PC 950, colorimeter). The strong magnetic feature of such MSPB can be easily found from the aggregation of MSPB visualized as the complete transparency of the NP solution (the light transmisttance (%T) was increased to 100%). As the magnetic field was removed and the particles were redispersed for 5 min under sonication, the light transmittance of the milky solution was significantly reduced. The hydrodynamic sizes are almost unchanged within the experimental error after six cycles of aggregation and redispersion as determined by DLS, indicating the excellent redispersibility of MSPB. 3.2. Adsorption of Enzyme in MSPB. 3.2.1. Isothermal Titration Calorimetry. ITC was used to quantitatively investigate the nonspecific interaction between amyloglucosidase and SPB. Figure 4a presents the ITC raw data as

3. RESULTS AND DISCUSSION 3.1. Characterization of MSPB. The obtained MSPB were observed by HRTEM as shown in Figure 3a. Magnetic iron oxide NPs shown in Figure 3a inset were successfully incorporated in MSPB and mainly aggregated in the center of polystyrene cores. This is because that water-soluble initiator (KPS) was used and KPS dissolved in water which initiated the polymerization from out to center pushing the MNP to the center of PS core.29 The core of MSPB shows a well-defined spherical structure and a uniform size distribution although the PAA brush is invisible in the dry state. The diameters of the polystyrene core particles and MSPB are shown in Figure 3b, which was in accordance with the results of HRTEM. XRD confirms the crystal structure of the magnetic NPs as shown in Figure 3c. The characteristic peaks appearing at 30.2° (220), 35.6° (311), 43.3° (400), 53.6° (422), 57.1° (511), 62.8° (440), and 74.1° (533) correspond to the face-center-cubic

Figure 4. Isothermal titration calorimetry results as amyloglucosidase binds onto PAA-MSPB at pH 6.1 in 5 mM MES buffer. Peaks in (a) are raw data of ITC experiment. (b) Integrated heat of each injection (solid square) and one-site binding fitting (solid line) are shown in the lower panel. A 200 μL of 3.2 × 10−6 mM SPB solution was titrated with enzyme solutions (40 μL of 0.41 mM).

amyloglucosidase binds to MSPB at the “wrong side of protein pI”. This is because of the existence of “charge patches”36−39 (charge anisotropy) or “charge regulation”40−43 on the enzyme surface even though both enzyme and polymer brushes are negative (around 60% of dissociation of carboxylic groups, Figure S1 in Supporting Information) at this pH.24,44 pH 6.1 was chosen to avoid the phase separation. This pH ensures only soluble complexes form between enzyme and MSPB. The 11159

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Table 1. Binding Parameters of Amyloglucosidase onto MSPB n (1.4 ± 0.2) × 10

3

Kb (M−1)

ΔH (cal/mol)

TΔS (cal/mol)

(1.9 ± 0.5) × 105

(−1.1 ± 0.2) × 105

−9.9 × 104

process implies that the binding between enzyme and MSPB is still electrostatic driven. As reported, protein binding to charged nanoparticles commonly leads to an increase of entropy due to the counterion or water release.48 The water reorganization means that external energy has to be supplied to release the water molecules which are confined inside the brush structure. However, the protein immobilization onto MSPB certainly reduces the entropy due to the constrain of the protein molecules. The net entropy change should be the combination of these two processes. As determined by ITC experiment, the overall entropic term is negative because the entropic reduction originated from enzyme binding is larger than entropic gain originating from counterions or water releasing. Therefore, in this system, enzyme binding to MSPB is the primary effect and counterion releasing is the secondary effect. 3.2.2. Adsorption Experiment. ITC experiments were only carried out under a defined pH and ionic strength conditions to study the binding nature of the system, while adsorption experiments are able to reveal more systematic information on the immobilization of amyloglucosidase under a full range of conditions. Figure 5a shows the amount of adsorbed amyloglucosidase by MSPB (τads) as a function of concentration of original enzyme solution (Cp) at different pH values. The adsorption experiments were performed at 4 °C instead of room temperature in order to prevent the enzyme from structure change and loss of activity during the long time equilibrium with MSPBs. The amount of adsorbed amyloglu-

binding event apparently shows an exothermic process in contrast to our previous binding between cationic gold nanoparticles and proteins even though it is commonly believed that electrostatic interactions are usually exothermic. Reversal of the charge polarity of the brushes might lead to the reversal of the sign of enthalpy, but it is still controversial regarding the factors controlling the exothermic or endothermic features. Nevertheless, the exothermic binding process likely indicates that such interaction is the primarily electrostatic driven as we noticed before for other proteins.45 Integration of the negative exothermic peaks provides a binding isotherm (Figure 4b) of the enzyme binding to MSPB. In order to simplify the binding model, each protein binding can be assumed to have the same enthalpy (ΔH) but is unrelated to the binding extent for a small protein binding to a large size MSPB. In this case, MSPB was proposed to have multiple identical binding sites. The binding result was analyzed by an independent binding model, and the description of this model is46 Kb

site + enzyme ↔ complex

(1)

where “complex” stands for 1:1 binding between an enzyme molecule and each independent binding site on MSPB. The total released heat (Q) in each injection at any site during the titration should be proportional to the complex concentration, i.e., Q = a[complex], where a is a constant. The concentration of total binding sites is n[MSPB]tot, where n is the number of independent binding site on each NP. The binding constant therefore can be described as shown in eq2 Kb = =

[complex] [site free][proteinfree] ([site]tot

Q /a − Q /a)([protein]tot − Q /a)

(2)

Solving for Q Q=

⎧ a ⎪⎛ 1 ⎞ ⎨⎜[protein]tot + n[MSPB]tot + ⎟ 2 ⎪⎝ Kb ⎠ ⎩ −

⎫ 2 ⎛ ⎪ 1 ⎞ ⎟ − 4n[protein]tot MSPBtot ⎬ ⎜[protein]tot + n[MSPB]tot + Kb ⎠ ⎪ ⎝ ⎭

(3)

Binding isotherms were derived from this binding model. The complete binding parameters are included in Table 1. In addition to the enthalpy term, the entropy term is also negative which is similar to our recent findings for bovine serum albumin (BSA) and β-lactoglobulin (BLG) binding to negatively charged spherical polymer brushes (SPBs) but is different from our previous work on protein binding on positively charged gold NPs.46 Reversal of the charge polarity seems reverse the sign of entropy term as well. The clear explanation of the reversal of the sign of for enzyme−MSPB interaction may attribute to many factors including conformation change, hydrophobic interactions, etc., as we pointed out before.47 However, the larger enthalpy term and exothermic

Figure 5. Adsorbed amount of amyloglucosidase τads per unit mass of MSPB as a function of the original enzyme concentration in solution Cenzyme. (a) Symbols denote various pH conditions: (□) pH 4, (○) pH 4.5, and (△) pH 5 at ionic strength of 10 mM. (b) Symbols denote various ionic strengths: (□) 10 mM, (○) 50 mM, and (△) 100 mM at pH 4. 11160

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should be involved. In the current MSPB system, high loading of enzyme molecuels can reduce the diffusion rate of reactant into MSPB; hence, the apparent catalytic rate decreases as illustrated below:

cosidase by MSPB decreases with pH. This is because the amount of positive charges of amyloglucosidase decreases with pH leading to a weaker electrostatic interaction between amyloglucosidase and MSPB. The adsorption increases and gets to a plateau at high protein concentration where saturation of adsorption is reached.20,21,49,50 Figure 5b shows the ionic strength effect on enzyme adsorption. In general, the ionic strength has much more eminent effects than pH which further supports the electrostatic nature of the interactions. The adsorption of amyloglucosidase by MSPB decreases as ionic strength is increased. It also shows a plateau of saturation when the surface charge of MSPB is neutralized by proteins. The simple Coulombic screening effect can explain the ionic strength influence on the electrostatic attraction between amyloglucosidase and MSPB. The time-dependent enzyme activity in the presence and absence of MSPB is shown in Figure 6. Apparently, enzyme in

Figure 7. Cleavage kinetics of starch by amyloglucosidase immobilized in MSPB particles with different immobilization amount at 60 °C. The immobilization amount of amyloglucosidase τads per unit mass MSPB is (○) 510 mg/g MSPB, (□) 340 mg/g MSPB, and (△) 150 mg/g MSPB. The slopes of the three lines are 0.010 (○), 0.012 (□), and 0.021 (△), respectively. The particles immobilized with various amounts of amyloglucosidase were diluted by MES to a final concentration of 0.01 g/L.

Figure 6. Cleavage of starch by amyloglucosidase immobilized in MSPB particles at 60 °C, pH 5, and ionic strength of 10 mM. The measured absorbance of 3-amino-5-nitrosalicylic acid versus amylolysis time was determined by UV−vis spectroscopy. (○) Free amyloglucosidase, (△) amyloglucosidase immobilized in MSPB. The rate of enzymatic cleavage was calculated from the slopes of these lines (see ref 19). The slopes of the two lines are 0.0016 (○) and 0.0218 (△), respectively.

The temperature effect on enzyme acitivity was then investigated as demonstrated in Figure 8. Enzymatic activity

MSPB generates 3-amino-5-nitrosalicylic acid (catalytic product) 30% faster than free enzyme in solution as shown by the slopes of these two lines. These lines directly related to the rate of enzymatic cleavage (k) (see ref 19). Experimental results demonstrate that the immobilization of amyloglucosidase in MSPB does not cause the loss of activity of enzyme but displays a higher enzymatic cativity after immobilization into MSPB than free enzyme. Because MSPBs can self-control and keep ionic strength and pH stable in a certain range within the shell layers because of the Donnan effect,51−54 the shell layers of MSPB provide the optimal conditions for amyloglucosidase to catalyze the amylolysis. In addition, the strong electrostatic interactions between enzyme and MSPB might stabilize the transition states when enzyme H-bonded to substrate during the catalysis, leading to a higher catalytic rate as explained by Welsch et al.22 The concentration dependence of catalytic rate was also performed as shown in Figure 7. However, the lowest loaded (150 mg/g) MSPB has higher activity. This indicates that overloading of the MSPB with enzyme employs a negative effect on enzyme activity. As is known, in a catalytic process, the two most important stepsdiffusion and activation

Figure 8. Glucose concentration versus amylolysis time at three different temperatures. Symbols denote (○) 20, (□) 40, and (△) 60 °C.

inside the brushes refected by the glucose cocentration is increased upon increasing amylolysis temperature from 20 to 60 °C. In fact, since TΔS is negative in such system, increase of temperature below denaturing point leads to a less negative ΔG and therefore weaker binding of proteins. Such results are consistent with the fact that amyloglucosidase has the optimal 11161

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catalytic activity at 60 °C,55 and the temperature dependent features are well retained in MSPB. 3.4. Reversible Adsorption of Enzyme by MSPB. In such electrostatically driven SPB−enzyme nanocomposite system, enzyme immobilization is supposed to be fully reversible as shown in Figure 9. The turbidity of the

tion,24,56,59 magnetic field-guided protein delivery,60 disease diagnosis/treatment, etc.

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ASSOCIATED CONTENT

* Supporting Information S

Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (X.G.). Author Contributions

Y.X. and S.W. contributed equally to this work. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (No. 51273063, 11076002/A06, and 21306049), the Fundamental Research Funds for the Central Universities, the higher school specialized research fund for the doctoral program (20110074110003 and 222201314029), and the China Postdoctoral Science Foundation (12Z102060005) is gratefully acknowledged.

Figure 9. Turbidity of enzyme−MSPB mixture as a function of pH in 10 mM NaCl solution. Symbols denote (○) the dependence of solution turbidity on pH from 2 to 12 upon addition of 0.1 M NaOH and (□) pH from 12 to 2 upon addition of 0.1 M HCl to the enzyme− MSPB mixture.

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enzyme−MSPB mixture solution shows a marked increase from pH 3.2 to 4.5, corresponding to the association of MSPB−enzyme complexes while a significant decreased turbidity corresponding to the dissociation of the aggregates or clusters appears upon increasing pH from 4.5 to 5.5 where turbidity curve returns to “baseline”. The pH dependence of turbidity for the enzyme−MSPB mixture shows almost the same results with slight hysteresis when pH was decreased from 12 to 2. Apparently, enzyme molecules interact with MSPB in the pH range from 3 to 6, including different interacting stages including soluble complex and associated aggregate regions as described in our previous studies for other SPB−protein systems.24,56 It reveals the reversible adsorption and desorption of enzymes from MSPB by modulating pH. The adsorption of enzymes by MSPB can be controlled simply by changing pH or ionic strength. Good redispersibility in addition to the quick magnetic response guarantees the easy and effective recycling of MSPB from an industrial aspect.

4. CONCLUSIONS A novel nano-size bioreactor has been developed by introducing magnetic control of spherical polyelectrolyte brushes to achieve controllable enzyme catalysis. MSPB serve as ideal carriers for immobilization of enzymes. The immobilization of enzymes in MSPB preserves the native tertiary and secondary structure of enzyme. Moreover, the increase of local enzyme concentration inside brushes was achieved. The preservation of structure and the strong electrostatic interaction may result in much greater enhancement of enzymatic activity in contrast to unbound enzyme. On the other hand, the adsorption of enzyme by MSPB can be adjusted by pH and ionic strength, and the immobilized enzyme hence can be recycled. This study provides a practical strategy for cost-effective applications of MSPB in biocatalysis57,58 in addition to their applications in protein separa11162

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