Immobilization of Alkaline Protease on Amino-Functionalized

Apr 13, 2015 - School of Biosciences & Bioengineering, South China University of Technology, Guangzhou 510006, China. ‡Laboratory of Applied Biocata...
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Immobilization of Alkaline Protease on Amino-Functionalized Magnetic Nanoparticles and Its Efficient Use for Preparation of Oat Polypeptides Teng-Gen Hu,†,‡ Jian-Hua Cheng,∥ Bo-Bo Zhang,‡ Wen-Yong Lou,*,‡,§ and Min-Hua Zong§ †

School of Biosciences & Bioengineering, South China University of Technology, Guangzhou 510006, China Laboratory of Applied Biocatalysis, §State Key Laboratory of Pulp and Paper Engineering, College of Light Industry and Food Sciences, and ∥College of Environment and Energy, South China University of Technology, Guangzhou 510640, China

‡

S Supporting Information *

ABSTRACT: Alkaline protease was successfully immobilized onto amino-functionalized Fe3O4 nanoparticles. The enzyme loading was 388.2 mg of protein/g of support and the activity recovery was more than 54.2%. After immobilization, the affinity of alkaline protease toward substrate and its stability were significantly enhanced. The immobilized enzyme still retained 50.1% of its initial activity after 10 cycles of successive reuse, exhibiting excellent operational stability. The immobilized enzyme was capable of efficiently catalyzing hydrolysis of oat bran into oat polypeptides. Under the optimized conditions, the maximum DPPH radical scavenging rate (antioxidant activity) of oat polypeptides (8.4 mg/mL) was 82.3%, which was much higher than the reported result. Moreover, the prepared oat polypeptides by immobilized enzyme showed higher antioxidant activity than those prepared by free enzyme, owing to an increase of relatively hydrophobic components of oat polypeptides. Furthermore, the immobilized enzyme was demonstrated to be very promising for production of oat polypeptides on a preparative scale. attachment.6 Noncovalent adsorption immobilizes enzymes onto a support by a variety of adsorbents.7 It involves physical surface interactions8−10 and ion exchange adsorption.11,12 The method immobilizes enzymes in mild conditions, which is simple and does not cause enzyme degeneration or inactivation. Although the carrier is cheap and readily available, these physical interactions alone are often not enough to keep the enzyme from desorbing from the support during catalysis.6 Encapsulation is embedding enzymes into a host support matrix, which can reduce leaching and excessive denaturing of enzymes.13 The support material and enzyme size must be carefully considered. If the size of the host system is too large or small, enzymes easily leach from the support or cannot load on the support. Covalent attachment links the enzyme and supports through covalent binding of amino acid residues (−NH2, −SH) of the enzyme. This method relies on the bifunctional cross-linking agents such as glutaraldehyde,14−17 maleic anhydride,18−20 and genipin21−23 to immobilize enzymes. It is an effective immobilization technique which can increase the operational stability of enzymes and be conducive to their recovery and recycling.24,25 In the recent years, because of the unique structure of nanomaterials, it shows great advantages in terms of enzyme immobilization in industry and biological sciences.26 Magnetic nanomaterials are nontoxic and easy to separate from reaction medium which can eliminate the need for centrifugation and thus solve the problem of excessive squeezing on account of

1. INTRODUCTION Oat polypeptides, which are extracted by hydrolyzing oat protein, are a kind of bioactive peptides. Their structures are similar to natural moisturizing factor (NMF). They possess excellent moisturizing performance, which can rapidly penetrate the skin to perform deep moisturizing. They can effectively block the harmful chain reaction of skin, so as to maintain the complete structure of skin, inhibit skin roughness, and enhance skin elasticity.1,2 Oat polypeptides also have high antioxidant activity, and therefore can well scavenge radicals, reactivate skin, and reduce skin roughness.3 Besides, oat polypeptides exhibit inhibitory activity toward the angiotensin converting enzyme (ACE) and consequently play an important role in lowering blood pressure.4 Oat polypeptides were usually prepared by biological protease degradation due to the great advantage of the biological approach. On the one hand, the reaction conditions were mild, low in energy consumption, and friendly to the environment. On the other hand, the productivity of the objective peptide fragments could be improved by controlling the reaction conditions. As an important biological catalyst, alkaline protease is a kind of serine endopeptidase and widely used in food, medicine, brewing, silk, leather, and other industries.5 It is commonly used in the efficient preparation of bioactive peptides including high performance oat polypeptides. However, the disadvantages of the free alkaline protease, such as poor mechanical stability, unrecyclability and difficulties in separating from products, hinder its application in industry. To overcome the shortcomings, immobilization techniques are widely employed. There are three general approaches for enzyme immobilization, including noncovalent adsorption, encapsulation, and covalent © XXXX American Chemical Society

Received: November 29, 2014 Revised: March 9, 2015 Accepted: April 13, 2015

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Industrial & Engineering Chemistry Research centrifugations.27,28 Currently, the magnetic nanomaterials as efficient support materials for enzyme immobilization have gained increasing and increasing attention.29,30 The whole process of enzyme immobilization involves the addition of amino-functionalized magnetic nanoparticles into enzyme solution, then the cross-linking of enzyme and magnetic nanoparticles by a bifunctional cross-linker. Because of the magnetic properties of these particles, immobilized enzymes can be easily separated through the addition of a magnetic field rather than centrifugation. Can et al.31 used aminosilanemodified superparamagnetic magnetite nanoparticles to immobilize albumin. The maximum bounding protein was around 71.0%, indicating that it was a good support for enzyme immobilization. In the study of Talekar et al.,32 α-amylase was successfully immobilized on the amino-functionalized magnetic particles with high activity recovery, which demonstrated the good biocompatibility of the support with α-amylase. Li et al.33 reported the immobilization of alkaline protease onto magnetic nanoparticles modified by amino-silane, and thus significantly improved the stability and recyclability of the enzyme. Wang et al.34 successfully employed magnetic chitosan nanoparticles to immobilize alcalase with a moderate recovery of enzyme activity, which was able to effectively catalyze hydrolysis of soy protein. Therefore, it was clearly shown that the magnetic nanoparticles had great potential for enzyme immobilization. In the present study, magnetic (Fe3O4) nanoparticles were successfully prepared and modified with 3-amino-propyltriethoxysilane (APTES) on the surfaces, and the resulting aminofunctionalized Fe3O4 nanoparticles were further structurally characterized. Alkaline protease from Bacillus licheniformis (Novozym 37071) was efficiently immobilized onto the amino-functionalized Fe3O4 nanoparticles by cross-linking with relatively high protein loading and activity recovery (Figure 1). Also, the catalytic characteristics of the resulting immobilized alkaline protease were investigated systematically, and its reusability was examined. Furthermore, the immobilized alkaline protease onto this magnetic support was, for the first time, tested for its potential for the enzymatic hydrolysis of oat bran into oat polypeptides with high antioxidant activity

(Figure 1), and the effects of several vital variables on the reaction were described. The index of scavenging effects on α,α-diphenyl-β-picrylhydrazyl (DPPH) radical was here used to measure the oxidation resistance of the prepared oat polypeptides, since DPPH is a stable free radical which would be scavenged when it encounters a proton-donating substance and is widely used for predicting antioxidant activity of various compounds.35 In addition, the efficient hydrolysis of oat bran into oat polypeptides catalyzed by the immobilized alkaline protease was evaluated on a preparative scale. The results presented in this paper clearly showed that the protease immobilized onto amino-functionalized Fe3O4 nanoparticles is very competitive and promising for the preparation of oat polypeptides with excellent antioxidant activity.

2. MATERIALS AND METHODS 2.1. Materials. Alkaline protease (Novozym 37071, 180 U/ mL) from Bacillus licheniformis was purchased from Novozyme (Bagsvard, Denmark). 3-Aminopropyltriethoxysilane (APTES, 98% purity) was obtained from Guangzhou Mofei Instrument Co. Ltd., China. α,α-Diphenyl-β-picrylhydrazyl (DPPH, ≥ 96% purity) was from Shanghai Jianglai Biochemical Reagent Co. Ltd. Oat bran (containing 23.4% (w/w) oat protein) was purchased from Shijiazhuang Lingfeng Agricultural and Sideline Products Development Co., Ltd. All other chemicals were from commercial sources and were of analytical grade. 2.2. Preparation and Modification of Fe3O4 Nanoparticles. Fe3O4 particles were prepared and modified with amino groups by the method of Reza et al.36 with slight modifications. To a mixture of 1.3510 g of ferric chloride (FeCl3·6H2O) and 0.6852 g of ferrous sulfate (FeSO4·7H2O) in 25 mL of deionized water, ammonium hydroxide (NH4OH) was added to synthesize magnetic particles at 25 °C. Fe3O4 was separated by adding a magnetic field and washing the particles several times with deionized water until a pH value of 7.0 was reached. Then, to obtain amino-functionalized Fe3O4 nanoparticles, the particles were modified with APTES by a silanization reaction. The process involves adding APTES (100 μL), Fe3O4 (0.02 g), and deionized water (25 μL) to methanol (2.5 mL), followed with ultrasound of the mixture for 30 min. After that, glycerol (1.5 mL) was added to the mixture, and then the mixture was heated at 90 °C for 6 h with mechanical agitation. 2.3. Characterization of Amino-Functionalized Fe3O4 Nanoparticles. The modified Fe3O4 nanoparticles were characterized with transmission electron microscopy (TEM, JEOL, Japan) operated at 200.0 kV and Fourier transformed infrared spectroscopy (FTIR, Bruker, Germany). Fe3O4 and amino-functionalized Fe3O4 nanoparticles were confirmed by surface analysis with X-ray photoelectron spectroscopy (XPS, Multi-Technique ESCA systems), X-ray diffraction (XRD) (Bruker/D8 Advance, Germany), vibrating sample magnetometer (VSM, PPMS-9, Quantum Design) at room temperature. 2.4. Immobilization of Alkaline Protease onto AminoFunctionalized Fe3O4 Nanoparticles. The immobilized alkaline protease was prepared as follows. In a typical experiment, 3.6 mL of phosphate buffer (200 mM, pH 7.5) containing 50−200 mg of the prepared amino-functionalized Fe3O4 nanoparticles and 72 U free alkaline protease (180 U/ mL) was added to a 10 mL Erlenmeyer flask capped with a septum. Subsequently, the cross-linker glutaraldehyde with various concentrations (19−31 mM) was added into the mixture to immobilize the enzyme onto the magnetic support

Figure 1. Schematic immobilization of alkaline protease onto aminofunctionalized Fe3O4 nanoparticles and its catalyzing hydrolysis of oat bran into oat polypeptides. B

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Vmax (kinetic parameters) values were calculated according to Hanes-Woolf plots. 2.7.3. Stability. pH stability: A certain amount of immobilized alkaline protease or free enzyme were incubated in phosphate buffer (200 mM) with different pH values at 25 °C. Sampling was conducted every 2 h and the residual activity of the enzyme samples was determined as above. Thermal stability: A certain amount of immobilized alkaline protease or free enzyme were added and incubated in phosphate buffer (200 mM, pH 7.5) at various temperatures (40, 60, 80 °C). Sampling was conducted every 2 h and the residual activity of the enzyme samples was assayed. 2.7.4. Reusability. In the system, the reaction proceeded in buffer (pH 7.5) at 65 °C and was repeated over 10 batches (2 h per batch). Between batches, the immobilized alkaline protease was recovered by magnet and washed three times with phosphate buffer (200 mM, pH 7.5). Then the recovered immobilized alkaline protease was added again in a fresh reaction medium. The relative activity of the immobilized alkaline protease of the first batch was defined as 100%. 2.8. General Procedure for Enzymatic Hydrolysis of Oat Bran into Oat Polypeptides with Immobilized Alkaline Protease. In a typical experiment, 10 mL of phosphate buffer (pHs 6.5−8.5, 200 mM) containing a predetermined quantity of oat bran (300−700 mg) was added to a 50 mL Erlenmeyer flask capped with a septum, and preincubated in a water-bath shaker at an appropriate temperature (40−60 °C) and a shaking rate of 200 rpm for 0− 3 h. The reactions were initiated by adding immobilized alkaline protease (0.25−1.25 U/mL) to the mixture and performed for 0−3 h. Details about amount of enzyme, concentration of oat bran, temperature, and buffer pH are specified for each case. The antioxidant activity of the prepared oat polypeptides was determined by measuring the DPPH radical scavenging rate of oat polypeptides according to the reported method39 with slight modifications. The sample solution (2 mL) was added to 2 mL of 0.1 mM DPPH in ethanol (50%), while the sample blank was performed using 2 mL of ethanol to replace DPPH. The control was determined by adding 2 mL of distilled water into 2 mL of DPPH. Before their absorbance was measured at 517 nm on a UV spectrophotometer, these mixtures were kept in the dark for 30 min. The components of the prepared oat polypeptides were analyzed using high performance liquid chromatography (HPLC, Waters 1525) with a UV detector at 220 nm on a Symmetry C18 column (4.6 mm × 75 mm, 3.5 μm, Waters, USA). The mobile phase was the mixture of water and acetonitrile (95/5, v/v) at 1 mL/min. All data were averages of experiments performed at least in triplicate, with no more than 1% standard deviation. 2.9. Preparative Scale Biotransformation with Alkaline Protease Immobilized on Amino-Functioned Fe3O4 Nanoparticles. The hydrolysis of oat bran was also applied on a 250 mL preparative scale by adding 187.5 U immobilized alkaline protease and 12.5 g of oat bran to phosphate buffer (200 mM, pH 7.5). The reaction was carried out for 2 h at 50 °C and 200 rpm, and the immobilized protease was removed by magnetic force. The antioxidant activity of the prepared oat polypeptides was measured as above.

materials. Then, the mixture was incubated in a water-bath shaker for a specified time (2−6 h) of cross-linking at 25 °C and 200 rpm. Afterward, immobilized alkaline protease was separated by magnetic field, washed for three times with phosphate buffer (200 mM, pH 7.5) and stored in phosphate buffer (200 mM, pH 7.5) at 4 °C for subsequent use. Details about the ratio of enzyme to support materials, cross-linker concentration, and cross-linking time are specified for each case. 2.5. Adsorption of Alkaline Protease onto AminoFunctioned Fe3O4 Nanoparticles without Addition of Cross-Linker. The support material, amino-functioned Fe3O4 nanoparticles (100 mg), was dispersed in 3.6 mL of phosphate buffer solution (200 mM, pH 7.5), and then 0.4 mL of free alkaline protease solution (180 U/mL) was added. The mixture was incubated at 25 °C for 12 h. Then, the magnetic support material was separated by magnetic field. The amount of the enzyme loading onto the support material by absorption in the absence of the cross-linker glutaraldehyde and the enzyme activity were determined according to the subsequent methods described below. Similarly, the concentration and activity of the enzyme in the material-free residual supernatant were assayed. 2.6. Assay of Enzyme Activity and Protein Concentration. Protein concentration was determined according to the Lowry method using bovine serum albumin as standard.37 The activities of free enzyme and immobilized alkaline protease were determined using N-α-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) as a substrate.38 The amount of Nα-benzoyl-L-argininie (BA) released was measured using UV spectrophotometer at 254 nm. The catalytic activity of the samples was determined as the production of 1 μmol of BA by 1 mL of enzyme in 1 min. And the activity recovery of immobilized enzyme and the enzyme loading on the support materials were calculated, respectively, as follows: activity recovery (%) =

total activity of immobilized enzyme (U) 100 total activity of free enzyme used for immobilization (U)

enzyme loading(%) =

total enzyme content of immobilized enzyme (mg) 100 total content of enzyme used for immobilization (mg)

2.7. Characteristics of Free and Immobilized Alkaline Protease. We use BAEE as a model substrate to investigate the characteristics of free and immobilized alkaline protease. 2.7.1. Optimal pH and Temperature. The optimal temperature of the free enzyme and immobilized alkaline protease was determined by adding the enzyme into the substrate solution in 200 mM phosphate buffer pH 7.5 at different temperatures (50−75 °C). The optimal pH was determined by adding the enzyme into the substrate solutions of different pH (7.0−9.0) at 60 °C. The activities of immobilized enzyme and free enzyme samples were determined. 2.7.2. Kinetic Parameters. The apparent kinetic parameters (Km and Vmax) for enzyme were determined under its optimum reaction conditions. The Km and Vmax values for free enzyme and immobilized alkaline protease were determined with varied concentrations of BAEE from 1.0 μM to 1.0 mM in phosphate buffer (200 mM, pH 7.5) at each optimum reaction temperature (60 °C for free enzyme and at 65 °C for immobilized enzyme, respectively) and under the same enzyme activity units for each form of enzyme. The apparent Km and C

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during the modified processes.42 Additionally, Figure S2 (see Supporting Information) showed the presence of nitrogen and silicon after APTES modification, which was consistent with FTIR. The X-ray diffraction spectrum reflected the planes of Fe3O4 and amino-modified Fe3O4 nanoparticles (Figure S3 shown in the Supporting Information) were consistent with the standard pattern for Fe3O4.43 This suggested that the crystalline structure of amino-modified Fe3O4 nanoparticles was not changed during the modification process, indicating that the physical properties of the magnetite particles were not affected by the surface modification and conjugation of the Fe3O4 particles. To study the magnetic properties of magnetite particles before and after APTES modification, a vibrating-sample magnetometer was used to measure the hysteresis loops of magnetite particles and APTES modified magnetic particles at room temperature (Figure S4). The saturation magnetizations for Fe3O4 and amino-modified Fe3O4 nanoparticles were found to be 60 and 45 emu/g, respectively. Consistent with previous studies, the saturation magnetization was affected by the process of modification on the surface of the Fe3O4.44 Both of the two magnetic particles showed negligible coercivity (Hc) and remanence, which suggested the features of superparamagnetic materials.45,46 The phenomenon that immobilized alkaline protease dispersed in reaction medium and was attracted by magnetism was shown in Figure S5, which provided the evidence of easy separation of immobilized alkaline protease from the reaction medium. 3.2. Immobilization of Alkaline Protease onto AminoFunctionalized Fe3O4 Nanoparticles. To achieve an effective alkaline protease immobilized on the prepared amino-functionalized Fe3O4 nanoparticles, it was important to study the effects of several key immobilization parameters, such as the ratio of the amount of the support to enzyme, crosslinker concentration, and cross-linking time, on the enzyme activity recovery and the enzyme loading. As evident from the data illustrated in Figure 3a, the enzyme activity recovery increased with the increasing ratio of the amount of aminofunctionalized Fe3O4 nanoparticles to enzyme from 1/5 to 1/4 (w/v). As the ratio of the amount of the support to enzyme was above 1/4 (w/v), a further increase in the ratio of the amount of the support to enzyme led to a drop in enzyme activity recovery. The enzyme loading was increased with the rise of the ratio. When the amount of amino-functionalized Fe3O4 nanoparticles to enzyme was 1/4 (w/v), the activity recovery of alkaline protease attained 50.2% and the enzyme loading was 67.1%. If the ratio increased, the support materials were easy to assemble. When the ratio became smaller, the loading of enzyme on the surface of magnetic nanoparticles was lower, which resulted in the lower activity recovery. Therefore, the optimal ratio of support to enzyme was considered to be 1/4 (w/v) for immobilization of the alkaline protease onto the amino-functionalized Fe3O4 nanoparticles. Cross-linkers generally play an important role in enzyme immobilization by cross-linking.14−17 Glutaraldehyde was commonly employed as a preferable cross-linker for immobilization of a number of enzymes,33,34 and consequently was applied for immobilization of alkaline protease on the prepared magnetic support materials. As expected, glutaraldehyde concentration showed a significant influence on catalytic performance of the immobilized enzyme. As shown in Figure 3b, the enzyme activity recovery in immobilized alkaline

3. RESULTS AND DISCUSSION 3.1. Characterization of Amino-Functionalized Fe3O4 Nanoparticles. The morphology and the size of the aminofunctionalized Fe3O4 nanoparticles were examined using transmission electron microscopy (TEM) (Figure S1 in Supporting Information). The average particle size ranged from 10 to 15 nm, which was smaller than the critical size (20− 25 nm), thus indicating that the obtained magnetic particles were superparamagnetic.40 Figure 2 showed the Fourier transform infrared (FTIR) spectra of both Fe3 O 4 and amino-functionalized Fe 3 O 4

Figure 2. FTIR spectra of Fe3O4 particles (a) and amino-functionalized Fe3O4 nanoparticles (b).

nanoparticles. It was found that the vibration of Fe−O stretching, O−H stretching, and O−H deformation were near 568, 3410, and 1620 cm−1, respectively. The significant features observed in Figure 2b were the appearance of the peaks at 992 cm−1 (Si−O stretching) and 2923 cm−1 (−CH2 stretching),41 which was formed by the silanization reaction and from APTES, respectively. The peak at 3393 cm−1 in Figure 2b, which was overlapped by the O−H stretching vibration, was probably the free amino groups. This evidence proved that the amino groups were successfully attached to the surface of Fe3O4 particles. The achieved amino-modified Fe3O4 nanoparticles can be further used to covalently immobilize alkaline protease with glutaradehyde. XPS survey spectra and high resolution spectra of the Fe 2p1, Fe 2p3, C 1s, O 1s, N1s, and Si 2p orbitals of Fe3O4 and aminomodified Fe3O4 nanoparticles were obtained. Table 1 gives an overview of the chemical composition (atom %) data for Fe3O4 and amino-modified Fe3O4 nanoparticles. Carbon impurity was observed on the bare magnetic nanoparticles and remained Table 1. Chemical Composition (atom %) of Fe3O4 and Amino-functionalized Fe3O4 Nanoparticles Based on High Resolution XPS Analysis Fe 2p O 1s C 1sa N 1s Si 2p a

Fe3O4 particles

amino-functionalized Fe3O4 nanoparticles

7.63 21.48 70.89 NDb NDb

5.37 20.79 67.67 1.92 4.25

Impurity. bND = not detect. D

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immobilization. Figure 3c depicts the effect of cross-linking time on enzyme activity recovery and enzyme loading. The enzyme activity recovery increased significantly with increasing cross-linking time up to 4 h, and then decreased afterward, which was possibly due to the partial inactivation of the enzyme within a relatively longer cross-linking time (above 4 h). It was clearly seen that the enzyme loading increased when the crosslinking time was prolonged. Obviously, the optimum crosslinking time was 4 h. Under the optimized conditions described above (the ratio of support to enzyme, cross-linker concentration, and cross-linking time was 1/4 (w/v), 28 mM, 4 h), the enzyme activity recovery and the corresponding enzyme loading were 54.2% and 61.3% (equivalent to 388.2 mg of enzyme loading/g support), respectively. On the basis of the amount of the enzyme loading onto the support materials, the activity loss for the immobilized enzyme could be calculated to be only around 11% of its initial enzyme activity, implying that the prepared amino-functionalized Fe3O4 nanoparticles have good biocompatibility with alkaline protease. The results presented here were superior to those reported previously (enzyme loading, 18.3 mg/g; the loss of enzyme activity, 24.3%).47 Furthermore, Bayramoglu et al.48 reported the immobilization of trypsin onto magnetic poly(GMA-MMA)-gMMA beads through adsorption, and afforded about 123.2 mg/ g of enzyme loading and 15.8% of enzyme activity loss. Besides, the enzyme immobilized onto the amino-functionalized Fe3O4 nanoparticles by cross-linking were seen to be more stable than the enzyme immobilized onto the above-described poly(GMAMMA)-g-MMA beads by adsorption. According to the absorption experiment without the addition of the cross-linker glutaraldehyde, the amount of alkaline protease loading onto the magnetic support materials by physical absorption and the corresponding enzyme activity were less than around 1.5% and 1.2%, respectively, relative to initial enzyme amount and activity. Most of the enzyme (more than 98.0%) remained in the support-free residual supernatant. It was concluded that the enzyme was immobilized onto the amino-functionalized Fe3O4 nanoparticles by cross-linking with glutaraldehyde and the physical attachment of the enzyme by the materials was fully negligible. 3.3. Characteristics of Free and Immobilized Alkaline Protease. 3.3.1. Optimal pH and Temperature. As shown in Figure 4a, both the highest activity of immobilized alkaline protease and free enzyme displayed at pH 7.5, which may be due to the little change of electronic valence around the enzyme active center after immobilization. However, immobilized alkaline protease retained more than 92.5% of its relative activity at pH values ranging from 7.5 to 8.5, which was much higher than that of the free enzyme, indicating that the immobilized alkaline protease was more resistant to alkaline conditions. It implied that covalent bonds or hydrogen bonds formed during the process of cross-linking made the immobilized enzyme more stable and have a stronger pH tolerance. Referring to the relative activity of alkaline protease (Figure 4b), the optimal temperature for the free enzyme was 60 °C while it shifted to 65 °C for the immobilized enzyme. It may be due to the covalent bond formation between proteins or protein and support caused by glutaraldehyde, which might increase the conformational inflexibility of enzyme and prevent it from distortion or damage by heat exchange.49,50 This improvement also may be caused by a mass of covalent cross-

Figure 3. Effects of several immobilization variables on activity recovery and enzyme loading of alkaline protease supported by aminofunctionalized Fe3O4 nanoparticles: (□) activity recovery; (○) binding protein. (a) Effect of ratio of enzyme to support, immobilization conditions: 28 mM glutaraldehyde, 6 h cross-linking, 25 °C. (b) Effect of glutaraldehyde concentration, immobilization conditions: 400 μL free enzyme, 100 mg support materials, 6 h cross-linking, 25 °C. (c) Effect of cross-linking time, immobilization conditions: 400 μL free enzyme, 100 mg support materials, 28 mM glutaraldehyde, 25 °C.

protease was enhanced with increasing glutaraldehyde concentration up to 28 mM. Subsequently, a further rise in glutaraldehyde concentration (above 28 mM) led to a fall in the enzyme activity recovery, probably because of the partial inactivation of enzyme caused by excess glutaraldehyde. With the range of the examined glutaraldehyde concentration, the enzyme loading increased with the increase of glutaraldehyde concentration. The relatively lower loading and activity recovery at a lower concentration of glutaraldehyde (below 28 mM) was caused by the insufficient cross-linking of the enzyme. Taking into account the enzyme activity recovery, the optimum glutaraldehyde concentration was 28 mM for enzyme E

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Figure 4. Influences of various pH values (a) and temperatures (b) on activity of immobilized alkaline protease and free enzyme: (□) relative activity of free enzyme; (○) relative activity of immobilized enzyme. The highest activity of immobilized or free enzyme was defined as 100%.

Figure 5. Stabilities of immobilized alkaline protease and free enzyme. (a) Effect of buffer pH. Open symbols (□,○,△), immobilized alkaline protease; solid symbols (■,●,▲), free enzyme: (□, ■) pH 7.0; (○, ●) pH 9.0; (△, ▲) pH 11.0. (b) Effect of temperature. Open symbols, (□,○,△); immobilized alkaline protease; solid symbols (■,●,▲), free enzyme: (□, ■) 40 °C; (○, ●) 60 °C; (△, ▲) 80 °C.

linking between enzyme and amino-functionalized Fe3O4 nanoparticles. 3.3.2. Kinetics Study. Apparent kinetic parameters of free enzyme and immobilized alkaline protease were comparatively studied by measuring initial reaction rates for different concentrations of BAEE at each optimum reaction temperature (60 °C for free enzyme and at 65 °C for immobilized enzyme, respectively). It was shown that the Km value of immobilized alkaline protease was lower than that of the free enzyme (1.7 μM vs 7.5 μM), suggesting an increased affinity of enzyme to substrate. The Vmax of immobilized alkaline protease was 62.5 μM/min, which was lower than that of free enzyme (250 μM/ min). It helps the active site of the enzyme be more suitable toward the substrate due to the conformational changes through immobilization.51 The catalytic efficiency (Vmax/Km) of immobilized alkaline protease was higher than that of the free enzyme (37.0 vs 33.3 min−1), indicating that the alkaline protease immobilized on amino-functionalized Fe3O4 nanoparticles had relatively high catalytic efficiency. The active site of free enzyme was chaotic and trended to different directions; however, after immobilization, the active site of enzyme was orderly arranged, which could be more ready and efficient for the combination with substrate. 3.3.3. Stability. To better understand the characteristics of the immobilized alkaline protease, its stability under different pH levels and temperatures were investigated and the results are shown in Figure 5. It was found that immobilized alkaline protease was more stable in a range of pH (7.0, 9.0, 11.0) than the free enzyme (Figure 5a). The immobilized alkaline protease retained 69.4% of its initial activity, while the relative activity of free enzyme was only around 53.9% for 10 h at each pH. This

improvement in pH stability of immobilized alkaline protease may be due to the covalent cross-linking among enzyme aggregates or enzyme and amino-functionalized Fe3O4 nanoparticles.52 The incubated time course curves of immobilized alkaline protease and free enzyme at different temperatures (40, 60, 80 °C) were shown in Figure 5b. It demonstrated that the temperature tolerance of immobilized alkaline protease was stronger than that of the free enzyme. Because of the poor stability of free enzyme, it deactivated after 8 h at 80 °C. In the tested temperatures, the activities of immobilized alkaline protease were higher than those of the free enzyme. The higher thermal tolerance might be attributed to the immobilization of enzyme to the supports. 3.3.4. Reusability. The reusability of immobilized enzyme is a key factor for its industry applications. So the recycling performance of a biocatalyst is an important index of immobilized enzyme.53 The reusability of the immobilized alkaline protease was investigated using the biocatalytic hydrolysis of BAEE as a model reaction. As shown in Figure 6, after being used repeatedly for 5 and 10 batches in phosphate buffer, the immobilized alkaline protease still remained more than 79.9% and 50.1%, respectively, of their initial activity. As to the properties of easy separation, great stability, and reusability about immobilized alkaline protease, it possesses tremendous potential for the biocatalytic hydrolysis in industry. 3.4. Enzymatic Hydrolysis of Oat Bran into Oat Polypeptides with Immobilized Alkaline Protease. To get a deeper insight into the enzymatic hydrolysis of oat bran into oat polypeptides catalyzed by alkaline protease immobiF

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As depicted in Figure 7a, the amount of immobilized alkaline protease manifested significant influence on the enzymatic reaction. The maximum DPPH radical scavenging rate of oat polypeptides was enhanced remarkably with an increasing amount of enzyme up to 0.75 U/mL, and the corresponding concentration of oat polypeptides attained was also increased significantly. Despite a slight increase of the corresponding concentration of oat polypeptides (from 7.8 to 8.1 mg/mL), a further rise in the amount of enzyme (from 0.75 to 1.25 U/mL) led to an appreciable fall in the maximum DPPH radical scavenging rate (from 79.8% to 74.5%). It is worth noting that the maximum DPPH radical scavenging rate is not always observed with a higher concentration of oat polypeptides, because of different components of oat polypeptides attained at different reaction conditions. So it can be concluded that the DPPH radical scavenging rate of oat polypeptides is related to but not directly proportional to their concentrations under their different components. Obviously, the optimum amount of the used immobilized enzyme was 0.75 U/mL, at which the observed maximum DPPH radical scavenging rate and the corresponding concentration of oat polypeptides obtained at reaction time of 2 h were 79.8%, and 7.8 mg/mL, respectively. Figure 7b illustrated the significant effect of substrate concentration on the enzymatic hydrolysis of oat bran. The maximum DPPH radical scavenging rate was markedly raised when the concentration of oat bran increased from 30 to 50 mg/mL, and further increasing substrate concentration above 50 mg/mL resulted in a significant drop in the maximum DPPH radical scavenging rate. Among the tested range of substrate concentration, the corresponding concentration of oat

Figure 6. Reusability of immobilized alkaline protease for enzymatic hydrolysis of BAEE. Reaction conditions: 2 mL phosphate buffer (200 mM, pH 7.5), 1 mM BAEE, 2.5 mg immobilized alkaline protease, 65 °C, 200 rpm.

lized onto amino-functioned Fe3O4 nanoparticles and enhance the antioxidant activity of oat polypeptides, a systematic investigation was made of the effects of several crucial variables such as amount of immobilized alkaline protease, concentration of oat bran, reaction temperature, and buffer pH on the enzymatic reaction (Figure 7). The DPPH radical scavenging rate was taken as a measure of the antioxidant activity of the attained oat polypeptides, and was determined with the change of reaction time. Also, the concentration of oat polypeptides achieved was assayed when the DPPH radical scavenging rate reached the maximum value.

Figure 7. Effects of several variables on enzymatic hydrolysis of oat bran into oat polypeptides by alkaline protease immobilized onto aminofunctionalized Fe3O4 nanoparticles. (a) Effect of amount of enzyme, reaction conditions: 10 mL (100 mM, pH 7.5) phosphate buffer, 50 mg/mL oat bran, 55 °C, 200 rpm. (b) Effect of substrate concentration, reaction conditions: 10 mL of (100 mM, pH 7.5) phosphate buffer, 0.75 U/mL immobilized enzyme, 55 °C, 200 rpm. (c) Effect of temperature, reaction conditions: 10 mL of (100 mM, pH 7.5) phosphate buffer, 0.75 U/mL immobilized enzyme, 50 mg/mL oat bran, 200 rpm. (d) Effect of buffer pH, reaction conditions: 10 mL of (100 mM, various pHs) phosphate buffer, 0.75 U/mL immobilized enzyme, 50 mg/mL oat bran, 50 °C, 200 rpm. (∗) Reaction time. G

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biocompatibility with alkaline protease. So the magnetic support has a huge potential for immobilization of enzyme. 3.5. Preparative Scale Biotransformation with Alkaline Protease Immobilized on Amino-Functioned Fe3O4 Nanoparticles. To show the applicability of biocatalytic hydrolysis of oat bran to oat polypeptides catalyzed by immobilized alkaline protease on a larger scale, we also performed the biotransformation reaction on a 250 mL scale under the optimum reaction conditions described above (0.75 U/mL immobilized alkaline protease, 2 h reaction time, 50 mg/ mL oat bran, 50 °C, buffer pH 7.5). The concentration and antioxidant activity of the obtained oat polypeptides on the 250 mL scale were marginally lower than the corresponding values on the 10 mL scale (8.1 vs 8.4 mg/mL oat polypeptide concentration; around 80.4% vs 82.3% DPPH radical scavenging rate), but were much better than that reported previously.3 Furthermore, no emulsification in the reaction system was observed on a preparative scale, so the immobilized enzyme on amino-functioned Fe3O4 nanoparticles and the products oat polypeptides could be readily separated from the reaction system by magnetic force and centrifugation, respectively. In particular, alkaline protease immobilized on the promising support of magnetic nanoparticles can be repeatedly employed with no significant activity loss in the next batch, reducing overall cost of the enzymatic process. Hence, the biocatalytic process with alkaline protease immobilized on amino-functioned magnetic nanoparticles is very competitive.

polypeptides attained at the maximum DPPH radical scavenging rate was considerably higher with increasing concentration of oat bran. The optimal substrate concentration was shown to be 50 mg/mL. As can be seen in Figure 7c, the maximum DPPH radical scavenging rate increased from 73.7% to 81.2% with increasing reaction temperature from 40 to 50 °C, and the corresponding concentration of oat polypeptides increased from 7.6 to 8.1 mg/mL. However, when the reaction temperature was increased from 50 to 60 °C, the maximum DPPH radical scavenging rate decreased to 74.5%. Thus, 50 °C was chosen as the optimum reaction temperature for the enzymatic reaction. As illustrated in Figure 7d, various buffer pH values (6.5− 8.5) exhibited significant impact on the enzymatic hydrolysis of oat bran to oat polypeptides. Lowering buffer pH from 7.5 to 6.5 gave rise to the decrease of both the maximum DPPH radical scavenging rate (from 82.3% to 67.5%) and the corresponding concentration of oat polypeptides (from 8.4 to 7.5 mg/mL), possibly because of partial loss of alkaline protease at a relatively low pH level. When buffer pH was raised from 7.5 to 8.5, similarly, the maximum DPPH radical scavenging rate and the corresponding concentration of oat polypeptides obtained decreased significantly. Thus, the optimal buffer pH was shown to be 7.5. Under the above-optimized reaction conditions (0.75 U/mL immobilized enzyme, 50 mg/mL oat bran, 50 °C, buffer pH 7.5) for the hydrolysis of oat bran catalyzed by alkaline protease immobilized onto amino-functionalized Fe3O4 nanoparticles, the maximum DPPH radical scavenging rate of oat polypeptides attained at a reaction time of 2 h was as high as 82.3% and the corresponding concentration of oat polypeptides recorded was 8.4 mg/mL, which were higher than the corresponding values (DPPH radical scavenging rate, 71.9%; concentration oat polypeptides, 7.5 mg/mL) with free enzyme (alkaline protease) under its own optimal reaction conditions (1.0 U/mL free enzyme, 60 mg/mL oat bran, 45 °C, buffer pH 7.5). Furthermore, the antioxidant activity of oat polypeptides prepared by immobilized alkaline protease was much higher than that reported previously.3,54 Interestingly, the DPPH radical scavenging rate of oat polypeptides prepared by immobilized alkaline protease was superior to that by free enzyme (76.5% vs 67.8%) at the same concentration of oat polypeptides (around 7.0 mg/mL), showing the enhanced antioxidant activity of oat polypeptides prepared by immobilized alkaline protease. This might be attributable to the different components of oat polypeptides between using immobilized alkaline protease and free enzyme. Subsequently, the components of oat polypeptides achieved by immobilized and free enzymes, respectively, were subjected to HPLC analysis, as indicated in Supporting Information, Figure S6. It was found that the immobilized alkaline protease afforded a higher content of relatively hydrophobic components of oat polypeptides than the free enzyme (about 44.2% vs 16.6%), which might contribute to the enhanced antioxidant activity of oat polypeptides attained.55 Each component of the prepared oat polypeptides and its effect on the antioxidant activity need further investigations, and these are underway in our laboratory. Alternately, it is generally difficult to separate the enzyme immobilized onto nonmagnetic supports from the reaction system through centrifugation or filtration, while the magnetic supports can solve the problem easily through extra magnetic field. Moreover, the prepared magnetic support showed good

4. CONCLUSIONS Alkaline protease was successfully immobilized onto aminofunctioned magnetic nanoparticles, which solved the problems of enzyme recycling, and substrate and product separation. The immobilized enzyme possessed greater pH and thermal stabilities. The kinetic study for both immobilized and free enzymes showed that the immobilized alkaline protease had relatively high catalytic efficiency. The oat polypeptides obtained with the immobilized enzyme displayed higher antioxidant activity than that with the free enzyme. Besides, the protease immobilized onto amino-functionalized Fe3O4 nanoparticles was demonstrated to be very promising for the preparation of oat polypeptides on a preparative scale.

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ASSOCIATED CONTENT

S Supporting Information *

TEM image of amino-functionalized Fe3O4 nanoparticles (Figure S1); XPS survey of Fe3O4 particles (a) and aminofunctionalized Fe3O4 nanoparticles (b) (Figure S2); X-ray diffraction spectra of Fe3O4 particles (a) and amino-functionalized Fe3O4 nanoparticles (b) (Figure S3); hysteresis loops of Fe3O4 particles (a) and amino-functionalized Fe3O4 nanoparticles (b) (Figure S4); dispersion of immobilized alkaline protease in reaction medium (a) and its separation from reaction system by magnetic force (b) (Figure S5); HPLC analysis for the components of oat polypeptides that were prepared by immobilized alkaline protease (a) and free enzyme (b), respectively (Figure S6). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-20-22236669. Fax: +86-20-22236669. E-mail: [email protected]. H

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We wish to thank the National Science Foundation for Excellent Young Scholars (21222606), the State Key Program of National Natural Science Foundation of China (21336002), the NSFC (21376096), the Key Program of Guangdong Natural Science Foundation (S2013020013049), and the Fundamental Research Funds for SCUT (2013ZG0003) for partially funding this work.

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